Function and targets of the

Urm1/Uba4 conjugation

machinery in Drosophila

melanogaster

Behzad Khoshnood

Department of Molecular Biology

Umeå 2017

This work is protected by the Swedish Copyright Legislation (Act 1960:729)

Dissertation for PhD

Umeå University Medical Dissertations New Series No. 1939

ISBN: 978-91-7601-815-6

ISSN: 0346-6612

Cover photo by Behzad Khoshnood, inspired by the art work from Nicolle Ragera

from National Science Foundation of USA. The image belongs to public domain.

Electronic version available at: http://umu.diva-portal.org/

Printed by: UmU print service, Umeå University

Umeå, Sweden 2017

رهزگ دل من ز علم محروم نشد

کم ماند ز ارسار هک معلوم نشد

هفتاد و دو سال فکر رکدم شب و روز

معلومم شد هک چیه معلوم نشد

خیام

My heart was never deprived of the passion for science

There was little of the mysteries that I did not understand

Days and nights, my whole life I have contemplated

Only to realise how little I know

Omar Khayyam 1048-1131

i

Table of Contents

1. Abstract ........................................................................................ iii

2. Papers included in this thesis ........................................................ v

3. Abbreviations ............................................................................... vi

4. Introduction ................................................................................... 1 4.1. Drosophila melanogaster, serving science as a model system ................................ 1

4.1.1 History ................................................................................................................ 1 4.1.2 Significance of using Drosophila as a model system ......................................3

4.2. Development and life cycle of Drosophila ............................................................... 4 4.2.1 Embryonic development ................................................................................... 5 4.2.2 Larval development and metamorphosis....................................................... 6 4.2.3 Eclosure and adult life ...................................................................................... 7

4.3 Neuromuscular junctions (NMJs) ............................................................................. 7 4.3.1 Structure and classification of NMJs .............................................................. 8 4.3.2 Activity and development of NMJs ................................................................. 9

4.4 Posttranslational modification and UBLs ............................................................... 10 4.4.1 Ubiquitin .......................................................................................................... 11 4.4.2 Ubiquitin-like modifiers (UBLs) ..................................................................... 12

4.5 Urm1 - Ubiquitin related modifier 1 ........................................................................ 13 4.5.1 Urm1 from evolutionary perspective ............................................................. 13 4.5.2 Structure and Biochemistry ........................................................................... 14 4.5.3 Urm1 in tRNA modification ............................................................................ 15 4.5.4 Urm1 as a protein modifier ............................................................................ 17

4.6 Uba4 - The activating enzyme for Urm1 .................................................................. 18 4.6.1 Structure and function of Uba4 ...................................................................... 19

4.7 Oxidative stress ......................................................................................................... 21 4.7.1 Molecular mechanisms behind oxidation and reduction .............................. 21

4.8 The JNK pathway .................................................................................................... 22 4.8.1 The JNK pathway in Drosophila ................................................................... 23 4.8.2 The JNK pathway in stress responses and lifespan regulation .................. 25

4.9 Drosophila as a model to study Tax-mediated oncogenesis.................................. 26 4.9.1 Adult T-cell leukaemia/lymphoma ................................................................ 26 4.9.2 HTLV-1 and Tax .............................................................................................. 27 4.9.3 The NF-B pathway ....................................................................................... 28 4.9.4 Posttranslational modification of Tax1 ........................................................ 29

5. Results and Discussion ................................................................. 31 5.1. Paper I ...................................................................................................................... 31

Urm1: an essential regulator of JNK signaling and oxidative stress in Drosophila

melanogaster. ........................................................................................................... 31 5.2. Paper II.....................................................................................................................35

A proteomics approach to identify targets of the ubiquitin-like molecule Urm1 in

Drosophila melanogaster ........................................................................................35

ii

5.3. Paper III .................................................................................................................. 36 The HTLV-1 oncoprotein Tax is modified by the Ubiquitin related modifier 1 ... 36

5.4. Paper IV .................................................................................................................. 38 Deciphering a novel role of the Urm1/Uba4 conjugation machinery for

Neuromuscular Junction (NMJ) development in Drosophila melanogaster ...... 38 5.5. General discussion and future perspectives .......................................................... 42

6. Acknowledgements ..................................................................... 43

7. References ................................................................................... 46

iii

1. Abstract

Posttranslational modification (PTM) of proteins is essential to maintain

homeostasis and viability in all eukaryotic cells. Hence, besides the sequence and

3D folding of a polypeptide, modification by multiple types of PTMs, ranging

from small molecular groups to entire protein modules, adds another layer of

complexity to protein function and regulation. The ubiquitin-like modifiers

(UBLs) are such a group of evolutionary conserved protein modifiers, which by

covalently conjugating to target proteins can modulate the subcellular

localization and activity of their targets. One example of such a UBL, is the

Ubiquitin related modifier 1 (Urm1). Since its discovery in 2000, Urm1 has been

depicted as a dual function protein, which besides acting as a PTM, in addition

functions as a sulfur carrier during the thio-modification of a specific group of

tRNAs. Due to this dual capacity, Urm1 is considered as the evolutionary ancestor

of the entire UBL family. At present, it is well established that Urm1, with help of

its dedicated E1 enzyme Uba4/MOCS3, conjugates to multiple target proteins

(urmylation) and that Urm1 thus plays important roles in viability and the

response against oxidative stress.

The aim of this thesis has been to, for the first time, investigate the role of Urm1

and Uba4 in a multicellular organism, utilising a multidisciplinary approach that

integrates Drosophila genetics with classical biochemical assays and proteomics.

In Paper I, we first characterized the Drosophila orthologues of Urm1 (CG33276)

and Uba4 (CG13090), verified that they interact physically as well as genetically,

and that they together can induce urmylation in the fly. By subsequently

generating an Urm1 null Drosophila mutant (Urm1n123), we established that

Urm1 is essential for viability and that flies lacking Urm1 are resistant to oxidative

stress. Providing a molecular explanation for this phenotype, we demonstrated

an involvement of Urm1 in the regulation of JNK signaling, including the

transcription of the cytoprotective genes Jafrac1 and gstD1. Besides the

resistance to oxidative stress, we have moreover (Manuscript IV) made an in-

depth investigation of another phenotype displayed by Urm1n123 mutants, an

overgrowth of third instar larval neuromuscular junctions (NMJs), a phenotype

which is shared also with mutants lacking Uba4 (Uba4n29).

To increase the understanding of Urm1 in the fly, we next employed a proteomics-

based approach to identify candidate Urm1 target proteins (Paper II). Using this

strategy, we identified 79 Urm1-interacting proteins during three different stages

of fly development. Of these, six was biochemically confirmed to interact

covalently with Urm1, whereas one was found to be associated with Urm1 by non-

covalent means. In Manuscript III, we additionally identified the virally encoded

iv

oncogene Tax as a target of Urm1, both in Drosophila tissues and mammalian cell

lines. In this study, we established a strong correlation between Tax urmylation

and subcellular localisation, and that Urm1 promoted a cytoplasmic

accumulation and enhanced signalling activity of Tax, with implications for a

potential role of Urm1 in Tax-induced oncogenesis.

Taken together, this thesis provides a basic understanding of the potential roles

and targets of Urm1 in a multicellular organism. The four studies included cover

different aspects of Urm1 function and clearly points towards a highly dynamic

role of protein urmylation in fly development, as well as in adult life.

v

2. Papers included in this thesis

I. Khoshnood, B., Dacklin, I. and Grabbe, C. (2016). Urm1: an

essential regulator of JNK signaling and oxidative stress in

Drosophila melanogaster. Cell Mol Life Science. 73:1939-1954.

II. Khoshnood, B., Dacklin, I. and Grabbe, C. (2017). A proteomics

approach to identify targets of the ubiquitin-like molecule Urm1 in

Drosophila melanogaster. PLoS One. 2017 Sep 27;12(9):e0185611.

III. Hleihel R*, Khoshnood B.*, Dacklin I, Omran H, Mouawad C,

Dassouki Z, El-Sabban M, *Shirinian M, *Grabbe C and *Bazarbachi

A. (2017) The HTLV-1 encoded oncoprotein Tax is modified by the

Ubiquitin related modifier 1 (Urm1). Submitted manuscript.

IV. Khoshnood, B., Dacklin, I. and Grabbe, C. (2017). Deciphering a

novel role of the Urm1/Uba4 conjugation machinery during

neuromuscular junction (NMJ) development in Drosophila

melanogaster. Manuscript.

vi

3. Abbreviations

AHP Alkyl hydroperoxide reductase

AIDS Acquired immune deficiency syndrome

ALDH Aldehyde dehydrogenases

AP-1 Activator protein 1

aPKC Atypical protein kinase C

AS2O3 Arsenic trioxide

ATAC Ada two a containing

ATF cAMP-dependent transcription factor

ATG Autophagy-related protein

ATL Adult T-cell leukaemia/lymphoma

ATP Adenosine triphosphate

cAMP Cyclic adenosine monophosphate

CD Cluster of differentiation

CDK Cyclin-dependent kinase

CG Computed gene

CNS Central nervous system

CREB cAMP response element-binding protein

CRISPR Clustered regularly interspaced short palindromic repeats

dMKK Drosophila MAP kinase kinase

DNA Deoxyribonucleic acid

DTT Dithiothreitol

DUB Deubiquitinating enzyme

Eip71CD Ecdysone-induced protein 28/29 kd

ELP Elongator protein

ERK Extracellular signal-regulated kinases

ESCRT Endosomal-sorting complex required for transport

FAT HLA-F-adjacent transcript

FUB Function of boundary

GAL4 Galactose-responsive transcription factor 4

GFP Green fluorescent protein

GG Glycine-glycine

GILT1 Gamma-interferon-inducible lysosomal thiol reductase 1

GST Glutathione S-transferase

vii

GstD1 Glutathione S transferase D1

H2O2 Hydrogen peroxide

HA Human influenza hemagglutinin

HAM/TSP HTLV-1-associated myelopathy/tropical spastic paraparesis

HCF Host cell factor

Hep Hemipterous

HIV Human immunodeficiency virus

Hiwi Highwire

Hsp Heat shock protein

HTLV Human T-lymphotropic virus

IFN Interferon

IKK IκB kinase

IL Interleukin

ISG15 Interferon-induced 15 kda protein

IκB Inhibitor of kappa B

JNK c-Jun N-terminal kinase

JNKK c-Jun N-terminal kinase kinase

JNKKK c-Jun N-terminal kinase kinase kinase

JNKKKK c-Jun N-terminal kinase kinase kinase kinase

K Lysine

LOF Loss of function

MAPK Mitogen-activated protein kinase

MBIP MAPK binding inhibitory protein

MCM5 5-methoxycarbonylmethyl

MCM5S2U 5-methoxycarbonylmethyl-2-thiouridine

MEKK Mitogen-activated protein kinase kinase kinase

MKP Map kinase phosphatase

mM Millimolar

MoaD Molybdenum cofactor biosynthesis protein D

MoaE Molybdenum cofactor biosynthesis protein E

MoCo Molybdenum cofactor

MOCS Molybdenum cofactor synthesis

MoeB Molybdenum cofactor biosynthesis protein B

MPT Molybdopterin

mRNA Messenger RNA

MVB Multivesicular bodies

viii

NADPH Nicotinamide adenine dinucleotide phosphate

NCS Nucleobase cation symporter

NEDD8 Neural precursor cell expressed, developmentally down-regulated 8

NF-κB Nuclear factor kappa-light-chain-enhancer of activated B cells

NLS Nuclear localization signal

NMJ Neuromuscular junction

NMR Nuclear magnetic resonance

NQO1 Nad(p)h quinone dehydrogenase 1

Nrf2 Nuclear factor (erythroid-derived 2)-like 2

O2- Superoxide anion

OH- Hydroxyl radical

ORF Open reading frame

pHgpX Phospholipid-hydroperoxide glutathione peroxidase

PI3K Phosphatidylinositol-3 kinase

pJNK PhosphoJNK

PML Promyelocytic leukemia

PRX Peroxiredoxin

PTM Posttranslational modification

RHD Rhodanese homology domain

RING Really intresting new gene

RNA Ribonucleic acid

RNAi RNA interference

RNF Ring finger protein

ROS Reactive oxygen species

S Sulfur

SAMP Small archaeal modifier protein

SAPK Stress-activated protein kinase

SSR Subsynaptic reticulum

SUMO Small ubiquitin-like modifier

TAK Transforming growth factor beta-activated kinase

TGFβ Transforming growth factor beta

tRNA Transfer RNA

U34 Uridine 34

UAS Upstream activation sequence

UBA Ubiquitin activating enzyme

UBC Ubiquitin conjugating enzyme

ix

UBL Ubiquitin like protein

UFM Ubiquitin fold modifier

URM Ubiquitin related modifier

UV Ultra violet

1

4. Introduction

4.1. Drosophila melanogaster, serving science as a model

system

If we set world domination as the ultimate aim of life, there are only a few living

organisms that can compete with the six-legged creatures that are found in most

eco-systems on earth, the insects. Insects are the most diverse group of animals

on earth, with a number of ~1.5 million species described to date and an estimated

number of 10 quintillion individuals alive at any given time point (Larsen et al.,

2017). As one of the most ancient groups of animals, their origin of evolution

dates back to 479 million years ago (Misof et al., 2014).

Even though insects may be small and appear unimportant, they contribute

significantly to human life in both good and bad ways. Insects can function as

vectors for transferring human diseases such as malaria, but are at the same time

indispensable for the pollination of fruits and nuts that are essential for our health

and diet.

For more than a century, one specific species of insects has captured the attention

of scientists. Heavily used as a model system in biological science, Drosophila

melanogaster, in plain language known as the fruit fly, has significantly

contributed to our understanding of cell and molecular processes, as well as

genetics. D. melanogaster belongs to the “old-world” clade of the Sophophora

subgenus within the Drosophilidae family (Van der linde et al., 2010), are roughly

2-2.5 mm in length, yellow-brown in colour with black stripes across the

abdomen, and have brick read eyes. There are many reasons for why the fruit fly

has become such a successful model system in research. Partly, it may be due to

the rather fast and high reproduction rate of insects, compared to other animals,

together with the low cost of maintaining and handling fly stocks. Moreover, the

risk factors associated with handling mice or rats, namely biting or inter-species

diseases, is virtually absent in the fly. In the following sections, I will describe

more beneficial aspects of D. melanogaster as a model system and highlight the

importance of Drosophila research in modern science.

4.1.1 History

The very first steps of using Drosophila for research were taken in Thomas Hunt

Morgan’s lab at Columbia University, the USA. His isolation of the first naturally

2

occurring mutation, in the white gene locus in D. melanogaster (Morgan 1910),

marks the clear starting point for decades of follow-up research. A few years later,

Morgan and his three famous students, A. H. Sturtevant, C. B. Bridges and H. J.

Muller, provided the first ground-breaking proof for the chromosomal

inheritance theory, which was in agreement with Mendelian genetics (Sturtevant,

1913; Morgan, Sturtevant and Bridge, 1920)

One of the most important findings from those revolutionary years, was when

Muller discovered that X-ray irradiation can induce mutations in the fly genome

and thereby exploited as a tool in research. Following his presentation at the Fifth

International Congress of Genetics in 1927, entitled “The problem of Genetic

Modification”, he became one of the most respected scientists of the 20th century.

The significance of Muller’s work was further acknowledged in 1946, when he

similar to his mentor (T. H. Morgan), was awarded the Nobel Prize in Physiology

or Medicine.

Throughout the years, many researchers have contributed to expanding the

Drosophila research toolbox. The earliest example of these tools was based on the

discovery that X-ray induced chromosomal inversions can suppress the crossover

of genes (Muller, 1918). This finding resulted in the generation of the first

balancer chromosome, today widely used to maintain recessive mutations in fly

stocks (Bentley Glass, 1933; Araye and Sawamura, 2013). Another key event in

the development of tools to study fly genetics, was the introduction of P elements

for mutagenesis and transgenesis, reported in (Spradling and Rubin, 1982). By

learning the skill of mobilizing non-autonomous P elements by introduction of an

exogenous transposase (e.g. Δ2-3), Drosophila researchers got their hands on a

multi-application gadget that could be employed for both chromosome

engineering and gene manipulation (Ryder and Russell, 2003).

In 1993, Brand and Perrimon published a paper describing the UAS/GAL4

system, which revolutionized the Drosophila field yet again (Brand and

Perrimon, 1993; Pan et al., 2009) (Figure 1). By integrating knowledge from yeast

(S. cerevisiae) genetics into Drosophila, in which the GAL4 transcription factor

induces transcription by binding to UAS (upstream activating sequence)

sequences, the UAS/GAL4 system was launched as a technique to control gene

expression in a tissue-specific manner. Since then, the UAS/GAL4 system has

been applied in numerous different ways and today it offers scientists a

methodology to perform sophisticated in vivo experiments. At present, there are

numerous cell- and tissue-specific GAL4 drivers and an extensive amount of UAS

transgenic constructs available to the fly community, which has resulted in that

there nowadays are hardly any Drosophila labs that do not employ the

UAS/GAL4 system in their research.

3

Figure 1. The UAS/GAL4 system.

4.1.2 Significance of using Drosophila as a model system

I personally think that the modern era of Drosophila genetics began in the year

2000, when the entire genome of D. melanogaster was sequenced (Adams et al.,

2000). This fascinating source of information revealed that the whole genome of

Drosophila is composed of roughly 14 000 genes, compiled into only four

chromosomes. Moreover, despite the lower complexity of the fly genome, flies

still share many similarities with humans, such as genetic networks and

physiological functions, as well as more complex behaviors such as memory, sleep

and addiction. One important characteristic of the Drosophila genome, which has

contributed to the success of the fruit fly as a model system, is that it in general

contains a lower number of genes in each gene class, resulting in a low genetic

redundancy. This feature makes the interpretation of mutant phenotypes more

straightforward and simplifies the analysis of systemic loss-of-function (LOF)

mutations.

In recent years, the introduction of the CRISPR/Cas system into fruit fly field (Yu

et al., 2013; Gratz et al., 2015) has made life much easier for Drosophila

researchers. This relatively easy and fast methodology to induce genetic

manipulations, combined with the fast-result-scoring nature of the fruit fly, has

made the Drosophila an interesting model to study almost all aspects of genetics

and physiology.

4

Despite its small genome, the adult fly is a fairly sophisticated and complex

organism. The Drosophila displays many tissues that function in a similar

manner as their mammalian counterparts, such as the heart, gut, and

reproductive tract, as well as a complex and fascinating brain made of a

remarkable number of more than 100,000 neurons. Together with the

continuous verification of gene functions that are conserved from flies to humans,

this encourages scientists to continue using flies as a model to study human

biology and disease. Throughout the years, a large number of researchers have

employed Drosophila to address complex scientific questions concerning

immunity, drug development, and cancer, making it obvious that this little fruit

fly is a potent model organism that has contributed a lot to our understanding of

biological life.

4.2. Development and life cycle of Drosophila

Partly, the feasibility of using Drosophila as a model system comes from its rather

fast development and high reproduction rate. The life cycle of the Drosophila

starts with the fertilized egg, which after 24 hours of embryonic development

hatch into a 1 mm long larvae. This larva is basically an eating machine that

during five to six days of larval development increases its size 200 times. During

this process the larva molts three times, marking the onset of three separate larval

stages, the first, second and third instar stages (Church and Robertson, 1966). By

the end of the third instar stage, the larva forms a pre-pupa and eventually a pupa

(Figure 2), in which the fly goes through a complete metamorphosis. During

metamorphosis, most larval tissues are degraded and the adult fly forms from

information laid down in the larval imaginal discs. After 4-5 days of pupal

development, the fly finally ecloses from the pupa and after a few hours of

maturation, the adult fly is ready for mating and reproduction.

The life cycle and the distinctive stages of fly development strongly contributes to

why Drosophila is such a powerful model system, since it offers multiple different

angles of approach for scientific work, yet they are all Drosophila. For example,

Drosophila embryos are excellent tools to answer developmental questions, while

the larvae and adults can be used for physiological and behavioral investigations.

In addition, there is plenty of potential to perform in vitro experiments in fly cell

lines, such as the Drosophila Schneider (S2) cells.

Each cycle of development, from egg laying to maturation of the adult fly, takes

roughly 10 days to complete (at 25C). It is also worth noting that a female fly can

5

Figure 2. Life cycle of Drosophila melanogaster.

produce up to 500 (Ashburner, Golic and Hawley, 2005) offspring during her life

time. In spite of the rather short period of development, Drosophila

melanogaster is still a very complex organism with diverse anatomical

properties. The specific details regarding development of the nervous system,

heart, muscles, digestive tract etc. of the fruit fly have been extensively described

in multiple reviews (Jennings, 2011; McGurk, Berson and Bonini, 2015; Yaniv

and Schuldiner, 2016; Liu and Jin, 2017; Piper and Partridge, 2017). However, I

will below give a brief description of each period of the fly development.

4.2.1 Embryonic development

Within two hours after fertilization and 13 mitotic divisions, each Drosophila

zygote contains roughly 6000 nuclei that share a single cytoplasm, thus forming

a syncytium. Already upon egg-laying, the oocyte inherits a huge supply of mRNA

molecules from the female. These “maternally contributed” mRNA molecules

control protein expression in early embryonic stages, until the zygotic

transcription starts to take over. Since there are no cell membranes separating

the cells in the syncytial embryo, the maternal factors can freely diffuse in the

syncytium, and thereby form morphogenic gradients. These gradients are

6

responsible for setting up the polarity and provide cues for cellular identity in the

early embryo, defined by the differential localization and concentration of these

mRNA molecules. The Nobel Prize winning work of Christiane Nüsslein-Volhard

and Eric Wieschaus, together with Edward B. Lewis, revolutionized the field of

embryonic development. Together, they identified many genes important for

early development, among which the morphogen-encoding genes that defines the

anterior-posterior patterning, and the homeotic genes the orchestrates

segmentation of the embryo, may be most important (Garcia-Bellido and Lewis,

1976; Nüsslein-volhard and Wieschaus, 1980; Lewis, 1982, 2007). In early

embryos bicoid and hunchback mRNAs determine the formation of the head and

thorax, while the protein products of nanos and caudal mRNAs are critical for

formation of the abdominal segments (Zaffran, Robrini and Bertrand, 2014).

After the first 13 divisions, each nuclei in the syncytial embryo becomes an

individual cell (Mahowald 1963), whereupon gastrulation begins. Later, the

embryonic ectoderm is divided into visible segments that will define the

segmented structure of the adult insect (Levine, 2008).

The whole process of embryonic development takes around 24 hours at room

temperature, and has been divided into 17 distinct stages (Campos-Ortega 1985).

By the end of stage 17, the foundation of all critical organs required for larval

development, such as body wall muscles, the intestinal tract, trachea and most

importantly the nervous system, has formed.

4.2.2 Larval development and metamorphosis

In the final stage of embryogenesis (stage 17) the tracheal tree gets filled with air

and 1-2 hours later the first instar larvae hatches out from the egg. The sole

purpose of this embryo-sized wormlike creature is eating and growing, passing

through the three instar larval stages.

Third instar larvae are heavily used in fly labs. Besides being semi-transparent,

which facilitates the investigation of living animals under a fluorescent

microscope, the internal organs are rather accessible and easy to dissect.

Furthermore, the presence of fascinating tissues such as salivary glands with

giant cells and polytene chromosomes, is a prime feature for researchers.

Another interesting feature of the larval stages is the presence of imaginal discs,

the epithelial sac-like structures of dividing cells, which are the primordia of

many of the major adult insect structures, such as wings, legs, halters, antennae

and genitalia. Therefore, many of the morphological phenotypes of the adult fly

have its origin in these tissues. When reaching a critical weight, associated with a

distinctive pulse of the fly hormone Ecdysone, the third instar larvae crawls out

7

of the food in search of a dry place to pupariate (Berreur et al., 1984). During the

pupal stages, a majority of the adult structures are developed. Inside the pupa,

the Drosophila undergoes a complete metamorphosis, where most of the larval

tissues, except the central nervous system (CNS), are degraded and the insect is

rebuilt following the instructions laid down in the imaginal discs (Campos-

Ortega, 1997; Brody, 1999; Grumbling, 2006)

4.2.3 Eclosure and adult life

Adult fruit flies emerge from the pupal case after 3-4 days of pupal development.

In the last step of morphogenesis, the wings are expanded and the fly acquires its

final shape. The mean lifespan of an adult fruit fly is 2-3 months, when kept at

room temperature and with sufficient supply of food (Helfand and Rogina, 2003).

Apart from genetic factors, diet and food intake are critical determinants of fly

longevity (Piper et al., 2011). However, adult life for a fruit fly is much more

challenging out in nature, where it in order to survive needs to compete for

resources and overcome environmental stressors and other threats, resulting in

a significantly shorter life span.

4.3 Neuromuscular junctions (NMJs)

The Drosophila larval nervous system is an invaluable model to study the

development and growth of neuromuscular junctions (NMJs), the insect

equivalent to mammalian motor end plates. Simplicity aside, it is relatively easy

to access the large, individually specified NMJs of a larvae in order to visualize or

record their activity (Menon, Carrillo and Zinn, 2013). Similar to synapses in

vertebrates, Drosophila NMJs contain glutamatergic synapses and exhibit

functional plasticity, supporting the relevance of using Drosophila NMJs to

understand neural development and function in general (Fernandes and

Keshishian, 1999; Menon, Carrillo and Zinn, 2013).

The larval body is composed of three head segments, three thoracic segments (T1-

T3) and eight abdominal segments (A1-A8). Of these, the larval locomotory

system is formed by the body wall musculature in the thoracic and abdominal

segments, together with the motor neurons that extends from the ventral nerve

cord to innervate them (Kohsaka et al., 2012). There are 32 motor neurons in

each abdominal hemisegment, each one innervating its specific target muscle.

The rather stereotyped pattern of connectivity in the neuromuscular system is

laid down already during embryogenesis, but the NMJs undergo extensive

changes in size and morphology also during postembryonic development

8

(Griffith and Budnik, 2006; Kohsaka et al., 2012; Menon, Carrillo and Zinn,

2013). In the following sections of this chapter I will briefly review the current

knowledge about Drosophila NMJs. (Figure 3)

Figure 3. Neuromuscular anatomy of Drosophila third instar larvae, displaying the dissected body wall musculure (marked by phalloidin in green) and the motor neurons

innervating them (marked by Cy3- conjugated anti-HRP antibodies in red). A close-up of the NMJ on muscle 4, abdominal segment 3, indicates the bouton morphology and

active zones (marked by anti-Brp antibodies).

4.3.1 Structure and classification of NMJs

Each Drosophila NMJ consists of a stereotyped number of boutons (10-50 per

muscle), i.e. points of contact between the motor neurons and the body wall

musculature. The boutons are oval-shaped structures that house the synapses,

which are concentrated at a number of neurotransmitter release sites, called

active zones. In these active zones, proteins such as Bruchpilot (Brp) are

specifically important for an effective organization and clustering of Ca2+

channels (Fouquet et al., 2009), and can also be used as markers for visualization

of the active zones. On the postsynaptic side, another structural feature of the

9

boutons can be found, the subsynaptic reticulum (SSR). The SSR is a highly

folded specialized membrane domain that contains neurotransmitter receptors,

ion channels, scaffolding proteins and postsynaptic signaling complexes. A key

protein present in the SSRs is Discs large (Dlg), which is essential for controlling

the shape, size and function of the synaptic structure. Loss of Dlg results in severe

defects in SSR expansion (Lahey et al., 1994).

The Drosophila NMJ boutons are classified into three categories, type I, II and

III, which differ in morphology and amount of SSR, as well as their type of

neurotransmitter. Type I boutons are further divided into two classes, type Ib

(big) and type Is (small), which primarily differ in size and the amount of Dlg that

is accumulated on the post-synaptic side (Menon, Carrillo and Zinn, 2013).

4.3.2 Activity and development of NMJs

As indicated earlier, the development of the fly NMJs begins during

embryogenesis (stage 13-15). Throughout the development, however, there are

three major stages of synaptic refinement. The first synaptic refinement occurs in

late embryonic stages, when misplaced synapses on non-target muscles are

eliminated (Keshishian et al., 1993; Keshishian, Chang and Jarecki, 1994; Jarecki

and Keshishian, 1995; Carrillo et al., 2010). The next refinement occurs during

larval development, primarily to compensate for the tremendous increase in

muscle size. During this period, boutons are continuously added to (and removed

from) the NMJs, meanwhile the number of active zones at each bouton are also

increasing up to tenfold (Atwood, Govind and Wu, 1993; Davis, Schuster and

Goodman, 1996). The last round of synapse refinement takes place during

metamorphosis, where dismantling of synapses occurs via signaling pathways

instructed by postsynaptic side (Liu et al., 2010).

Over the past decades a large set of genes, including spastin, futsch and shaggy,

have been characterized as important for the regulation of synaptic targeting,

growth and refinement of Drosophila NMJs (Menon, Carrillo and Zinn, 2013;

Vonhoff and Keshishian, 2017). Among these, one group of genes contribute to

the regulation of NMJ growth by specifically altering the neuronal terminal

cytoskeleton and positively regulating the bouton number. A few examples of

such genes are futsch (Roos et al., 2000), which acts through atypical protein

kinase C (aPKC) (Ruiz-Canada et al., 2004) as well as arrow (arr) and

dishevelled (dsh), which act through the Wnt signaling pathway (Miech et al.,

2008). In contrast, genes such as the E3 ubiquitin ligase highwire (hiw) have

been shown to negatively regulate NMJ growth, resulting in a NMJ overgrowth

phenotype in LOF mutants (Wan et al., 2000; DiAntonio et al., 2001; Wu, 2005).

10

One major signaling pathway involved in the regulation of synaptic growth is the

Jun N-terminal kinase (JNK) pathway (Sanyal et al., 2002, 2003; Collins et al.,

2006). In fact, research has shown that the underlying reason for the NMJ

overgrowth displayed by hiw mutants is a hyperactivation of the AP-1

heterodimeric transcription factor, via the JNK pathway (Collins et al., 2006;

Shen and Ganetzky, 2009) resulting in a transcriptional upregulation of a panel

of downstream genes that affect synaptic growth and activity. Complementary

work by Etter and co-workers (Etter et al., 2005) has in addition identified a list

of genes that are transcriptionally upregulated in neurons with hyperactive JNK

signaling. Because of the significance of the JNK pathway in oxidative stress,

lifespan and neuromuscular junction development, I have dedicated chapter 4.8

of this thesis to discuss the JNK signaling in more detail.

4.4 Posttranslational modification and UBLs

Posttranslational modification (PTM) is a molecular event, in which amino-acid

residues of a protein can be modified, often resulting in a change in the target

protein, such as altered structural conformation or subcellular localization,

increased or decreased activity, or alteration of protein stability (Prabakaran et

al., 2012). To rapidly change the properties of target proteins by the addition of

PTMs, is a powerful tool for regulating biological processes in all organisms

across evolution, from bacteria to humans (Grangeasse, Stülke and Mijakovic,

2015). While some prevalent PTMs such as phosphorylation and ubiquitination

are dynamic and reversible, others are thought to be irreversible or more

accurately put, “not known to be reversible” and possess limited processing

capacity (Prabakaran et al., 2012). For example, the anchoring of signal-

transduction molecules such as Src and Ras to the cytoplasmic side of the plasma

membrane is facilitated by the covalent attachment of lipid groups, which are not

readily reversible (Berg et al., 2002).

To date, more than 200 types of PTMs has been identified (Prabakaran et al.,

2012), which all rely on one or more enzymes to catalyse the attachment of the

modifying agent to the target protein. Based on the nature of the modifying

element, there are several kinds of PTMs. Firstly, target proteins can be modified

by the attachment of a small molecular moiety, which occurs in the case of

phosphorylation and acetylation. In the example of protein phosphorylation, the

balance in the activity of kinases and phosphatases regulates the addition (and

removal) of a phosphate group to the target protein, often resulting in a

conformational and functional change of the target (Cieśla, Fraczyk and Rode,

2011). Besides small chemical groups, proteins can also be modified by entire

11

polypeptides, such as ubiquitin or any of the ubiquitin-like proteins (UBLs)

(Schwartz and Hochstrasser, 2003; Prabakaran et al., 2012).

4.4.1 Ubiquitin

The most common type of polypeptide-based posttranslational modification is

ubiquitination. Ubiquitin is a small protein, composed of 76 amino acids (roughly

8.5 kDa), which form a β-grasp fold structure. Besides this globular fold, the most

characteristic structural feature of ubiquitin is the glycine-glycine (GG) motif

used for conjugation in the C-terminal tail, together with the conserved lysine

residues that can be used to build ubiquitin chains, namely K6, K11, K27, K29,

K33, K48 and K63 (in addition to the starting methionine (M1)). Ubiquitin is

highly conserved among eukaryotic species (yeast and human ubiquitin share

96% sequence identity), and is in addition a very stable protein that can withstand

a large variety of different pH values and temperatures (Vijay-Kumar et al., 1985;

Wilkinson, 2005; Kimura and Tanaka, 2010; van der Veen and Ploegh, 2012).

Ubiquitin was first described in 1975 (Goldstein et al., 1975) and since then there

have been thousands of studies linking ubiquitination to a wide range of cellular

processes, such as gene expression, DNA repair, protein degradation and

apoptosis (Schwartz and Hochstrasser, 2003).

Ubiquitination (also called ubiquitylation) is the process in which ubiquitin is

covalently attached to a target protein via a cascade of enzymatic activity, driven

by ATP hydrolysis. From a mechanistic point of view, the transfer of ubiquitin to

its substrate is initiated by an E1 activating enzyme. Following activation,

ubiquitin is transferred onto an E2 enzyme (ubiquitin conjugating enzyme). The

subsequent and final step in the cascade is handled by an E3 enzyme (ubiquitin

ligase), whereby the target protein and the conjugating enzyme are recruited to

the E3, to enable conjugation of ubiquitin to a specific lysine residue in the target

protein (Glickman and Ciechanover, 2002). Considering that ubiquitination is a

reversible process, a fourth class of enzymes can be added to the ubiquitin

machinery, the deubiquitinating enzymes (DUBs). DUBs release the modified

protein from ubiquitin, by catalysing the breakage of the peptide bond between

ubiquitin and the ubiquitinated protein (Wilkinson, 2000).

One of the most extensively studied outcomes of ubiquitination is the

proteasomal degradation of target proteins. Most commonly, the targeting of

proteins for degradation by the proteasome requires a modification of the target

by an ubiquitin chain, in which multiple ubiquitins are linked to each other via

covalent conjugation of the K48 residue in one ubiquitin, to the carboxyl group of

G76 in the adjacent ubiquitin molecule in the chain. Besides these branched K48-

linked chains, ubiquitin chains with other linkages, as well as modification by one

12

single ubiquitin moiety (monoubiquitination), can instead of instructing protein

degradation, translate into several different scenarios for the target. One common

example is the monoubiquitination of histone proteins, which is associated with

chromatin remodeling and transcriptional regulation (Busch and Goldknopf,

1981; Briggs et al., 2002). Another important function of ubiquitination is the

sorting of cargo proteins along the endocytic pathway, which is predominantly

regulated by monoubiquitination and K63-linked ubiquitin chains, but may also

involve other linkages such as K11, K29 and K48 chains (Shields and Piper, 2011).

During endocytic sorting, ubiquitin is involved in most steps, from the initial

internalization of the cargo, to the sorting into multivesicular bodies (MVBs)

through activation of the endosomal-sorting complex required for transport

(ESCRT) machinery (Teo, Veprintsev and Williams, 2004).

The ubiquitin system is fairly well conserved throughout evolution, but the

composition of E1, E2 and E3 enzymes differ between species. However, in

general terms, most eukaryotes display hundreds of E3 encoding genes, whereas

the number of E2s is considerably lower and E1 often exists as a single gene

(Hicke, Schubert and Hill, 2005; Petroski and Deshaies, 2005).

4.4.2 Ubiquitin-like modifiers (UBLs)

The ubiquitin-like proteins (UBLs) is a family of small globular proteins that

similar to ubiquitin displays the structural feature of a β-grasp superfold and C-

terminal GG-motif. To date the list of identified UBLs includes Smt3 (SUMO-1, -

2, -3, -4), NEDD8, FUB1, FAT10, ISG15, Atg12, Atg8, UFM1 and Urm1

(Hochstrasser, 2009; Swatek and Komander, 2016; Cappadocia and Lima, 2017).

Among the UBLs, SUMO is the most well studied modifier, which also seems to

affect the largest number of target proteins, compared with the rest of UBL family

members (Meulmeester and Melchior, 2008). In spite of only 18% sequence

identity with ubiquitin, the three-dimensional structures of SUMO and ubiquitin

are highly similar (Bayer et al., 1998). SUMO is primarily recognized for its role

in nuclear functions including transcriptional regulation, nuclear transport and

genome integrity, but is also involved in cytoplasmic signal transduction

pathways (Rodríguez, 2014).

The UBL that shares the highest sequence identity with ubiquitin (approximately

60%) is NEDD8. NEDD8 conjugation shares the similar mechanistic features as

ubiquitin, involving the employment of NEDD8-specific E1 (Uba3), E2 (Ubc12)

and E3 (ROC1) enzymes (Kumar, Yoshida and Noda, 1993). Modification by

NEDD8, referred to as neddylation, has been shown to regulate the

13

multifunctional transcription factor NF-κB and in this way affect immune

responses and apoptotic cell death (Kawakami et al., 2001).

During the past decades, essential functions have been ascribed also to many of

the other UBLs, such as Atg8 and Atg12 in autophagy (Hanada and Ohsumi,

2005), as well as Fat10 in the regulation of the cell cycle (Ren et al., 2006;

Lukasiak et al., 2008; Merbl et al., 2013). Nevertheless, the depth of our

understanding of UBLs is still very shallow and requires further research.

4.5 Urm1 - Ubiquitin related modifier 1

Ubiquitin related modifier 1 (Urm1) is one of the least studied of the UBLs. Urm1

has been established to be the most ancient member in the UBL family and is

therefore considered as the evolutionary origin of polypeptide-based

posttranslational modifications (Iyer, Burroughs and Aravind, 2006; Pedrioli,

Leidel and Hofmann, 2008).

The study that led to the identification of Urm1 was performed by Furukawa and

co-workers, in which Urm1 was characterized as a protein modifier based on a

search of the yeast genome for proteins with sequence similarity to prokaryotic

sulfur carriers. This study further identified Uba4 as the specific E1 activating

enzyme for Urm1 in S. cerevisiae (Furukawa et al., 2000) and established

Urm1/Uba4 as the fifth protein conjugation system in yeast. Further studies

revealed that Urm1 has inherited the sulfur transfer activity from its prokaryotic

ancestors (Huang, Lu and Bystrom, 2008; Nakai, Nakai and Hayashi, 2008;

Schlieker et al., 2008; Leidel et al., 2009; Noma, Sakaguchi and Suzuki, 2009),

and thus reinforced the conclusion that Urm1 is indeed the “molecular fossil” that

links the ancient UBL progenitors to eukaryotic protein modifiers (Iyer,

Burroughs and Aravind, 2006; Schlieker et al., 2008).

4.5.1 Urm1 from evolutionary perspective

Similar to other members of the UBL family, Urm1 has the ability to form

conjugates with other proteins. However, upon identification, it was noticed that

S. cerevisiae Urm1p shares ~40% sequence identity with the E.coli proteins of

MoaD and ThiS, which are involved in the biosynthesis of molybdopeterin and

thiamine, respectively. (Furukawa et al., 2000; Goehring, Rivers and Sprague,

2003a). These early studies of Urm1 in yeast linked Urm1 to nutrient sensing,

budding, rapamycin hypersensitivity and impaired growth at high temperatures

(Furukawa et al., 2000; Goehring et al., 2003; Goehring, Rivers and Sprague,

14

2003a). A few years later, Urm1 was in addition demonstrated to function in the

modification of cytosolic tRNAs, specifically displaying a key role as a sulfur

carrier in tRNA thiolation (Huang, Lu and Bystrom, 2008; Nakai, Nakai and

Hayashi, 2008; Schlieker et al., 2008; Leidel et al., 2009; Noma, Sakaguchi and

Suzuki, 2009). Apart from its role during cytokinesis in HeLa cells (Schlieker et

al., 2008), Urm1 has in recent years primarily been considered as a protein

important for the cellular response against oxidative stress (A. S. Goehring,

Rivers and Sprague, 2003; Goehring, Rivers and Sprague, 2003b; Van der Veen

et al., 2011).

Multiple species of archaea, which phylogenetically are placed closer to

eukaryotes than prokaryotes, also express UBL proteins that display a dual ability

to function in tRNA thiolation and protein conjugation (Humbard et al., 2010;

Miranda et al., 2011; Ranjan et al., 2011; Anjum et al., 2015), suggesting that the

dual functionality of UBLs initially appeared in archaea. However, this theory was

recently challenged by the discovery that the UBL TtuB in prokaryotic T.

thermophiles, which in addition to acting as a sulfur carrier in tRNA modification,

also has the capacity to conjugate to target proteins (Shigi, 2012; Maupin-Furlow,

2014).

Another model organism in which Urm1 has been studied is the plant

Arabidopsis thaliana, which encodes two genes that are considered as

orthologues of Urm1; Urm11 and Urm12, both activated by the ubiquitin-

activating enzyme-like protein Cnx5 (Nakai et al., 2012). Urm11 and Urm12 are

highly similar to each other and exhibit 55% and 54% sequence identity to human

Urm1, respectively. Interestingly, also in plants, loss of the Urm1 results in severe

organ development defects, as well as impaired modification of tRNAs (Nakai et

al., 2012).

4.5.2 Structure and Biochemistry

Urm1 displays the typical UBL β-grasp structural fold and conserved C-terminal

tail that ends with the characteristic GG-motif (Pedrioli, Leidel and Hofmann,

2008; Wang et al., 2011) In 2006, the structural properties of Urm1 in Mus

musculus (AAH26994.1) was studied in detail by NMR spectroscopy. Similar to

MoaD, ThiS and ubiquitin, M. musculus Urm1 was indeed found to exhibit the

conserved β-grasp topology and GG-motif, however, it showed a stronger

sequence identity to its prokaryotic orthologous (14%-22%), as compared with

the other UBLs (6%-14%) (Singh et al., 2005). Hence, this structural analysis of

Urm1 yet again supported the hypothesis that eukaryotic UBLs have evolved, via

Urm1, from sulfur carrier proteins in prokaryotes.

15

Urm1 is a dual function protein. On one hand it functions as a protein modifier

by covalently conjugating to target proteins, and on the other hand it has a well-

defined role as a sulfur carrier during tRNA modification. Nevertheless, these two

processes show striking similarities, primarily since the initial step in both

pathways involves activation of Urm1 by acyl adenylation of its C-terminus and

formation of a complex with Uba4. The subsequent steps however, are different,

as shown in Figure 4 and described in detail below. Following activation, Urm1

can either be utilized as a sulfur carrier in the thiolation of lysine, glutamine or

glutamate tRNA, or conjugate to cellular target proteins.

4.5.3 Urm1 in tRNA modification

Between the years 2008 and 2009 a chain of independent publications linked

Urm1 to tRNA modification (Huang, Lu and Bystrom, 2008; Nakai, Nakai and

Hayashi, 2008; Schlieker et al., 2008; Leidel et al., 2009; Noma, Sakaguchi and

Suzuki, 2009). tRNA molecules contain several non-canonical nucleotides that

are under control of posttranscriptional modifications, which affect the folding,

stability and turnover of tRNAs (Wang, Yan and Guan, 2007; Agris, 2008). These

modified nucleotides can also regulate the interaction between mRNAs and the

ribosome scaffold, and therefore alter the effectiveness of protein translation

(Ghosh, 1976; Bjork et al., 2007; Johansson et al., 2008).

One of the best characterized tRNA modifications is the modification of the

wobble uridine 34 (U34) in the tRNA molecules tRNALys (UUU), tRNAGlu

(UUC) and tRNAGln (UUG) (Gustilo, Vendeix and Agris, 2008; Phizicky and

Hopper, 2010; Laxman et al., 2013). In general, there are two sets of

modifications that target U34. These include mcm5U34, where a methoxy-

carbonyl-methyl group is added to position 5 of U34, an event that often is

followed by a second modification, namely the replacement of the oxygen at

position 2 by a sulfur atom, resulting in mcm5s2U34 (Laxman et al., 2013).

However, it should be noted that these conserved modifications can also exist

separately on their own (Yarian et al., 2002; Chen, Huang, Eliasson, et al., 2011).

Modification by mcm5 is mediated by the Elongator protein (ELP) complex

together with the methyltransferase Trm9p and its associated protein Trm112p

(Kalhor and Clarke, 2003; Huang, 2005; Begley et al., 2007; Chen, Huang,

Anderson, et al., 2011), whereas thiolation is achieved by a different set of

proteins, including Nfs1, Tum1, Ncs2, Ncs6, Uba4 and Urm1. In the latter

pathway, Urm1 functions as a sulfur carrier, which transfers sulfur derived from

a cysteine in the sulfur donor protein Nfs1, onto U34 (Goehring, 2003; Nakai et

al., 2004; Nakai, Nakai and Hayashi, 2008; Schlieker et al., 2008; Leidel et al.,

2009; Noma, Sakaguchi and Suzuki, 2009; Juedes et al., 2016). Specifically, the

sulfur rely that leads to tRNA thiolation begins with the transfer of a sulfur atom

16

Figure 4. Urm1 is involved in UBL conjugation, as well as sulfur rely in tRNA

modification.

from a cysteine residue in the desulfurase enzyme Nfs1 onto Uba4, which occurs

with help of the sulfur transferase Tum1. Next, Uba4 forms a complex with Urm1,

leading to the activation of Urm1. Following a reduction of the acyl-disulfide bond

between Urm1 and Uba4, the thiolase enzymes Ncs2 and Ncs6 orchestrate the

transfer of sulfur onto the specific tRNA. (Figure 4)

It is evident that U34 modifications modulate translation by enhancing the

codon-anticodon interaction and facilitating the mRNA-tRNA translocation on

the ribosome, allowing the next codon to move into the decoding center.

Importantly, these modifications allow the translation of AGA and AGG codons

during DNA damage (Yarian et al., 2002; Murphy et al., 2004; Phelps et al.,

2004; Begley et al., 2007). In recent years, it has further been shown that nutrient

availability, in particular of sulfur containing resources, affect tRNA thiolation,

and that the levels of sulfur-containing amino acids is a determinative factor for

the occurrence of s2U34 modifications (Laxman et al., 2013).

17

4.5.4 Urm1 as a protein modifier

Even though Urm1 shares minimal sequence similarity with ubiquitin, it can

similar to ubiquitin attach to and modify other proteins in a process known as

urmylation (Furukawa et al., 2000; Goehring, Rivers and Sprague, 2003; Van der

Veen et al., 2011). As indicated earlier, several of the mechanistic properties of

UBL conjugation are comparable to the events that occur during the biosynthesis

of sulfur-containing cofactors (Rudolph et al., 2001). The first protein to be

identified as a binding partner of Urm1 was Uba4, picked up in a yeast-2-hybrid

assay (Furukawa et al., 2000).

A few years after the Urm1-Uba4 conjugation machinery was discovered in yeast,

Alkyl hydroxide reductase 1 (Ahp1p) was pinpointed as the first bona fide target

of urmylation. Besides providing evidence for a covalent conjugation of Urm1 to

Ahp1, this study demonstrated that apart from temperature sensitivity, the

urmylation pathway displays anti-oxidant roles (A. S. Goehring et al., 2003). In

subsequent reports, it was further confirmed that following exposure to oxidative

stress agents such as H2O2 and diamide, yeast cells show a striking elevation of

Urm1 conjugation (Van der Veen et al., 2011).

As demonstrated by Furukawa et al already in 2000, the interaction between

Urm1 and its activating enzyme Uba4 is sensitive to the presence of reducing

agents such as DTT, suggesting that a thioester bond is linking the two proteins.

However, this conclusion was later challenged by Schmitz and co-workers, who

demonstrated the presence of a thiocarboxylate at the C-terminus of Urm1p, thus

implicating that the Urm1-Uba4 interaction is more likely mediated via a

disulfide bridge (Schmitz et al., 2008).

To date there are no reports on any Urm1 specific E2 or E3 enzymes. However,

given the presence of an RHD domain in Uba4, it has been proposed that Uba4

may function as an E1-E2 hybrid protein (Hochstrasser, 2000). This idea is

supported also by the identification of an acyl-disulfide bond between Urm1 and

the active-site cysteine in Uba4, which is located in the RHD domain (Schmitz et

al., 2008). In keeping with this hypothesis, acyl-disulfide bonds are frequently

observed between E2 conjugating enzymes and their corresponding UBLs

(Pedrioli, Leidel and Hofmann, 2008).

For a long time Uba4 and Ahp1 were the only known Urm1-associated proteins.

However, by employing a proteomics approach, Van der Veen et al., identified 21

novel Urm1 targets in HeLa cells, which were conjugated by Urm1 in response to

oxidative stress. Importantly, this study suggested a variety of different molecular

processes that putatively could be affected by urmylation, including nuclear

18

transport, apoptosis, DNA repair, and ubiquitination (Van der Veen et al., 2011).

Another important contribution to our current knowledge about the targets of

urmylation originates from the archaea field, where Humbard and co-workers

have identified a rather extensive number of target proteins in H. volcanii,

including proteins involved in Ubl-/S- chemistry, stress responses and

metabolism, as well as DNA replication, transcription, translation and mRNA

processing (Humbard et al., 2010). Interestingly, this study indicates that of the

two UBLs present in H. volcanii, SAMP1 targets proteins for degradation in

proteasomes and that SAMP2, similar to ubiquitin, can form chains and possibly

compete with SAMP1 (Humbard et al., 2010). Similarly, the Urm1 orthologue in

S. acidocaldarius has also been found to utilize urmylation to induce proteasomal

degradation of its targets (Anjum et al., 2015).

It is estimated that in a yeast cell under normal conditions, ∼60% of Urm1 can be

found it its thiocarboxylated form. This suggests that Urm1 activation is necessary

but not sufficient for its conjugation, and therefore that urmylation most likely

requires some type of stimulation (Petroski, Salvesen and Wolf, 2011). It is not

clear to what extent target proteins are modified by Urm1, yet according to the

analogy with other UBLs, and the example of Ahp1, only a small amount of the

total population of the target proteins is likely to undergo modification at a given

time point (Van der Veen et al., 2011).

4.6 Uba4 - The activating enzyme for Urm1

The mechanistic similarities between UBL conjugation and the biosynthesis of

sulfur-containing co-factors originates in the initial step of the two processes,

which begins with adenylation of the C-terminal glycine of the UBL molecule. In

both prokaryotes and eukaryotes, this event is handled by proteins in the E1-like-

enzyme superfamily (Burroughs, Iyer and Aravind, 2009). The activity of Urm1,

whether it is acting as a sulfur carrier or a UBL, is dependent on its dedicated

activating enzyme Uba4, known as MOCS3 (Molybdenum Synthesis Cofactor 3)

in mammals (Furukawa et al., 2000).

Uba4 is a highly conserved protein and its orthologues are universally present in

phylogeny, from ancient prokaryotes to man. In parallel to the suggestion that

Urm1 and the UBL superfamily originated from MoaD and ThiS, the proteins

MoeB and ThiF were put forward as the evolutionary progenitors of the E1

superfamily (Iyer, Burroughs and Aravind, 2006). Besides acting as the Urm1-

specific E1-activating enzyme, human MOCS3 in addition plays a well-

characterized role in the biosynthesis of the molybdenum co-factor (MoCo),

19

which is required for the activity of a group of enzymes called molybdoenzymes

(Schwarz and Mendel, 2006).

4.6.1 Structure and function of Uba4

Structurally, Uba4 consists of two specific domains, an E1-like domain and a

rhodanese homology domain (RHD), located in the N-terminal and C-terminal

part of the protein, respectively. Even though Uba4 resembles the bacterial

proteins ThiF and MoeB, the sequence similarity shared between these

prokaryotic ancestors and human MOCS3 is limited to the N-terminus of the

protein, where the E1 domain is located (Andreas Matthies, Manfred Nimtz and

Silke Leimkühler, 2005). Nevertheless, within the eukarya domain, the

Uba4/MOCS3 is highly conserved, as indicated by a sequence identity of 36%

shared between yeast Uba4 and human MOCS3 (Schmitz et al., 2008). Another

critical structural characteristic of Uba4/MOCS3 is the presence of two strictly

conserved cysteine residues within the conserved domains. These catalytic

cysteines are essentially the active sites for the enzymatic activity of Uba4

(Krepinsky and Leimkühler, 2007; Schmitz et al., 2008). The C-terminal RHD

domain displays rhodanese activity. Rhodaneses are sulfur carrier enzymes that

catalyse the transfer of sulfane (thiosulfoxide) sulfur atoms from thiosulfate to

cyanide, and can be found as extensions to other proteins, as single-domain

proteins or as tandem repeats (Bordo and Bork, 2002; Schmitz et al., 2008).

Sulfane sulfurs function in co-factor synthesis, tRNA sulfuration, enzyme activity

modulation and regulation of the redox environment in the cell (Toohey and

Cooper, 2014).

The dual functions of Uba4 are mediated via two distinct pathways (Figure 5).

Firstly, by activating Urm1, Uba4 initiates protein urmylation, as well as tRNA

modification. Secondly, Uba4 can also contribute to the biosynthesis of MoCo

(molybdenum co-factor), by activating another UBL protein, Mocs2A.

Interestingly, there is a clear homologue of Mocs2A also in the D. melanogaster

genome, CG42503-R), which displays 40% amino acid sequence identity to

human Mocs2A. While Urm1 and Uba4 have shown to be essential for invasive

vegetation and budding in yeast, S. cerevisiae is in fact one of few organisms that

does not employ molybdoenzymes, and therefore the role of Mocs2A and MoCo

can be disregarded in yeast. In contrast, human MOCS3 has retained the ability

to activate both Urm1 and Mocs2A (Schmitz et al., 2008).

20

Figure 5. Molecular mechanism underlying the dual activities of Uba4, mediated via

interaction with Urm1 and Mocs2A, respectively.

In humans, loss of MOCS3 is strongly implicated in the aetiology of the rare

disease molybdenum co-factor deficiency, an inborn condition that also is

associated with the loss of other key enzymes in the MoCo biosynthesis pathway,

namely MOCS1, and MOCS2 (in addition to MOCS3). Patients suffering from

molybdenum co-factor deficiency are severely affected during the neonatal period

and display abnormalities such as seizures and skeletal dysmorphic features. In

most cases the disease causes mortality within a few months after birth (Atwal

and Scaglia, 2016; Huijmans et al., 2017).

21

4.7 Oxidative stress

Oxygen molecules are essential for aerobic life. In eukaryotic cells, the major

consumption of oxygen occurs in mitochondria, during the production of ATP in

the cellular respiration pathway. An unavoidable by-product of oxygen

metabolism in different cellular compartments is the generation of reactive

oxygen species (ROS), such as superoxide anions (O2−), hydrogen peroxide

(H2O2) and hydroxyl radicals (OH−). Production of ROS can also be induced by

environmental stressors such as molecular pollutants, UV radiation and

hyperthermia (Finkel and Holbrook, 2000; Taylor and Pouyssegur, 2007; Zaher

et al., 2007).

Accumulation of ROS leads to oxygen toxicity (e.g. oxidative stress) and hence, it

is critical for every cell to be able to counteract oxidative stress in order to survive.

Excessive ROS can directly damage cellular macromolecules, such as DNA,

proteins and lipids, which may result in cell cycle arrest and ultimately cell death

(Cacciuttolo et al., 1993). On a larger scale, in humans, inadequate responses to

oxidative stress contributes to ageing, as well as the initiation and progression of

disease, such as cancer and diabetes (Zhang, Wang and Su, 2009; Le Lay et al.,

2014)

4.7.1 Molecular mechanisms behind oxidation and reduction

Over the course of evolution, cells have developed several different strategies to

defend themselves against oxidative stress. One of the most important families of

anti-oxidant proteins are the peroxiredoxins, which act by catalyzing the

reduction of hydrogen peroxide to water. The peroxiredoxins (Prxs) are one of the

most abundant protein families and are, based on the number and position of Cys

residues that participate in catalyzing reduction, historically classified into three

groups, including the; 1) Typical 2-Cys Prxs, 2) Atypical 2-Cys Prxs, and the 3) 1-

Cys Prxs (Sue, Ho and Kim, 2005).

Another mechanism of counteracting oxidative stress is the upregulation of

cytoprotective genes, including glutathione S-transferases (GSTs), aldehyde

dehydrogenases (ALDH) and NAD(P)H:quinone oxido-reductases (NQO1)

(Taylor and Pouyssegur, 2007; Jones, 2008; Zhang, Wang and Su, 2009). These

proteins function by maintaining a cellular buffer of redox proteins such as

glutathione, which detoxify ROS through a non-enzymatic mechanism (Le Lay et

al., 2014).

Several signalling pathways collaborate in order to induce the appropriate

responses needed to enable a cell to withstand oxidative stress. Among these the

22

JNK, p38 MAPK, p53, NF-κB and Nrf2 pathways have been recognized as most

important. The activity of these pathways is by no means specific to oxidation-

reduction responses, as they are known to have crucial roles in the response also

to other stressors, as well as in normal development, growth and metabolism. It

is also needless to say that the nature and duration of the stress inducer, together

with the cell type, are also important factors that determine the degree of

activation and the outcome of these pathways (Finkel and Holbrook, 2000).

Drosophila has been extensively used in research to understand the mechanisms

and cellular defense programs against oxidative stress. One of the most

commonly used ROS inducers in Drosophila labs is paraquat (1,1′dimethyl-4-

4′bipyridynium dichloride), a quaternary nitrogen herbicide, that is highly toxic

to humans and animals (Arking et al., 1991; Wang, Bohmann and Jasper, 2003).

The toxicity of paraquat is caused by the generation of ROS, such as hydroxyl

radicals and hydrogen peroxide, which may result in acute poisoning, multiple-

organ failure and ultimately death (Suntres, 2002; Sittipunt, 2005; Raghu et al.,

2013). In flies, paraquat exposure leads to locomotor defects and shortened

lifespan, and is on the cellular level associated with increased levels of apoptotic

death and activation of the JNK pathway (Peng et al., 2004; Klintworth et al.,

2007; Jordan et al., 2012).

4.8 The JNK pathway

Stress-activated protein kinases (SAPKs) are evolutionary conserved signaling

molecules that enable living organisms to respond to intrinsic and extrinsic

stimuli in the form of stress. One of the most versatile and ubiquitous stress

sensors, is the c-Jun N-terminal kinase (JNK) signaling pathway, which is

triggered by a wide range of environmental insults including UV radiation,

inflammatory cytokines, DNA damage, heat stress, bacterial antigens, and

perhaps most importantly, ROS (Biteau et al., 2011).

Classified as one of the mitogen-activated protein kinase (MAPK) pathways, the

JNK pathway is a pleiotropic signaling cascade with well-established roles in the

response to stress signals, as well as proliferation, apoptosis, motility,

metabolism and DNA repair (Biteau et al., 2011). Therefore, it is no surprise that

aberrant activity of JNK signaling is involved in human pathogenesis, linked to

multiple birth defects and diseases such as neurodegeneration, chronic

inflammation and cancer (Biteau et al., 2011).

23

As the name indicates, JNK activity results in the phosphorylation of c-Jun,

specifically at residues Ser63 and Ser73, located in the N-terminal part of the

protein (Behrens, Sibilia and Wagner, 1999). Together with Fos, Maf and ATF, c-

Jun contributes to the formation of the major JNK-induced AP-1 heterodimeric

transcription factor complex, which upon phosphorylation mediates gene-

specific transcriptional control (Behrens, Sibilia and Wagner, 1999; Shaulian and

Karin, 2001; Johnson and Nakamura, 2007).

The canonical JNK pathway is built on a chain of phosphorylation events that is

triggered by a stimulus-induced, selective activation of a JNK kinase kinase

(JNKKK), which phosphorylates a JNK kinase (JNKK) that subsequently induces

phosphorylation of JNK. Following activation, JNK in turn affects its downstream

targets, the c-Jun and Fos components of the AP-1 complex, as well as the

transcription factor Forkhead Box O (FoxO), subsequently triggering a chain of

events that lead to changes in the cellular transcriptome (Johnson and

Nakamura, 2007; Weston and Davis, 2007) (Figure 6).

4.8.1 The JNK pathway in Drosophila

In contrast to vertebrates, where the components of the JNK pathway are

represented by a rather large set of genes, the low redundancy in the fly genome

renders a Drosophila JNK pathway that is far less complex and much easier to

study (Biteau et al., 2011). In mammals, there are three JNK proteins, encoded

by three separate genes (Bsk1, Bsk2 and Bsk3), whereas in flies there is only one

known JNK homologue, basket (bsk), which regulates the fly AP-1 complex,

consisting of Jra (dJun) and Kayak (dFos). Upstream of Drosophila Bsk, there is

one major JNK kinase, Hemipterous (Hep), together with the less-characterized

JNKK, dMKK4 (Davis, 2000; Boutros, Agaisse and Perrimon, 2002; Chen, White

and Cobb, 2002; Geuking et al., 2009). In Drosophila, the AP-1 complex have

been shown to regulate the transcription of a battery of stress-associated genes,

as exemplified by hsp68, gstD1, fer1HCH, and mtnA (Wang, Bohmann and

Jasper, 2003). Another important target gene of the fly AP-1 complex is puckered

(puc), a phosphatase that targets JNK itself and thereby downregulates its activity

in a negative transcriptional feedback loop (Martín-Blanco et al., 1998; McEwen,

2005). Despite its relative simplicity, JNK signaling in Drosophila remains highly

diverse and context-dependent, involved in an extensive array of physiological

events, such as embryonic development, immunity, stress responses and

longevity (Riesgo-Escovar et al., 1996; Sluss et al., 1996).

24

Figure 6. The JNK pathway in Drosophila.

Mutation in any of the genes encoding the major components of the JNK pathway

(e.g. hep, bsk, kay, jra and even puc), severely affects embryonic development in

Drosophila. Commonly, they all cause lethality due to a disruption of dorsal

closure, pinpointing that a strictly regulated JNK signaling is required for proper

development of the fly (Martín-Blanco et al., 1998; Agnès, Suzanne and Noselli,

1999; Noselli and Agnès, 1999; Stronach and Perrimon, 1999; McEwen, 2005).

Furthermore, there have been many additional studies, in which critical roles of

the JNK pathway have been reported in pupal thorax closure, endoderm

patterning, wing patterning and eye development (Kockel, Homsy and Bohmann,

2001). All these functions are clearly in agreement with the established role of

JNK signaling in vertebrates, including cell migration, wound healing, and bone

morphogenesis, as well as neuronal cell death and differentiation, strongly

indicating an evolutionary conserved role for the JNK pathway in development

(Martin, 2004).

25

As briefly mentioned previously in the NMJ growth context, JNK signaling is

involved in the formation of neuronal circuits in fruit fly. In third instar larvae,

neuronal AP-1 positively regulates synapse formation and growth (e.g. bouton

count), as well as synaptic strength (Sanyal et al., 2002; Etter et al., 2005). Hence,

simultaneous neuronal overexpression of Jun and Fos results in an increased

bouton count, whereas expression of dominant negative variants of the proteins

induces the reciprocal effect. In agreement with being a downstream event of JNK

activity, this neuronal function of AP-1 is antagonized by the expression of Puc

(Sanyal et al., 2002). Molecularly, the outcome of neuronal hyperactivation is a

transcriptional upregulation of multiple downstream genes (Etter et al., 2005),

and even if the biological importance of these have not been determined, it is

obvious that many rapid changes in gene expression occurs during the refinement

of NMJs (Zhang et al., 2016).

Interestingly, JNK activity at NMJs has previously been shown to be regulated by

the ubiquitin system, where the E3 ubiquitin ligase Highwire (Hiw) targets the

JNKKK Wallenda (Wnd) for ubiquitination and subsequent degradation in the

proteasome (Collins et al., 2006). As a result, highwire (hiw) loss-of-function

mutants display an enhanced JNK/AP-1 activation, resulting in a significant NMJ

overgrowth (Collins et al., 2006; Shen and Ganetzky, 2009). Altogether, it can be

concluded that increased activity of the JNK pathway triggers NMJ overgrowth,

regardless of the underlying cause. In support of this theory, a rather recent study

demonstrated a role of oxidative-stress-induced JNK activity in promoting NMJ

growth (Milton et al., 2011).

4.8.2 The JNK pathway in stress responses and lifespan

regulation

According to the “free radical theory of aging” (Harman, 1956), an organism’s

lifespan is determined by its genetic ability to counteract oxidative stress.

Consequently, it is common that the signal transduction pathways that promote

protection of cellular macromolecules during oxidative stress, also positively

influence the lifespan of an animal (Nathan and Ding, 2010; Sykiotis and

Bohmann, 2010; Biteau et al., 2011). In agreement with its role as a key player in

the network of cytoprotective genes, the JNK pathway mediates protection

against oxidative damage, as well as increased lifespan, which at least in part is

due to the expression of anti-oxidant proteins (Wang, Bohmann and Jasper,

2003, 2005). This is particularly important in neuronal cells, where the

generation of ROS is high and the levels of anti-oxidant proteins are low (Lin and

Beal, 2006).

26

As discussed earlier, peroxiredoxins have important functions in the cellular

response against oxidative stress and play a crucial role in ROS reduction. It is

therefore important to note that one of the prominent targets that is upregulated

in response to JNK activation is the peroxiredoxin Jafrac1 (also known as PrxII).

Indeed, Jafrac1 overexpression has been shown both to increase the resistance

against oxidative stress and extend lifespan in flies, whereas Jafrac1 knockdown

by RNAi results in reciprocal phenotypes (Lee et al., 2009).

4.9 Drosophila as a model to study Tax-mediated

oncogenesis

When the sequencing of the human genome was completed, the similarities

between the human and D. melanogaster genomes strengthened the role of the

fruit fly as a model system for understanding human biology and disease, as well

as for the development of drugs. Importantly, it is estimated that approximately

75% of the disease-related genes in humans, have functional orthologues in

Drosophila (Reiter et al., 2001; Pandey and Nichols, 2011). In recent years, many

researchers have employed Drosophila to address questions in the cancer

research field, extensively described in multiple reviews (Potter, Turenchalk and

Xu, 2000; Halder and Mills, 2011; Miles, Dyson and Walker, 2011). Similar to

these studies, we utilized the fruit fly in the third study (submitted manuscript)

included in this thesis, to increase the understanding of how the viral oncogene

Tax1 is regulated by posttranslational modifications (e.g. Urm1) in Adult T-cell

leukemia/lymphoma (ATL).

4.9.1 Adult T-cell leukaemia/lymphoma

Adult T-cell leukemia/lymphoma (ATL) is a rare and often aggressive T-cell

lymphoproliferative malignancy that can be found in the blood (leukemia), lymph

nodes (lymphoma), skin, and multiple other tissues of the human body (Matutes,

2006). ATL has been linked to infection by the Human T-cell lymphotropic virus

type 1 (HTLV-1), however, only 5-10% of the HTLV-1-infected individuals develop

ATL. It is also worth noting that the T-lymphocyte transformation commonly

occurs after 20-40 years of latency (Kannian and Green, 2010), suggesting that a

multistep process is likely to underlie the disease (Barmak et al., 2003; Hasegawa

et al., 2006; Matsuoka and Jeang, 2007). ATL almost exclusively affects adults,

with a median age of onset in the mid-sixties and no gender preferences (Matutes,

2006). According to present classification systems, ATL exists in four clinical

forms, including acute, chronic and smouldering ATL, as well as lymphoma.

27

Among these, the acute form is the most severe and also the most common type,

representing almost 65% of the total number of ATL cases. (Aouba et al., 2004;

Matutes, 2006)

4.9.2 HTLV-1 and Tax

HTLV-1 is a retrovirus that can be transmitted through sexual contact, blood

transfusions, and from mother to child via breast-feeding (Edlich, Arnette and

Williams, 2000; Matsuoka, 2003; Ohsugi and Koito, 2007). At present, it is

estimated that nearly 10 to 20 million people around the world are infected with

HTLV-1, predominantly in areas including Japan, the Caribbean, Africa, South

America and certain areas in the Middle East and Eastern Europe (Barmak et al.,

2003; Van Dooren et al., 2007; Rafatpanah et al., 2011).

HTLV-1 can be detected in cells from virtually all ATL patients and besides

causing ATL, HTLV-1 infection has in addition been associated with the

neuroinflammatory disease HTLV-1-associated myelopathy/tropical spastic

paraparesis (HAM/TSP) (Shimoyama, 1991; Shembade, 2010; Shirinian et al.,

2013). To date, four types of HTLV have been identified, numerically named as

HTLV-1 to HTLV-4. The most publicly known member of the group is HTLV-3,

which was later renamed as Human Immunodeficiency Virus (HIV), the virus

that causes AIDS. HTLV-1 and HTLV-2 share approximately 70% genome

homology and structural similarity, yet they are completely different in terms of

pathogenesis and disease manifestation (Roucoux and Murphy, 2004; Calattini

et al., 2005; Matsuoka and Jeang, 2007; Mahieux and Gessain, 2009).

The RNA genome of HTLV-1 is composed of 9032 nucleotides, encoding the

structural proteins of the viral core particle (Gag, Env, and Pol), as well as the

proteins of the retroviral enzymatic machinery (reverse transcriptase, integrase

and protease). In addition, the HTLV-1 genome displays a pX cluster with five

open reading frames (ORFs) that can produce accessory proteins such as Tax,

p12I, p27I, p13II, and p30II, through alternative splicing (Cereseto et al., 1997;

Grant et al., 2002; Azran, Schavinsky-Khrapunsky and Aboud, 2004).

The most extensively studied protein of HTLV-1 is Tax (e.g. Tax-1). Tax is a 40

kDa protein, which is both necessary and sufficient for tumor formation

(Shembade, 2010). However, the Tax protein encoded by HTLV-2, Tax-2, does

not show any of the oncogenic characteristics of Tax. From a molecular

perspective, expression of Tax disrupts the regulation of hundreds of cellular

genes, among which proto-oncogenes, cytokines, growth factor receptors, cyclin-

dependent kinases (CDKs), inhibitors of CDKs, DNA repair machineries and cell

adhesion molecules can be found (Ng et al., 2001; Grassmann, Aboud and Jeang,

28

2005). The pleiotropic function of Tax originates from its wide range of direct

interactions with cellular proteins, many of which are involved in signal

transduction pathways. These include the PI3K/Akt, NF-B, MAPK, TGFβ and

CREB (cyclic AMP responsive element binding)/ATF (activating transcription

factor) pathways, as well as multiple small G proteins that regulate cytoskeletal

organization and chemotaxis (Shirinian et al., 2013). Among these, one of the

most important signaling pathways that are directly influenced by Tax, is the

Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-B) pathway

(Suzuki et al., 1996; Neuveut et al., 1998; Haller et al., 2002; Wu et al., 2004).

4.9.3 The NF-B pathway

In mammals there are five members in the NF-κB protein family, including RelA

(p65), RelB, c-Rel, p50/p105 (NF-κB1) and p52/p100 (NF-κB2) (Napetschnig

and Wu, 2013). By forming homo- and heterodimeric complexes in response to a

variety of stimuli, the NF-κB proteins utilize their Rel homology domains to bind

DNA and regulate gene expression (Napetschnig and Wu, 2013). A constitutive

activation of NF-κB has been reported in many types of human cancers, including

ATL (Sun and Yamaoka, 2005). Activity of NF-κB is essential for the regulation

and maintenance of the immune system (Hayden and Ghosh, 2011), skeletal

system (Novack, 2011), and epithelial tissues (Wullaert, Bonnet and Pasparakis,

2011). At the cellular level, NF-κB functions to promote cell survival,

differentiation, and proliferation (Hayden and Ghosh, 2012). In Drosophila,

many studies have linked the NF-κB pathway to multiple events during

development, as well as functions in the immune system (Minakhina and

Steward, 2006; Hayden and Ghosh, 2012).

In the absence of activation, NF-κB proteins are kept inactive in the cytoplasm by

binding to a protein of the IκB family, which masks the nuclear localization signal

(NLS) of NF-κB (Napetschnig and Wu, 2013). NF-κB is activated following

phosphorylation of the IκB by the IKK complex (consisting of IKKα, IKKβ and

IKKγ/NEMO). Phosphorylation of IκB enables the subsequent ubiquitination

and proteasomal degradation of IκB, resulting in a release and translocation of

NF-κB into the nucleus, where it functions as a transcriptional regulator (Chen,

2005).

Tax has been shown to interact with several of the NF-κB proteins, including

RelA, p50, and p52 (Sun and Yamaoka, 2005), however, the key event in Tax-

mediated activation of NF-κB, is the binding of Tax to IKKγ/NEMO, one of the

three components of the IKK complex. Following the binding of Tax to the IKK

complex, Tax recruits kinases that normally act upstream of the IKK complex,

such as TAK1 (TGF-β activating kinase 1) and MEKK1 (MAPK/ERK kinase kinase

29

1) to the complex. These kinases phosphorylate and activate IKKα and IKKβ,

resulting in IκB degradation and nuclear translocation of NF-κB, independently

of external stimuli (Chu et al., 1999; Harhaj and Sun, 1999; Harhaj et al., 2000;

Xiao, Harhaj and Sun, 2000; Xiao et al., 2001; Harhaj, Sun and Harhaj, 2006;

Wu and Sun, 2007). In addition, Tax has been demonstrated to interact directly

with and activate the kinase activity of IKKα and IKKβ, independent of upstream

kinases (Chu et al., 1998). Studies of Tax in transgenic mouse models support the

finding that Tax maintains these oncogenic properties in vivo (Kwon et al., 2005;

Hasegawa et al., 2006; El Hajj et al., 2012). Recently, it was further shown by

Shirinian et al. that tissue specific expression of Tax in Drosophila plasmatocytes

(resembling mammalian leukocytes) and the compound eye, results in increased

plasmatocyte proliferation and ommatidial perturbation, respectively, leading to

the formation of a “rough-eye” phenotype (Shirinian et al., 2015). Interestingly,

the Tax-induced rough eye phenotype was found to be dependent on Kenny, the

Drosophila orthologue of IKKγ/NEMO, which is in keeping with the previous

finding that Tax activation of NF-κB is inhibited in T-cells deficient of

IKKγ/NEMO (Harhaj et al., 2000).

4.9.4 Posttranslational modification of Tax1

Tax is modified by a number of different posttranslational modifications,

including phosphorylation, acetylation, O-GlcNAcylation, ubiquitination, and

sumoylation. These modifications are important for Tax-mediated activation of

NF-κB signaling, inhibition of DNA repair, repression of the tumor suppressor

p53 and cell cycle control (Shembade, 2010; Shirinian et al., 2013).

Tax undergoes phosphorylation on multiple serine and threonine residues, of

which at least phosphorylation of Ser300 or Ser301 is necessary for the

translocation of Tax into nuclear bodies, as well as the activation of gene

expression via NF-κB (Bex et al., 1999; Durkin et al., 2006). Further boosting the

activity of Tax, ubiquitination has been demonstrated to result in a cytoplasmic

retention of Tax, which is essential for the activation of IKKs and NF-κB signaling

(Shembade, 2010). Primarily, Tax is conjugated by K63-linked polyubiquitin

chains that enhances Tax activity (Chiari et al., 2004; Shembade et al., 2007;

Kfoury et al., 2008), but it can also be modified by K48-linked chains, which in

contrast induce proteasomal degradation of Tax (Kfoury et al., 2008). In addition

to ubiquitin, Tax is also targeted by sumoylation, which results in a nuclear

retention of Tax and formation of promyelocytic leukemia nuclear bodies (PML

bodies) (Ariumi et al., 2003). Interestingly, the sites targeted by sumoylation and

ubiquitination partly overlap, indicating that a cross-talk between different

posttranslational modifications may determine the fate of Tax in the cell

(Lamsoul et al., 2005). In line with this hypothesis, Tax degradation was recently

30

associated with PML-dependent sumoylation, followed by ubiquitination

mediated by the ubiquitin E3 ligase RING finger 4 (RNF4) (Dassouki 2015).

Although there has been intense research on ATL, the prognosis and therapeutic

options are still very poor. A recent study of the available treatments and

outcomes among 1594 ATL patients reported that the median survival of acute,

lymphoma, chronic, and smoldering ATL subtypes were 8.3, 10.6, 31.5, and 55.0

months, respectively. This clearly illustrates that despite recent advances, the

current therapeutic options for the acute and lymphoma types of ATL are still

very poor (Katsuya et al., 2015; Watanabe, 2017). Currently ATL patients are

commonly treated with conventional chemotherapy, monoclonal antibodies

(against CD2, CD4, CD52, IL-2 and transferrin receptors), hematopoietic stem

cell (HSC) transplantation and anti-viral therapy methods (Bazarbachi et al.,

2011). However, our collaborators in Lebanon have demonstrated that treatment

with a combination of arsenic trioxide (As2O3) and interferon (IFN)-α can induce

cell-cycle arrest and apoptosis in HTLV-1–infected and freshly isolated leukemia

cells from ATL patients (Bazarbachi et al., 1999; El-Sabban et al., 2000; Kchour

et al., 2009; Dassouki et al., 2015). The arsenic/IFN treatment have yielded

promising results in mouse model of ATL (El Hajj et al., 2010) and seems to act

via a rapid shut-off of the NF-κB pathway and a delayed shut-off of cell cycle–

associated genes, in addition to triggering Tax degradation in the proteasome

(Mahieux et al., 2001; Nasr et al., 2003; Wu et al., 2016).

31

5. Results and Discussion

The seed for the concept of employing model organisms to understand biological

life was planted in the middle of the 19th century, by the work of scientists like

Charles Darwin and Gregor Mendel. Without doubt, the work on model

organisms have led to significant advancements in science and the understanding

of human biology. Taking these principles into account, the idea of taking the

knowledge from one organism (“a model organism”) and applying it on another

(humans) is the foundation of the work that has been done in this thesis.

The protein Urm1 and its specific E1 activating enzyme Uba4 lie at the heart of

the four papers/manuscripts included in this thesis. In the first paper, we provide

the first identification and characterization of Urm1 in Drosophila. While

confirming the identity of the annotated gene CG33276 as the fly homologue of

Urm1, this paper also provides a new perspective into the potential regulatory

roles of Urm1 in cellular signaling pathways (e.g. the JNK pathway). In the second

paper, we aimed at expanding the knowledge about the urmylation landscape, by

identifying novel targets of Urm1 in three developmental stages of the fly. The

work in the third paper takes the concept of urmylation to a new level, by

establishing a putative novel role for Urm1 in Tax-mediated oncogenesis. Finally,

in the fourth paper we have investigated the function of Urm1 and Uba4 in the

development and growth of Drosophila NMJs.

5.1. Paper I

Urm1: an essential regulator of JNK signaling and oxidative

stress in Drosophila melanogaster.

After the discovery of Urm1 by Furukawa and co-workers in 2000 (Furukawa et

al., 2000), a majority of the studies on Urm1 was performed in S. cerevisiae or

mammalian cells, without any insights into the role of Urm1 in a multicellular

organism. By analyzing the Drosophila genome, we found that it encodes one

single clear Urm1 homologue, the annotated gene CG33276, located on the third

chromosome (66A8). Drosophila Urm1 shares a striking 66% sequence identity

with its human counterpart and 37% sequence identity with the S. cerevisiae

Urm1p. The similarity is not limited to the sequence, as all the characteristics of

fly Urm1, including size, predicted structural folding and C-terminal GG-motif

are similar to the yeast and human homologues.

32

Having the right set of tools is a necessity for performing high quality research.

Therefore, the early years of this project was dedicated to building a toolbox for

studying Urm1 in flies. This included generation of an Urm1 specific antibody,

transgenic flies expressing tagged (Flag-, GFP-, HA-) wildtype Urm1, or Urm1

lacking the capacity to conjugate to target proteins (Urm1GG), and most

importantly a fly strain deficient of the Urm1 locus (the Urm1n123 null allele).

One of the main benefits of analyzing protein function in a multicellular

organism, such as Drosophila, compared to yeast, is the diversity of specified

tissues and developmental stages. While the yeast model provides less complexity

and facilitates conclusions to be made, the fact that a certain protein can display

different expression levels and perform different functions in specific tissues

and/or developmental stages, fails to be addressed. A clear illustration of the

latter is the apparent dynamics of Urm1 activity in flies, where there is a dramatic

difference in both the expression level and urmylation pattern of an early embryo,

compared to adult flies. Besides this temporal regulation of Urm1 expression and

activity, it is also clear that Urm1 is differentially expressed in different tissues

and at specific developmental stages. This is exemplified by the relatively high

levels of Urm1 that can be found in the wing discs of third instar larvae, compared

with the salivary glands, where it is hardly detectable (illustrated in Manuscript

III). Such diversity in expression can possibly translate into a stage- and/or

tissue-specificity of Urm1 targeting, which is highlighted in the second paper.

When discussing a posttranslational protein conjugation system, the first

question that comes to mind is the subject of protein modification itself. Similar

to findings in S. cerevisiae (Furukawa et al., 2000; Goehring, Rivers and Sprague,

2003; Goehring, Rivers and Sprague George F., 2003), we showed in Paper I that

Urm1 interacts with Uba4, and that co-expression of Urm1 and Uba4 can induce

urmlyation in Drosophila tissues. Importantly, we also demonstrated that

urmylation in flies is dependent on Uba4, since RNAi-mediated knockdown of

Uba4 resulted in significantly reduced levels of target protein urmylation.

Already from the early years, urmylation has been associated with oxidative

stress, given the identification of the anti-oxidant protein Ahp1 as a the first

identified target of Urm1, and the accumulation of urmylated targets detected in

response to exposure to diamide or H2O2 by Van der Veen et al. in 2011 (Van der

Veen et al., 2011). In agreement, we found Urm1 to physically interact with the fly

homologue of Ahp1, Peroxiredoxin 5 (Prx5), an interaction which seems to be

further boosted in response to paraquat-induced oxidative stress (to levels easily

detectable by Western blot). (Michalak, Orr and Radyuk, 2008; Radyuk et al.,

2009). This suggests that in non-stressed conditions, only a small portion of Prx5

is urmylated, whereas during oxidative stress the levels of urmylated Prx5 is

elevated. This finding was further supported in the second paper, where Prx5 was

33

identified among the targets of urmylation. Interestingly, Prx5 displays a

cytoprotective role against oxidative stress and apoptosis, and thereby promotes

longevity in flies.

A homozygous loss of function mutation in Urm1 (Urm1n123) is predominantly

lethal in flies, however, there appears to be certain factors that can contribute to

support the survival and completion of development into adulthood of at least a

small percentage of the mutant population. Analyzing the survival graph for

Urm1n123 mutants, it is noticeable that roughly 30% of the eggs do not hatch into

first instar larvae. This is especially interesting, since there is a high level of Urm1

activity during embryogenesis. In situ hybridization analysis of Urm1 mRNA

suggests that Urm1 is highly maternally contributed, which most likely enables

the survival of Urm1n123 animals. In agreement, if the maternal contribution of

Urm1 is removed through genetic approaches, the survival rates dramatically

drop to zero, with mutant eggs displaying severe early embryonic developmental

defects. Furthermore, in a mixed population of heterozygous Urm1n123 flies, it is

rare to come across the Urm1n123 mutant flies. However, if the mutant animals are

sorted and kept separately from the first instar stage, their survival rate increase,

which may indicate that homozygous mutants fail to compete in an environment

with heterozygous siblings. The small population of escapers that eventually

eclose into adults are valuable animals, since we can employ them to investigate

the effects of Urm1 loss of function (LOF) in adult life. Homozygous Urm1n123

escapers have short life span, are slightly smaller and skinnier than wild type flies,

and display a distinctive female sterility.

It was against our expectations to find that Urm1n123 mutant flies were not

sensitive, but instead more tolerant to paraquat-induced oxidative stress. Despite

confirming the evolutionary phenomenon of Urm1 conjugation to Prx5, we were

unable to establish any biological proof for a genetic interaction between Prx5

and Urm1, which could explain the stress resistance. Arguably, loss of Urm1 is

even beneficial for the stress sensitivity displayed by Prx5 null mutants (Figure

7), suggesting an involvement of a parallel pathway in oxidative stress tolerance.

Elevation of pJNK in Urm1n123 LOF clones is an unmistakable sign of JNK

pathway hyperactivation in the absence of Urm1 (Glise, Bourbon and Noselli,

1995; Stronach, 2005). This was further reinforced by an increased nuclear

accumulation of dJun, as well as upregulation of proteins that are

transcriptionally regulated by JNK, such as gstD1 and Jafrac1. This increased

JNK activity is a plausible explanation for the oxidative stress resistance in

Urm1n123 flies, but also raises an important question. JNK activity has been

associated with an enhanced longevity in flies (Wang, Bohmann and Jasper,

2003, 2005), then why is the lifespan shorter in Urm1n123 mutants? There are

several possible explanations and answers, like interference of Urm1 with other

34

signaling cascades, or activation of the pro-apoptotic role of the JNK pathway

(Igaki, 2009) in older adult flies, which could contribute to shortening the

lifespan and induce premature aging in Urm1n123 mutants, but the exact answers

remain unknown.

Figure 7. Loss of Urm1 enhances the tolerance of Prx5 mutants to oxidative stress and

increases their survival rate.

Other important questions in Paper I addresses the role of Urm1 in the regulation

of the JNK pathway, and whether any of the major components in the JNK

pathway are direct targets of urmylation. In our investigations, we have not

managed to prove any urmylation events in the pathway, but this may very well

be due to technical limitations and the absence of proper tools. There is published

data from other labs that may suggest Urm1 to function as a sulfur carrier in the

JNK pathway, since two established inhibitors of JNK signaling, Puckered and

MKP4, both are influenced by thiolation (Saitoh et al., 1998) and the cellular

redox status (Cavigelli et al., 1996; Kamata et al., 2005; Sun et al., 2008). Hence,

the exact molecular mechanism linking the JNK pathway and Urm1 remains to

be revealed.

35

5.2. Paper II

A proteomics approach to identify targets of the ubiquitin-like

molecule Urm1 in Drosophila melanogaster

To unravel potential Urm1 interacting regulators of the JNK pathway was

certainly a catalyst, but not the main motivation to perform the proteomics

analysis to find putative Urm1 targets. One of the ultimate goals of studying a

protein modification system, is to know the identity of the modified targets, since

they pinpoint the biological processes in which the modifier may be involved and

represent starting points for future studies. For Urm1 specifically, prior to our

study, the interacting network was quite small and not that many studies had

aimed at expanding it. Nevertheless, efforts by Goehring et al., Van der Veen et

al., and Humbard et. al. (reviewed in chapter, 4.5) should be recognized for their

pioneering work to discover target proteins of Urm1 (Goehring, Rivers and

Sprague, 2003; Humbard et al., 2010; Van der Veen et al., 2011). Hence, to

overall increase the knowledge about the targets of urmylation, and to specifically

identify Urm1-interacting proteins in a multicellular organism and at different

developmental stages, we utilized a proteomics-based approach to decipher

candidate targets of urmylation in Drosophila.

Since Paper II does not aim at making any functional conclusions regarding the

biological role of the interaction of Urm1 with the identified binding partners, the

take home massage from this paper is essentially a list of 79 Urm1-interacting

proteins, identified specifically at three developmental stages of Drosophila

development. A wide range of diversity in molecular processes is covered by these

candidate Urm1 targets, such as oxidative stress, tRNA modification, aging, DNA

damage response, and immunity. Importantly, among these, we have confirmed

five proteins, including Jafrac1, Ciao1, MsrA/EIP71CD, GILT1 and Crammer, in

addition to the previously studied Uba4 and Prx5, to interact physically with

Urm1. Interestingly, while the majority of these targets were found to behave as

urmylation targets that display a size shift following conjugation to Urm1, we

discovered that one of these Urm1-interacting proteins, Crammer, showed a non-

covalent interaction with Urm1. In contrast to the bona fide urmylation targets,

there is no size shift of Crammer on Western blots following immuneprecipitation

together with Urm1, suggesting a mode of binding that is sensitive to denaturing

conditions (i.e. a non-covalent interaction).

In light of the identification of novel Urm1-associated proteins in this paper, we

found more clues regarding the potential connection between JNK signaling and

Urm1. For instance, among the putative urmylation targets, we found Sac1, which

36

displays a reported role as a repressor of JNK pathway activation in flies (Wei et

al., 2003). Another protein that is interesting to highlight in this regard is Par-1,

a kinase with a regulatory function in the Wnt signaling pathway, and further

known to also actively block JNK signaling by acting as a kinase switch for the

downstream responses mediated by the JNK activator, Dishevelled (Dsh) (Sun et

al., 2001).

As the nature and functionality of many of the targets are adequately discussed in

the paper, I will not go through them one by one. Still, I think that some key

findings should be mentioned here. Except for the six targets that were found in

all stages (Uba4, PHGPx, Prx5, MsrA/Eip71CD, CG8078 and GILT1), the majority

of the identified proteins were stage-specific. However, considering that the

samples were extracted from whole animals, the possibility of having other Urm1-

associated proteins that are not predicted by our study cannot be excluded. Other

unanswered questions that can be raised in this aspect is firstly which of the

proteins that are posttranslationally modified by Urm1 and which ones that

interact with Urm1 by other means, and secondly in which tissues all these

interactions occur. Likewise, another important consideration is to assess which

amount of the target proteins that is modified by and/or interact with Urm1.

There are currently no studies addressing this issue, however, based on the

general analogy of UBLs, it is to be expected that only a small percentage of the

targets is modified. Anyhow, at least for one of the urmylation targets, Prx5, we

have noticed a change in modification status following oxidative stress,

emphasizing that signaling events or environmental clues most likely have the

potential to induce protein urmylation.

5.3. Paper III

The HTLV-1 oncoprotein Tax is modified by the Ubiquitin related

modifier 1

Studies on Tax and HTLV-1 have a long history. Since 1977, when the first clinical

cases of ATL were reported (Takatsuki, 2005), many researchers have dedicated

their time and effort to understand the disease and its underlying molecular

mechanisms. As discussed earlier in the introduction (chapter 4.9.2), the

causative agent that triggers ATL, the virally encoded oncoprotein, Tax, is at the

crossroad of protein modifications.

37

In Manuscript III of this thesis, we contribute to the understanding of ATL, by

adding yet another molecule to the list of PTMs that modify Tax, namely Urm1.

By collaborating together with established researchers in the field, Prof. Ali

Bazarbachi and Dr. Margret Shirinian at the American University of Beirut

(Lebanon), we discovered that Urm1 conjugates to Tax in HeLa cells, specifically

targeting lysine residues 4-8 in Tax. By following up these results with studies in

Drosophila, we could further confirm that Urm1 and Tax interact physically also

in fly tissues (in vivo), indicating that urmylation of Tax is evolutionary conserved

from fly to man.

Given that Tax is known to be modified by ubiquitin and SUMO and that

modification by these UBLs strongly affect the subcellular targeting of Tax, we

next investigated whether Urm1 also influence the localization of Tax. When

consequently co-expressing Tax together with excessive levels of Urm1, it was

obvious that Urm1 strongly promotes a cytoplasmic localization of Tax, in HeLa

cells as well as in vivo in Drosophila salivary glands. Interestingly, this

cytoplasmic accumulation of Tax was also observed upon expression of Urm1 in

HuT-102 cells, which are HTLV-1 positive cells derived from an ATL patient.

Further supporting a role of Urm1 in the subcellular targeting of Tax, we found

that a reduction of Urm1 levels by RNA interference in Drosophila, reciprocally

triggered a nuclear accumulation of Tax.

One of the most important cellular pathways that have been depicted as

responsible for driving the oncogenesis downstream of Tax, is the NF-B

pathway. Similar to the modification of Tax by ubiquitin and SUMO, we have in

this manuscript established that also the presence of Urm1 affects the ability of

Tax to activate NF-B signaling. Specifically, co-overexpression of Urm1 and Tax

results in enhanced activity of NF-κB signaling, as indicated by increased levels

of Diptericin mRNA, an antimicrobial peptide and classical downstream target of

NF-B in Drosophila. This finding further emphasizes a regulatory role of

urmylation, not only in Tax localization, but also for the oncogenic capacity of

Tax.

Considering that the same lysine residues that are targeted by ubiquitin and

SUMO, also appear to be targeted by Urm1, one possible explanation for the

differential localization of Tax may be the result of a competition between Urm1,

ubiquitin and SUMO for these lysine residues in Tax (Lamsoul et al., 2005; Nasr

et al., 2006). The level of Urm1, ubiquitin and SUMO could thus be a

determinative factor for ATL, since the localization, oncogenic activity and also

Tax degradation most likely is strongly dependent on the balance between these

three UBLs.

38

Taken together, the Tax-Urm1 interaction seems to represent an important event

in the regulatory network that orchestrates the oncogenic properties of Tax. These

preliminary results provide an excellent starting point for further investigations

about the regulation of Tax by urmylation and its involvement in ATL.

5.4. Paper IV

Deciphering a novel role of the Urm1/Uba4 conjugation

machinery for Neuromuscular Junction (NMJ) development in

Drosophila melanogaster

In Paper I, we established a close link between Urm1 and activation of the JNK

pathway in adult flies, focusing on its role in the response against oxidative stress.

However, this does not rule out that Urm1 could potentially also affect JNK

signaling in other developmental stages. The majority of Urm1n123 mutant

animals die in late pupal stages, which provides an opportunity to study

homozygous mutants during larval development. At first glance, Urm1n123 mutant

larvae do not show any obvious developmental defects up until pupariation,

however, a closer look at the neuromuscular junctions (NMJs) of third instar

larvae, reveals a striking overgrowth phenotype.

Similar to the deficiency of Urm1 protein in Urm1n123 mutants, RNAi-mediated

knockdown of Urm1 in both pre- and post-synaptic tissues also results in

comparable NMJ phenotypes (utilizing OK371-GAL4 and 24B-GAL4,

respectively). Attempt to rescue the NMJ overgrowth of Urm1n123 mutants by

overexpressing Urm1 in pre- or post-synaptic tissues, does indeed lead to a partial

rescue, confirming that this phenotype is specifically caused by Urm1, and is not

the result of a background genetic alteration. In agreement, Urm1rv164 larvae

display completely normal NMJs. This finding is particularly interesting, as

rescue attempts employing the ubiquitous GAL4 driver Actin-5C, fails to rescue

the NMJ overgrowth phenotype in Urm1n123 mutants, suggesting a high tissue-

specificity of Urm1 activities that is not satisfactory fulfilled by Actin-5C during

NMJ growth.

Based on our previous studies in adult flies, we were keen to investigate whether

the JNK pathway is involved also in causing the NMJ overgrowth phenotype in

Urm1n123 larvae. These investigations were further prompted by the multitude of

papers that depict an important role of JNK signaling during NMJ growth

39

(reviewed in the introduction). Our initial results in this regard clearly indicated

that the JNK pathway is involved in triggering the NMJ overgrowth caused by the

absence of Urm1, since downregulation of JNK activity in synaptic tissue can (at

least partially) suppress the phenotype induced by Urm1 knockdown. This further

confirms the genetic interaction between Urm1 and the JNK pathway and also

suggests that NMJ growth regulation by Urm1 occurs via regulation of the JNK

pathway.

In light of previously discussed role of Par-1 as a negative regulator of the JNK

pathway (Sun et al., 2001), one of the potential candidates that could explain the

NMJ overgrowth in Urm1n123 mutants, is Par-1. Par-1 is a kinase that regulates

the synaptic targeting of Dlg through phosphorylation, and thereby affects NMJ

growth. Loss of par-1 leads to increased synapse formation, together with an

abnormal synaptic ultrastructure and increased synaptic transmission, whereas

overexpression of Par-1 has the opposite effects (Zhang et al., 2007).

Arguably, since the function of Urm1 is dependent on its activating enzyme, Uba4,

it is equally important to characterize Uba4 and its interaction with Urm1, as it is

to investigate Urm1 itself. Similar to our research on Urm1, we started our

investigations of Uba4 by generating the required tools (including the mutant

strain Uba4n29) and confirmed its genetic and physical interaction with Urm1

(Papers I, II).

Similar to the Urm1n123 strain, Uba4n29 mutants exhibit a NMJ overgrowth

phenotype. However, the structural characteristics of the NMJs in Uba4n29

animals are slightly different from those displayed by Urm1 deficient flies,

particularly in terms of synapse branching. This can possibly be the result of a

different genetic background, or a demonstration that although Urm1 and Uba4

are acting in the same pathway, there are also other pathways (e.g. MoCo

biosynthesis) that are affected by lack of Uba4, which may also affect the synaptic

structure.

What makes the Uba4n29 mutants a very interesting strain, is the unusually long

extension of the larval stages, which is a hallmark for a disturbed hormonal

regulation. Considering the striking prolongation of the larval development up to

22 days after egg laying, the list of candidate mechanisms, which may be affected

by the absence of Uba4 is rather short. Several hormones and neuropeptides are

known to be involved in the regulation of developmental timing in Drosophila,

but the master regulator of the events leading to metamorphosis is evidently the

steroid hormone ecdysone (Marchal et al., 2010; Yamanaka, Rewitz and

O’Connor, 2013). We still do not know if Uba4 is associated with and/or affects

the ecdysone pathway, but it is indeed highly tempting to speculate that the

explanation for the disturbed timing of development in Uba4n29 mutants is a

40

dysfunctional regulation of ecdysone signaling. This hypothesis is to some degree

supported by the identification of ecdysone-induced peptides among the Urm1

target candidates described in paper II. However, since Urm1n123 mutants do not

show any prolonged larval phenotype and survive quite well in the third instar

larval stage, it is unlikely that the Urm1/Uba4 system directly interacts with the

ecdysone pathway and may suggest that the influence on ecdysone regulation

could be indirect.

Another possible explanation for the prolonged larval stage and lethality in

Uba4n29 mutants, may be an involvement of Uba4 in one or several of the major

signaling pathways that are important for sulfur metabolism. In this regard, it is

important to remember that Uba4 is a dual function protein, which besides

activating the Urm1 machinery, also is involved in MoCo biosynthesis. In the

MoCo biosynthesis pathway Uba4 interacts with another UBL protein, Mocs2A

(Chowdhury et al., 2012). Here again, a sulfur rely system is involved, as follows.

The MoCo pathway is initiated by the Molybdopterin (MPT) synthase, which

catalyzes the conversion of precursor Z to molybdopterin, a MoCo precursor, by

donating sulfur to form a dithiolene group and thereby coordinating the

molybdenum atom. Importantly, MoaD is the sulfur donor used during the

formation of the dithioloene group in molybdopterin (Rizzi and Schindelin,

2002). MoCo is essential for the function of multiple metabolic enzymes in the

xanthine oxidase and sulfite oxidase families (Hille, Nishino and Bittner, 2011).

In the absence of MoCo, these enzymes cannot function, leading to an

accumulation of toxic chemicals, including sulfite, S-sulfocysteine, xanthine, and

hypoxanthine, and ultimately early natal lethality. It is tempting to speculate that

a similar cause may underlie the lethality in Uba4n29 mutants.

Similar to the prokaryotic ancestors, mammalian MPT synthases are

heterotetramers, consisting of two MoaE and two MoaD subunits (Schwarz and

Mendel, 2006; Daniels et al., 2008). In Drosophila, the annotated genes

CG42503 and CG10238 encode the small (Mocs2A) and large (Mocs2B) subunits

of Mocs2 (dMoaE), respectively. We have generated Mocs2A mutant strains in

the lab, but due to lack of time these flies have not been studied in detail.

However, Mocs2A null mutants appear to be homozygous viable as adults, but

fail to be established as a homozygous mutant line, due to sterility.

The Drosophila counterpart of Mocs2B, CG10238, contains a C-terminal

sequence that codes for a MAPK upstream kinase (MUK)-binding inhibitory

protein (MBIP) (Suganuma et al., 2010). Interestingly, it was recently shown that

dMoaE (Mocs2) acts as a negative regulator of the JNK cascade, mediated via its

function as a subunit of the ATAC (Ada two A containing) histone

acetyltransferase complex. The ATAC complex directly interacts with the JNK

pathway, by acting as a transcriptional cofactor for Jun (Suganuma et al., 2008,

41

2010; Wang et al., 2008). This raises the possibility of a putative crosstalk

between the Urm1/Uba4 machinery and Uba4/Mocs2 complex involved in MoCo

biosynthesis, potentially offering a link between Urm1 and the JNK pathway, via

the ATAC complex. In support of this theory, one of the components of the ATAC

complex, HCF, has also popped up as an Urm1-interacting protein in our

proteomics study (Paper II). Nevertheless, making any detailed conclusions

about the relationship between the Urm1 pathway and developmental

phenotypes such as JNK-dependent NMJ overgrowth requires further studies.

42

5.5. General discussion and future perspectives

In this final part of the discussion, I would like to present my personal perspective

on my doctoral project and highlight some reflections and theories that may lie

outside the scope of the thesis work.

In the past six years, my understanding of the Urm1 field has led me to believe

that there is a huge gap between the different ideas that people in the field

express. On one side, there is one group of researchers who tend to argue that

Urm1 is only involved in tRNA modification, and that any protein modification

function merely is a result of the hyperactive nature of activated Urm1. Hence,

these people suggest that protein modification by Urm1, if anything, is a very

primitive system that was later specialized in the form of ubiquitin and other

UBLs. This may in fact actually be a probable theory, considering that Urm1 is

indeed a “molecular fossil”, phylogenetically placed between ancient sulfur

carriers and eukaryotic UBLs. On the other side, a second group of researchers

including our laboratory, instead believe that Urm1 truly is a dual function

protein, which in addition to the sulfur transfer activity displayed during tRNA

modification, has acquired the capacity to modify cellular proteins, thereby

suggesting that several diverse molecular functions are regulated by urmylation.

This hypothesis is supported by our work considering the tissue and

developmental-stage specificity of Urm1 interacting proteins, as well as the

genetic interaction of Urm1 with one of the major signaling pathways in the cell,

the JNK pathway.

Another theory that I would like to put forward here regards the JNK pathway

and how the Urm1/Uba4 machinery interacts with it. Considering that the two

UBL proteins Urm1 and Mocs2A both interact with Uba4, one hypothesis could

be that there exists a competitive relationship between them. Lack of Urm1 could

in that case potentially shift the balance towards increased levels of Mocs2A

bound to Uba4 and therefore a lower degree of ATAC complex assembly, which

could lead to the elimination of a key inhibitory mechanism of the JNK pathway.

I do not have much time left as a PhD student, but I sincerely hope that future

research will uncover the nature of the interaction and functions of the

Urm1/Uba4/Mocs2A cellular machinery and reveal the molecular mechanism

behind how it affects the JNK pathway.

43

6. Acknowledgements

Twelve years ago, when I decided to study cell and molecular biology at the

university, I set my goal to become a successful scientist. Those years are long

gone and now I have realized that becoming a scientist is a long journey and that

I am just at the beginning of the road, yet that there are checkpoints and subaims

to be reached along the way. Now when I am reaching one of these checkpoints

in my scientific career, I would like to take the time and thank the people who

have supported me during these years.

On top of the list is of course my kind supervisor, Dr. Caroline Grabbe. Thank

you for believing in me and giving me the opportunity to work in your lab. I have

always looked at you as a wise person, an intellectual boss, a good friend and a

role model. You are one of the most precise and organized people that I have ever

met. In the past 6 years you have literally raised me as a PhD student and I have

learned a lot from you. I wish you the best of luck in the future.

Our research group was a very small with only three people in the meetings for

the most part, however, having a speedster technical expert in our lab have

boosted our ability to perform experiments and do good research. Ingrid, I have

been really lucky to have someone like you as a colleague. Publishing the papers

included in this thesis would not have been possible without your effort. Thank

you for always being nice and helpful.

I would like to mention our former lab member, Henrik Lindehell, for his work

that contributed to generate the Mocs2A mutants, even if they were never really

studied in detail. I wish you good luck with your own PhD studies.

Next, I would like to thank our collaborators, Dr. Margret Shirinian and Prof.

Ali Bazarbachi, and all the people who contributed to the work of the Tax paper

in Lebanon.

Thanks also to my co-supervisor, Prof. Ruth Palmer, for participating in my

seminar committee multiple times even when she was away in Gothenburg. Also

thank you for giving me the chance to start working as a master student in your

lab back in 2011. The current and former members of the Palmer lab, Yasuo,

George, Kathrin and Patricia. I honesty miss the crowded fly room and the

journal clubs back in those days, when you were still at the Department of

Molecular biology. I wish you the best of luck in your experiments and a

successful career in Gothenburg (or wherever you will end up).

44

To Fredrik my very good colleague and friend. I have enjoyed your fellowship in

the past seven years. Thank you for helping me with fly crosses. We did not get to

finish those experiments, but the results show that you are a reliable person and

a compassionate friend. I wish you a successful career, and who knows, maybe

someday we will publish our own collaborative paper.

To the Åsa Rasmuson-Lestander lab, thanks for sharing the lab and office

space with us, especially you Sa, always friendly and kind. Thank you for all the

times that you shared your fly lines and antibodies with me, as well as fly food

and protocols….!! Always notifying me when there was cake in the kitchen.

Working alongside you was truly fun.

Next I want to thank the people in the Department of Molecular biology:

To Gunilla Jäger for helping us with the tRNA data and HPLC, the Fly food

and Media facility, who have a critical role in catalyzing the speed of research

at the department. To Johnny Stenman, for handling the orders, deliveries,

posts and literally solving any possible technical problem in the department, to

Marek Wilczynski for computer and IT support and finally the current and

former administration staff, Maria, Jannike, Patrik and Lina.

When I joined the Department of Molecular biology in 2011, I became a part of a

small but great community within the department, the fly floor. I think that our

common goal in fly floor is to prove that the Drosophila model system still has its

greatest days ahead. This being said, I would like to thank the fly people for nearly

seven years of friendliness.

Thanks also to the Epicon groups for the weekly journal clubs, which gave me

the chance to learn more about epigenetics and chromosomes. Prof. Jan

Larsson and Prof. Dan Hultmark for always smiling and agreeing to join my

seminar committee. Respectable Fly researchers Yuri Schwartz and Per

Stenberg for interesting scientific discussions in journal clubs and my dear fly

floor collogues, Jens-Ola, Inez, Anna-Mia, Masha, Tatiana, Alexander,

Jana, Juan, Edvin and Jacob, for friendly interactions in these years.

During my years here in Umeå, I have also made some valuable friendships with

some of my colleagues. One of them is my good friend Aman, one of the coolest

guys on the planet. Thank you for all of your ideas and support regarding the

bioinformatics. I guess that soon it will be your turn to write your PhD thesis, so

best of luck to you. My fellow poker buddy, Isaac, thank you for all the fly food

support, protocol advice and the brief scientific discussions that we have had here

and there. For you too, I wish the best of luck with your PhD thesis. One of my

very first friends in Umeå, Marie-Line, thank you for more than seven years of

45

friendship, I hope that you and Hasan and baby Tomas stay happy forever. Also,

I wish you too a good luck with your PhD studies.

Last but not least, I would like to thank the fellow colleagues with whom I had the

pleasure to share the teaching and also everyone else who I failed to mention here.

من معنا میده و هدف و نهایت تمام زندگیربای کسانی هک وجودشون هب رد انتها الزم میدونم هک چند خطی رو هم هب افرسی بنویسم تالش اهم خوشحالیه این زعزیاهن.

ی هب نوشتن این شماست. وقت خود گذشتگی از خارطب من زندگی موفقیت اهی هک تمام ،هم، ، هن فقط نوشتن این اپیان انپدر و مارد زعزیم سپ دارمار بودن زربای سپاسگ ل دلی ،ردیایی بی رکانربای گفتن و هب اندازه ی اقیانوسی از رحفمتن رسیدم متوهج شدم هک

. رمسی، بابت همه چیز.میکنم این ممهه لالهردرو اهم همه ی ان گفتهو تلخی اه و شادی اه و غم اه ختی اهتمام س رد ی زعزیم، تو امید و خوشبختی رو ربای من معنا رکدی. ازت ممنونم بابت اینکهزرها

ازت ممنونم بابت اینکه هستی. .سون رکدیآربام و سختیه رغبت رو حمایتم رکدیمن بودی و جور نبودن من رد . رمسی از اینکه پشتواهن یاحساس تنهایی نکردم رهزگبا داشتن رباردی مثل تو، من جان، بهنام

کنار لانواده رو هب دوش کشیدی.کانی میکنم رد نهایت و تنور دوستی یدت و هچ رد وطن من روهمراهی رکدرغب هک هچ رد صمیماهن رتین سپاس اه رو تقدیم هب شما دوستان و زندی و رگم نگه داشتید.رو معرفت

46

7. References

Adams, M. D. et al. (2000) ‘The genome sequence of Drosophila melanogaster.’, Science (New York, N.Y.), 287(5461), pp. 2185–95. doi: 10.1126/science.287.5461.2185.

Agnès, F., Suzanne, M. and Noselli, S. (1999) ‘The Drosophila JNK pathway controls the morphogenesis of imaginal discs during metamorphosis.’, Development (Cambridge, England), 126(23), pp. 5453–62.

Agris, P. F. (2008) ‘Bringing order to translation: The contributions of transfer RNA anticodon-domain modifications’, EMBO Reports, 9(7), pp. 629–635. doi: 10.1038/embor.2008.104.

Anjum, R. S. et al. (2015) ‘Involvement of a eukaryotic-like ubiquitin-related modifier in the proteasome pathway of the archaeon Sulfolobus acidocaldarius’, Nature Communications, 6, p. 8163. doi: 10.1038/ncomms9163.

Aouba, A. et al. (2004) ‘Hemophagocytic syndrome as a presenting sign of transformation of smoldering to acute adult T-cell leukemia/lymphoma: Efficacy of anti-retroviral and interferon therapy’, American Journal of Hematology, 76(2), pp. 187–189. doi: 10.1002/ajh.20065.

Araye, Q. and Sawamura, K. (2013) ‘Genetic decay of balancer chromosomes in Drosophila melanogaster.’, Fly. Taylor & Francis, 7(3), pp. 184–186. doi: 10.4161/fly.24466.

Ariumi, Y. et al. (2003) ‘Distinct nuclear body components, PML and SMRT, regulate the trans-acting function of HTLV-1 Tax oncoprotein’, Oncogene, 22(11), pp. 1611–1619. doi: 10.1038/sj.onc.1206244.

Arking, R. et al. (1991) ‘Elevated paraquat resistance can be used as a bioassay for longevity in a genetically based long‐ lived strain of Drosophila’, Developmental Genetics, 12(5), pp. 362–370. doi: 10.1002/dvg.1020120505.

Ashburner, M., Golic, K. G. and Hawley, R. S. (2005) Drosophilia: A Laboratory Handbook. Cold Spring Harbor Laboratory Press.

Atwal, P. S. and Scaglia, F. (2016) ‘Molybdenum cofactor deficiency’, Molecular Genetics and Metabolism, 117(1), pp. 1–4. doi: 10.1016/j.ymgme.2015.11.010.

Atwood, H. L., Govind, C. K. and Wu, C. ‐ F (1993) ‘Differential ultrastructure of synaptic terminals on ventral longitudinal abdominal muscles in Drosophila larvae’, Journal of Neurobiology, 24(8), pp. 1008–1024. doi: 10.1002/neu.480240803.

Azran, I., Schavinsky-Khrapunsky, Y. and Aboud, M. (2004) ‘Role of Tax protein in human T-cell leukemia virus type-I leukemogenicity.’, Retrovirology, 1(1), p. 20. doi: 10.1186/1742-4690-1-20.

Barmak, K. et al. (2003) ‘Human T cell leukemia virus type I-induced disease: Pathways to cancer and neurodegeneration’, Virology, 308(1), pp. 1–12. doi: 10.1016/S0042-6822(02)00091-0.

Bayer, P. et al. (1998) ‘Structure determination of the small ubiquitin-related modifier SUMO-1.’, Journal of molecular biology, 280(2), pp. 275–286. doi: 10.1006/jmbi.1998.1839.

Bazarbachi, A. et al. (2011) ‘How I treat adult T-cell leukemia/lymphoma’, Blood, 118(7), pp. 1736–1745. doi: 10.1182/blood-2011-03-345702.

Bazarbachi, a et al. (1999) ‘Arsenic trioxide and interferon-alpha synergize to induce cell

47

cycle arrest and apoptosis in human T-cell lymphotropic virus type I-transformed cells.’, Blood, 93(1), pp. 278–83.

Begley, U. et al. (2007) ‘Trm9-Catalyzed tRNA Modifications Link Translation to the DNA Damage Response’, Molecular Cell, 28(5), pp. 860–870. doi: 10.1016/j.molcel.2007.09.021.

Behrens, A., Sibilia, M. and Wagner, E. F. (1999) ‘Amino-terminal phosphorylation of c-Jun regulates stress-induced apoptosis and cellular proliferation’, Nature Genetics, 21(3), pp. 326–329. doi: 10.1038/6854.

Bentley Glass, H. (1933) ‘A study of dominant mosaic eye-colour mutants in Drosophila Melanogaster - II. Tests involving crossing-over and non-disjunction’, Journal of Genetics. Springer India, 28(1), pp. 69–112. doi: 10.1007/BF02981769.

Berg, J. M. (Jeremy M. et al. (2002) Biochemistry. W.H. Freeman.

Berreur, P. et al. (1984) ‘Ecdysteroids during the third larval instar in 1(3)ecd-1ts, a temperature-sensitive mutant of Drosophila melanogaster.’, General and comparative endocrinology, 54(1), pp. 76–84.

Bex, F. et al. (1999) ‘Phosphorylation of the human T-cell leukemia virus type 1 transactivator tax on adjacent serine residues is critical for tax activation.’, Journal of virology, 73(1), pp. 738–745.

Biteau, B. et al. (2011) ‘Regulation of Drosophila lifespan by JNK signaling’, Experimental Gerontology. NIH Public Access, 46(5), pp. 349–354. doi: 10.1016/j.exger.2010.11.003.

Bjork, G. R. et al. (2007) ‘A conserved modified wobble nucleoside (mcm5s2U) in lysyl-tRNA is required for viability in yeast’, Rna, 13(8), pp. 1245–1255. doi: 10.1261/rna.558707.

Bordo, D. and Bork, P. (2002) ‘The rhodanese/Cdc25 phosphatase superfamily. Sequence-structure-function relations’, EMBO Reports. European Molecular Biology Organization, 3(8), pp. 741–746. doi: 10.1093/embo-reports/kvf150.

Boutros, M., Agaisse, H. and Perrimon, N. (2002) ‘Sequential activation of signaling pathways during innate immune responses in Drosophila’, Developmental Cell, 3(5), pp. 711–722. doi: 10.1016/S1534-5807(02)00325-8.

Brand, A. H. and Perrimon, N. (1993) ‘Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.’, Development (Cambridge, England), 118(2), pp. 401–15. doi: 10.1101/lm.1331809.

Briggs, S. D. et al. (2002) ‘Trans-histone regulatory pathway in chromatin’, Nature, 418(6897), p. 498. doi: 10.1038/nature00970.

Brody, T. (1999) ‘The Interactive Fly: gene networks, development and the Internet.’, Trends in genetics  : TIG. Elsevier, 15(8), pp. 333–334. doi: 10.1016/S0168-9525(99)01775-8.

Burroughs, A. M., Iyer, L. M. and Aravind, L. (2009) ‘Natural history of the E1-like superfamily: Implication for adenylation, sulfur transfer, and ubiquitin conjugation’, Proteins: Structure, Function and Bioinformatics. NIH Public Access, 75(4), pp. 895–910. doi: 10.1002/prot.22298.

Busch, H. and Goldknopf, I. L. (1981) ‘Ubiquitin - protein conjugates’, Molecular and Cellular Biochemistry. Martinus Nijhoff/Dr. W. Junk Publishers, 40(3), pp. 173–187. doi: 10.1007/BF00224611.

Cacciuttolo, M. A. et al. (1993) ‘Hyperoxia induces DNA damage in mammalian cells’, Free

48

Radic Biol Med, 14(3), pp. 267–276.

Calattini, S. et al. (2005) ‘Discovery of a new human T-cell lymphotropic virus (HTLV-3) in Central Africa.’, Retrovirology, 2(1), p. 30. doi: 10.1186/1742-4690-2-30.

Campos-Ortega, J. A. (1997) ‘Neurogenesis in Drosophila: an historical perspective and some prospects.’, Perspectives on developmental neurobiology, 4(4), pp. 267–71.

Cappadocia, L. and Lima, C. D. (2017) ‘Ubiquitin-like Protein Conjugation: Structures, Chemistry, and Mechanism’, Chemical Reviews. American Chemical Society, p. acs.chemrev.6b00737. doi: 10.1021/acs.chemrev.6b00737.

Carrillo, R. A. et al. (2010) ‘Presynaptic activity and CaMKII modulate retrograde semaphorin signaling and synaptic refinement’, Neuron, 68(1), pp. 32–44. doi: 10.1016/j.neuron.2010.09.005.

Cavigelli, M. et al. (1996) ‘The tumor promoter arsenite stimulates AP-1 activity by inhibiting a JNK phosphatase.’, The EMBO journal, 15(22), pp. 6269–79.

Cereseto, A. et al. (1997) ‘Differential expression of alternatively spliced pX mRNAs in HTLV-I-infected cell lines.’, Leukemia, 11(6), pp. 866–70. doi: 10.1038/sj.leu.2400665.

Chen, C., Huang, B., Eliasson, M., et al. (2011) ‘Elongator complex influences telomeric gene silencing and DNA damage response by its role in wobble uridine tRNA modification’, PLoS Genetics, 7(9), p. e1002258. doi: 10.1371/journal.pgen.1002258.

Chen, C., Huang, B., Anderson, J. T., et al. (2011) ‘Unexpected accumulation of ncm5u and ncm5s2u in a trm9 mutant suggests an additional step in the synthesis of mcm5u and mcm5s2u’, PLoS ONE. Edited by L. Randau, 6(6), p. e20783. doi: 10.1371/journal.pone.0020783.

Chen, W., White, M. A. and Cobb, M. H. (2002) ‘Stimulus-specific requirements for MAP3 kinases in activating the JNK pathway’, Journal of Biological Chemistry, 277(51), pp. 49105–49110. doi: 10.1074/jbc.M204934200.

Chen, Z. J. (2005) ‘Ubiquitin signalling in the NF-κB pathway’, Nature Cell Biology, 7(8), pp. 758–765. doi: 10.1038/ncb0805-758.

Chiari, E. et al. (2004) ‘Stable ubiquitination of human T-cell leukemia virus type 1 tax is required for proteasome binding.’, Journal of virology, 78(21), pp. 11823–32. doi: 10.1128/JVI.78.21.11823-11832.2004.

Chowdhury, M. M. et al. (2012) ‘Dual role of the molybdenum cofactor biosynthesis protein MOCS3 in tRNA thiolation and molybdenum cofactor biosynthesis in humans’, Journal of Biological Chemistry. American Society for Biochemistry and Molecular Biology, 287(21), pp. 17297–17307. doi: 10.1074/jbc.M112.351429.

Chu, Z. L. et al. (1998) ‘The tax oncoprotein of human T-cell leukemia virus type 1 associates with and persistently activates IκB kinases containing IKKα and IKKβ’, Journal of Biological Chemistry, 273(26), pp. 15891–15894. doi: 10.1074/jbc.273.26.15891.

Chu, Z. L. et al. (1999) ‘IKKγ mediates the interaction of cellular IκB kinases with the Tax transforming protein of human T cell leukemia virus type 1’, Journal of Biological Chemistry, 274(22), pp. 15297–15300. doi: 10.1074/jbc.274.22.15297.

Church, R. B. and Robertson, F. W. (1966) ‘A biochemical study of the growth of Drosophila melanogaster’, Journal of Experimental Zoology. Wiley Subscription Services, Inc., A Wiley Company, 162(3), pp. 337–351. doi: 10.1002/jez.1401620309.

Cieśla, J., Fraczyk, T. and Rode, W. (2011) ‘Phosphorylation of basic amino acid residues in proteins: Important but easily missed’, Acta Biochimica Polonica, 58(2), pp. 137–148.

49

doi: 201114789 [pii].

Collins, C. A. et al. (2006) ‘Highwire Restrains Synaptic Growth by Attenuating a MAP Kinase Signal’, Neuron, 51(1), pp. 57–69. doi: 10.1016/j.neuron.2006.05.026.

Daniels, J. N. et al. (2008) ‘Crystal structure of a molybdopterin synthase-precursor Z complex: Insight into its sulfur transfer mechanism and its role in molybdenum cofactor deficiency’, Biochemistry. American Chemical Society , 47(2), pp. 615–626. doi: 10.1021/bi701734g.

Dassouki, Z. et al. (2015) ‘ATL response to arsenic/interferon therapy is triggered by SUMO/PML/RNF4-dependent Tax degradation’, Blood, 125(3), pp. 474–482. doi: 10.1182/blood-2014-04-572750.

Davis, G. W., Schuster, C. M. and Goodman, C. S. (1996) ‘Genetic dissection of structural and functional components of synaptic plasticity. III. CREB is necessary for presynaptic functional plasticity’, Neuron, 17(4), pp. 669–679. doi: 10.1016/S0896-6273(00)80199-3.

Davis, R. J. (2000) ‘Signal transduction by the JNK group of MAP kinases’, Cell, 103(2), pp. 239–252. doi: 10.1016/S0092-8674(00)00116-1.

DiAntonio, A. et al. (2001) ‘Ubiquitination-dependent mechanisms regulate synaptic growth and function’, Nature, 412(6845), pp. 449–452. doi: 10.1038/35086595.

Van Dooren, S. et al. (2007) ‘Phylogeny of primate T lymphotropic virus type 1 (PTLV-1) including various new Asian and African non-human primate strains’, Infection, Genetics and Evolution. Elsevier, 7(3), pp. 374–381. doi: 10.1016/j.meegid.2006.06.003.

Durkin, S. S. et al. (2006) ‘Site-specific phosphorylation differentiates active from inactive forms of the human T-cell leukemia virus type 1 tax oncoprotein’, Journal of Biological Chemistry, 281(42), pp. 31705–31712. doi: 10.1074/jbc.M607011200.

Edlich, R. F., Arnette, J. A. and Williams, F. M. (2000) ‘Global epidemic of human T-cell lymphotropic virus type-I (HTLV-I)’, The Journal of Emergency Medicine, 18(1), pp. 109–119. doi: 10.1016/S0736-4679(99)00173-0.

El-Sabban, M. E. et al. (2000) ‘Arsenic-interferon-alpha-triggered apoptosis in HTLV-I transformed cells is associated with tax down-regulation and reversal of NF-kappa B activation’, Blood, 96(8), pp. 2849–2855.

Etter, P. D. et al. (2005) ‘Synaptic and genomic responses to JNK and AP-1 signaling in Drosophila neurons.’, BMC neuroscience. BioMed Central, 6(1), p. 39. doi: 10.1186/1471-2202-6-39.

Fernandes, J. J. and Keshishian, H. (1999) ‘Development of the adult neuromuscular system.’, International review of neurobiology, 43, pp. 221–239. doi: 10.1016/S0074-7742(08)60547-4.

Finkel, T. and Holbrook, N. J. (2000) ‘Oxidants, oxidative stress and the biology of ageing’, Nature. Nature Publishing Group, 408(6809), pp. 239–247. doi: 10.1038/35041687.

Fouquet, W. et al. (2009) ‘Maturation of active zone assembly by Drosophila Bruchpilot’, Journal of Cell Biology, 186(1), pp. 129–145. doi: 10.1083/jcb.200812150.

Furukawa, K. et al. (2000) ‘A protein conjugation system in yeast with homology to biosynthetic enzyme reaction of prokaryotes’, Journal of Biological Chemistry. American Society for Biochemistry and Molecular Biology, 275(11), pp. 7462–7465. doi: 10.1074/jbc.275.11.7462.

Garcia-Bellido, A. and Lewis, E. B. (1976) ‘Autonomous cellular differentiation of homoeotic bithorax mutants of Drosophila melanogaster’, Developmental Biology.

50

Academic Press, 48(2), pp. 400–410. doi: 10.1016/0012-1606(76)90101-9.

Geuking, P. et al. (2009) ‘A non-redundant role for Drosophila Mkk4 and hemipterous/Mkk7 in TAK1-mediated activation of JNK’, PLoS ONE. Edited by A. Bergmann, 4(11), p. e7709. doi: 10.1371/journal.pone.0007709.

Ghosh, H. P. (1976) ‘Role of Modified Nucleosides in tRNA: Effect of Modification of the 2-Thiouridine Derivative Located at the 5’-End of the Anticodon of Yeast Transfer RNALys2’, Nucleic Acids Research, 3(3), pp. 523–536. doi: 10.1093/nar/3.3.523.

Glickman, M. H. and Ciechanover, A. (2002) ‘The Ubiquitin-Proteasome Proteolytic Pathway: Destruction for the Sake of Construction’, Physiological Reviews, 82(2), pp. 373–428. doi: 10.1152/physrev.00027.2001.

Glise, B., Bourbon, H. and Noselli, S. (1995) ‘hemipterous encodes a novel drosophila MAP kinase kinase, required for epithelial cell sheet movement’, Cell, 83(3), pp. 451–461. doi: 10.1016/0092-8674(95)90123-X.

Goehring, A. S. (2003) ‘Synthetic Lethal Analysis Implicates Ste20p, a p21-activated Protein Kinase, in Polarisome Activation’, Molecular Biology of the Cell, 14(4), pp. 1501–1516. doi: 10.1091/mbc.E02-06-0348.

Goehring, A. S., Rivers, D. M. and Sprague, G. F. (2003) ‘Attachment of the ubiquitin-related protein Urm1p to the antioxidant protein Ahp1p’, Eukaryotic Cell. American Society for Microbiology (ASM), 2(5), pp. 930–936. doi: 10.1128/EC.2.5.930-936.2003.

Goehring, A. S., Rivers, D. M. and Sprague George F. (2003) ‘Urmylation: A Ubiquitin-like Pathway that Functions during Invasive Growth and Budding in Yeast’, Molecular biology of the cell, 14(November), pp. 4329–4341. doi: 10.1091/mbc.E03-02-0079.

Goldstein, G. et al. (1975) ‘Isolation of a polypeptide that has lymphocyte-differentiating properties and is probably represented universally in living cells.’, Proceedings of the National Academy of Sciences. National Academy of Sciences, 72(1), pp. 11–15. doi: 10.1073/pnas.72.1.11.

Grangeasse, C., Stülke, J. and Mijakovic, I. (2015) ‘Regulatory potential of post-translational modifications in bacteria’, Frontiers in Microbiology. Frontiers, 6(MAY), p. 500. doi: 10.3389/fmicb.2015.00500.

Grant, C. et al. (2002) ‘Human T cell leukemia virus type I and neurologic disease: Events in bone marrow, peripheral blood, and central nervous system during normal immune surveillance and neuroinflammation’, Journal of Cellular Physiology, 190(2), pp. 133–159. doi: 10.1002/jcp.10053.

Grassmann, R., Aboud, M. and Jeang, K.-T. (2005) ‘Molecular mechanisms of cellular transformation by HTLV-1 Tax’, Oncogene, 24(39), pp. 5976–5985. doi: 10.1038/sj.onc.1208978.

Gratz, S. J. et al. (2015) ‘CRISPR-Cas9 genome editing in Drosophila’, Current Protocols in Molecular Biology, 2015(1), p. 31.2.1-31.2.20. doi: 10.1002/0471142727.mb3102s111.

Griffith, L. C. and Budnik, V. (2006) ‘Plasticity and Second Messengers During Synapse Development’, in International Review of Neurobiology. NIH Public Access, pp. 237–265. doi: 10.1016/S0074-7742(06)75011-5.

Grumbling, G. (2006) ‘FlyBase: anatomical data, images and queries’, Nucleic Acids Research, 34(90001), pp. D484–D488. doi: 10.1093/nar/gkj068.

Gustilo, E. M., Vendeix, F. A. and Agris, P. F. (2008) ‘tRNA’s modifications bring order to gene expression’, Current Opinion in Microbiology, 11(2), pp. 134–140. doi: 10.1016/j.mib.2008.02.003.

51

El Hajj, H. et al. (2010) ‘Therapy-induced selective loss of leukemia-initiating activity in murine adult T cell leukemia’, The Journal of Experimental Medicine, 207(13), pp. 2785–2792. doi: 10.1084/jem.20101095.

El Hajj, H. et al. (2012) ‘Animal models on HTLV-1 and related viruses: What did we learn’, Frontiers in Microbiology. Frontiers, 3(SEP), p. 333. doi: 10.3389/fmicb.2012.00333.

Halder, G. and Mills, G. B. (2011) ‘Drosophila in cancer research: To boldly go where no one has gone before’, Oncogene. Nature Publishing Group, 30(39), pp. 4063–4066. doi: 10.1038/onc.2011.128.

Haller, K. et al. (2002) ‘Physical interaction of human T-cell leukemia virus type 1 Tax with cyclin-dependent kinase 4 stimulates the phosphorylation of retinoblastoma protein’, Mol Cell Biol, 22(10), pp. 3327–3338.

Hanada, T. and Ohsumi, Y. (2005) ‘Structure-function relationship of Atg12, a ubiquitin-like modifier essential for autophagy.’, Autophagy, 1(2), pp. 110–118. doi: 10.4161/auto.1.2.1858.

Harhaj, E. W. et al. (2000) ‘Somatic mutagenesis studies of NF-kappa B signaling in human T cells: evidence for an essential role of IKK gamma in NF-kappa B activation by T-cell costimulatory signals and HTLV-I Tax protein.’, Oncogene, 19(11), pp. 1448–56. doi: 10.1038/sj.onc.1203445.

Harhaj, E. W. and Sun, S. C. (1999) ‘IKKgamma serves as a docking subunit of the IkappaB kinase (IKK) and mediates interaction of IKK with the human T-cell leukemia virus tax protein’, Journal of Biological Chemistry, 274(33), pp. 22911–22914. doi: 10.1074/jbc.274.33.22911.

Harhaj, N. S., Sun, S.-C. and Harhaj, E. W. (2006) ‘Activation of NF- B by the Human T Cell Leukemia Virus Type I Tax Oncoprotein Is Associated with Ubiquitin-dependent Relocalization of I B Kinase’, Journal of Biological Chemistry, 282(6), pp. 4185–4192. doi: 10.1074/jbc.M611031200.

Harman, D. (1956) ‘Aging: A Theory Based on Free Radical and Radiation Chemistry’, Journal of Gerontology, 11(3), pp. 298–300. doi: 10.1093/geronj/11.3.298.

Hasegawa, H. et al. (2006) ‘Thymus-derived leukemia-lymphoma in mice transgenic for the Tax gene of human T-lymphotropic virus type I’, Nature Medicine. Nature Publishing Group, 12(4), pp. 466–472. doi: 10.1038/nm1389.

Hayden, M. S. and Ghosh, S. (2011) ‘NF-κB in immunobiology’, Cell Research, 21(2), pp. 223–244. doi: 10.1038/cr.2011.13.

Hayden, M. S. and Ghosh, S. (2012) ‘NF-κB, the first quarter-century: Remarkable progress and outstanding questions’, Genes and Development, 26(3), pp. 203–234. doi: 10.1101/gad.183434.111.

Helfand, S. L. and Rogina, B. (2003) ‘Genetics of Aging in the Fruit Fly, Drosophila melanogaster’, Annual Review of Genetics, 37(1), pp. 329–348. doi: 10.1146/annurev.genet.37.040103.095211.

Hicke, L., Schubert, H. L. and Hill, C. P. (2005) ‘Ubiquitin-binding domains’, Nature Reviews Molecular Cell Biology, 6(8), pp. 610–621. doi: 10.1038/nrm1701.

Hille, R., Nishino, T. and Bittner, F. (2011) ‘Molybdenum enzymes in higher organisms’, Coordination Chemistry Reviews. NIH Public Access, 255(9–10), pp. 1179–1205. doi: 10.1016/j.ccr.2010.11.034.

Hochstrasser, M. (2000) ‘BIOCHEMISTRY: All in the Ubiquitin Family’, Science. American Association for the Advancement of Science, 289(5479), pp. 563–564. doi:

52

10.1126/science.289.5479.563.

Hochstrasser, M. (2009) ‘Origin and function of ubiquitin-like proteins’, Nature. NIH Public Access, 458(7237), pp. 422–429. doi: 10.1038/nature07958.

Huang, B. (2005) ‘An early step in wobble uridine tRNA modification requires the Elongator complex’, Rna, 11(4), pp. 424–436. doi: 10.1261/rna.7247705.

Huang, B., Lu, J. and Bystrom, A. S. (2008) ‘A genome-wide screen identifies genes required for formation of the wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridine in Saccharomyces cerevisiae’, Rna, 14(10), pp. 2183–2194. doi: 10.1261/rna.1184108.

Huijmans, J. G. M. et al. (2017) ‘Molybdenum cofactor deficiency: Identification of a patient with homozygote mutation in the MOCS3 gene’, American Journal of Medical Genetics, Part A, 173(6), pp. 1601–1606. doi: 10.1002/ajmg.a.38240.

Humbard, M. A. et al. (2010) ‘Ubiquitin-like small archaeal modifier proteins (SAMPs) in Haloferax volcanii’, Nature. Nature Publishing Group, 463(7277), pp. 54–60. doi: 10.1038/nature08659.

Igaki, T. (2009) ‘Correcting developmental errors by apoptosis: Lessons from Drosophila JNK signaling’, Apoptosis, 14(8), pp. 1021–1028. doi: 10.1007/s10495-009-0361-7.

Iyer, L. M., Burroughs, a M. and Aravind, L. (2006) ‘The prokaryotic antecedents of the ubiquitin-signaling system and the early evolution of ubiquitin-like beta-grasp domains.’, Genome biology. BioMed Central, 7(7), p. R60. doi: 10.1186/gb-2006-7-7-r60.

Jarecki, J. and Keshishian, H. (1995) ‘Role of neural activity during synaptogenesis in Drosophila.’, The Journal of neuroscience  : the official journal of the Society for Neuroscience, 15(12), pp. 8177–8190.

Jennings, B. H. (2011) ‘Drosophila-a versatile model in biology & medicine’, Materials Today. Elsevier, 14(5), pp. 190–195. doi: 10.1016/S1369-7021(11)70113-4.

Johansson, M. J. O. et al. (2008) ‘Eukaryotic Wobble Uridine Modifications Promote a Functionally Redundant Decoding System’, Molecular and Cellular Biology, 28(10), pp. 3301–3312. doi: 10.1128/MCB.01542-07.

Johnson, G. L. and Nakamura, K. (2007) ‘The c-jun kinase/stress-activated pathway: Regulation, function and role in human disease’, Biochimica et Biophysica Acta (BBA) - Molecular Cell Research. Elsevier, 1773(8), pp. 1341–1348. doi: 10.1016/j.bbamcr.2006.12.009.

Jones, D. P. (2008) ‘Radical-free biology of oxidative stress’, AJP: Cell Physiology. American Physiological Society, 295(4), pp. C849–C868. doi: 10.1152/ajpcell.00283.2008.

Jordan, K. W. et al. (2012) ‘Genome-wide association for sensitivity to chronic oxidative stress in Drosophila melanogaster’, PLoS ONE. Edited by P. Csermely, 7(6), p. e38722. doi: 10.1371/journal.pone.0038722.

Juedes, A. et al. (2016) ‘Sulfur transfer and activation by ubiquitin-like modifier system Uba4•Urm1 link protein urmylation and tRNA thiolation in yeast’, Microbial Cell. Shared Science Publishers, 3(11), pp. 554–564. doi: 10.15698/mic2016.11.539.

Kalhor, H. R. and Clarke, S. (2003) ‘Novel methyltransferase for modified uridine residues at the wobble position of tRNA’, Molecular and cellular biology, 23(24), pp. 9283–9292. doi: 10.1128/MCB.23.24.9283-9292.2003.

Kamata, H. et al. (2005) ‘Reactive oxygen species promote TNFα-induced death and sustained JNK activation by inhibiting MAP kinase phosphatases’, Cell. Cell Press, 120(5),

53

pp. 649–661. doi: 10.1016/j.cell.2004.12.041.

Kannian, P. and Green, P. L. (2010) ‘Human T lymphotropic virus type 1 (HTLV-1): Molecular biology and oncogenesis’, Viruses. Multidisciplinary Digital Publishing Institute (MDPI), 2(9), pp. 2037–2077. doi: 10.3390/v2092037.

Katsuya, H. et al. (2015) ‘Treatment and survival among 1594 patients with ATL’, Blood, 126(24), pp. 2570–2577. doi: 10.1182/blood-2015-03-632489.

Kawakami, T. et al. (2001) ‘NEDD8 recruits E2-ubiquitin to SCF E3 ligase’, EMBO Journal. European Molecular Biology Organization, 20(15), pp. 4003–4012. doi: 10.1093/emboj/20.15.4003.

Kchour, G. et al. (2009) ‘Phase 2 study of the efficacy and safety of the combination of arsenic trioxide, interferon alpha, and zidovudine in newly diagnosed chronic adult T-cell leukemia/lymphoma (ATL)’, Blood, 113(26), pp. 6528–6532. doi: 10.1182/blood-2009-03-211821.

Keshishian, H. et al. (1993) ‘Cellular mechanisms governing synaptic development in Drosophila melanogaster.’, Journal of neurobiology, 24(6), pp. 757–787. doi: 10.1002/neu.480240606.

Keshishian, H., Chang, T. N. and Jarecki, J. (1994) ‘Precision and plasticity during Drosophila neuromuscular development’, Faseb J, 8(10), pp. 731–737.

Kfoury, Y. et al. (2008) ‘Ubiquitylated Tax targets and binds the IKK signalosome at the centrosome’, Oncogene, 27(12), pp. 1665–1676. doi: 10.1038/sj.onc.1210804.

Kimura, Y. and Tanaka, K. (2010) ‘Regulatory mechanisms involved in the control of ubiquitin homeostasis’, Journal of Biochemistry, 147(6), pp. 793–798. doi: 10.1093/jb/mvq044.

Klintworth, H. et al. (2007) ‘Activation of c-Jun N-terminal protein kinase is a common mechanism underlying paraquat- and rotenone-induced dopaminergic cell apoptosis’, Toxicological Sciences, 97(1), pp. 149–162. doi: 10.1093/toxsci/kfm029.

Kockel, L., Homsy, J. G. and Bohmann, D. (2001) ‘Drosophila AP-1: Lessons from an invertebrate’, Oncogene. Nature Publishing Group, 20(19 REV. ISS. 2), pp. 2347–2364. doi: 10.1038/sj.onc.1204300.

Kohsaka, H. et al. (2012) ‘Development of larval motor circuits in Drosophila’, Development Growth and Differentiation, 54(3), pp. 408–419. doi: 10.1111/j.1440-169X.2012.01347.x.

Krepinsky, K. and Leimkühler, S. (2007) ‘Site-directed mutagenesis of the active site loop of the rhodanese-like domain of the human molybdopterin synthase sulfurase MOCS3: Major differences in substrate specificity between eukaryotic and bacterial homologs’, FEBS Journal. Blackwell Publishing Ltd, 274(11), pp. 2778–2787. doi: 10.1111/j.1742-4658.2007.05811.x.

Kumar, S., Yoshida, Y. and Noda, M. (1993) ‘Cloning of a cDNA which encodes a novel ubiquitin-like protein.’, Biochemical and biophysical research communications, 195(1), pp. 393–399. doi: 10.1006/bbrc.1993.2056.

Kwon, H. et al. (2005) ‘Lethal cutaneous disease in transgenic mice conditionally expressing type I human T cell leukemia virus tax’, Journal of Biological Chemistry. American Society for Biochemistry and Molecular Biology, 280(42), pp. 35713–35722. doi: 10.1074/jbc.M504848200.

Lahey, T. et al. (1994) ‘The drosophila tumor suppressor gene dlg is required for normal synaptic bouton structure’, Neuron, 13(4), pp. 823–835. doi: 10.1016/0896-

54

6273(94)90249-6.

Lamsoul, I. et al. (2005) ‘Exclusive ubiquitination and sumoylation on overlapping lysine residues mediate NF-kappaB activation by the human T-cell leukemia virus tax oncoprotein.’, Molecular and cellular biology, 25(23), pp. 10391–406. doi: 10.1128/MCB.25.23.10391-10406.2005.

Larsen, B. B. et al. (2017) ‘Inordinate Fondness Multiplied and Redistributed: the Number of Species on Earth and the New Pie of Life’, The Quarterly Review of Biology. University of Chicago PressChicago, IL, 92(3), pp. 229–265. doi: 10.1086/693564.

Laxman, S. et al. (2013) ‘XSulfur amino acids regulate translational capacity and metabolic homeostasis through modulation of tRNA thiolation’, Cell. NIH Public Access, 154(2), pp. 416–29. doi: 10.1016/j.cell.2013.06.043.

Le Lay, S. et al. (2014) ‘Oxidative stress and metabolic pathologies: From an adipocentric point of view’, Oxidative Medicine and Cellular Longevity, 2014, pp. 1–18. doi: 10.1155/2014/908539.

Lee, K. S. et al. (2009) ‘JNK/FOXO-mediated neuronal expression of fly homologue of peroxiredoxin II reduces oxidative stress and extends life span’, Journal of Biological Chemistry, 284(43), pp. 29454–29461. doi: 10.1074/jbc.M109.028027.

Leidel, S. et al. (2009) ‘Ubiquitin-related modifier Urm1 acts as a sulphur carrier in thiolation of eukaryotic transfer RNA’, Nature, 458(7235), pp. 228–232. doi: 10.1038/nature07643.

Levine, M. (2008) ‘A systems view of Drosophila segmentation’, Genome Biology. BioMed Central, 9(2), p. 207. doi: 10.1186/gb-2008-9-2-207.

Lewis, E. B. (1982) ‘Control of body segment differentiation in Drosophila by the bithorax gene complex.’, Progress in clinical and biological research, 85 Pt A, pp. 269–88.

Lewis, E. B. (2007) ‘A gene complex controlling segmentation in Drosophila’, Genes, Development, and Cancer: The Life and Work of Edward B. Lewis, 276(5688), pp. 229–242. doi: 10.1007/978-1-4020-6345-9_10.

Lin, M. T. and Beal, M. F. (2006) ‘Mitochondrial dysfunction and oxidative stress in neurodegenerative diseases’, Nature, 443(7113), pp. 787–795. doi: 10.1038/nature05292.

Van der linde, K. et al. (2010) ‘A supermatrix-based molecular phylogeny of the family Drosophilidae’, Genetics Research. Cambridge University Press, 92(1), p. 25. doi: 10.1017/S001667231000008X.

Liu, Q. and Jin, L. H. (2017) ‘Organ-to-Organ Communication: A Drosophila Gastrointestinal Tract Perspective’, Frontiers in Cell and Developmental Biology, 5, p. 29. doi: 10.3389/fcell.2017.00029.

Liu, Z. et al. (2010) ‘Distinct Presynaptic and Postsynaptic Dismantling Processes of Drosophila Neuromuscular Junctions during Metamorphosis’, Journal of Neuroscience, 30(35), pp. 11624–11634. doi: 10.1523/JNEUROSCI.0410-10.2010.

Lukasiak, S. et al. (2008) ‘Proinflammatory cytokines cause FAT10 upregulation in cancers of liver and colon’, Oncogene, 27(46), pp. 6068–6074. doi: 10.1038/onc.2008.201.

Mahieux, R. et al. (2001) ‘Arsenic trioxide induces apoptosis in human T-cell leukemia virus type 1- and type 2-infected cells by a caspase-3-dependent mechanism involving Bcl-2 cleavage.’, Blood, 98(13), pp. 3762–3769. doi: 10.1182/blood.V98.13.3762.

Mahieux, R. and Gessain, A. (2009) ‘The human HTLV-3 and HTLV-4 retroviruses: New

55

members of the HTLV family’, Pathologie Biologie, 57(2), pp. 161–166. doi: 10.1016/j.patbio.2008.02.015.

Mahowald, A. P. (1963) ‘Electron microscopy of the formation of the cellular blastoderm in Drosophila melanogaster’, Experimental Cell Research, 32(3), pp. 457–468. doi: 10.1016/0014-4827(63)90186-1.

Marchal, E. et al. (2010) ‘Control of ecdysteroidogenesis in prothoracic glands of insects: A review’, Peptides. Elsevier, 31(3), pp. 506–519. doi: 10.1016/j.peptides.2009.08.020.

Martín-Blanco, E. et al. (1998) ‘puckered encodes a phosphatase that mediates a feedback loop regulating JNK activity during dorsal closure in Drosophila’, Genes and Development, 12(4), pp. 557–670. doi: 10.1101/gad.12.4.557.

Martin, P. (2004) ‘Parallels between tissue repair and embryo morphogenesis’, Development, 131(13), pp. 3021–3034. doi: 10.1242/dev.01253.

Matsuoka, M. (2003) ‘Human T-cell leukemia virus type I and adult T-cell leukemia’, Oncogene, 22(33 REV. ISS. 2), pp. 5131–5140. doi: 10.1038/sj.onc.1206551.

Matsuoka, M. and Jeang, K.-T. (2007) ‘Human T-cell leukaemia virus type 1 (HTLV-1) infectivity and cellular transformation’, Nature Reviews Cancer, 7(4), pp. 270–280. doi: 10.1038/nrc2111.

Matthies, A., Nimtz, M. and Leimkühler, S. (2005) ‘Molybdenum cofactor biosynthesis in humans: Identification of a persulfide group in the rhodanese-like domain of MOCS3 by mass spectrometry’, Biochemistry. American Chemical Society , 44(21), pp. 7912–7920. doi: 10.1021/bi0503448.

Matutes, E. (2006) ‘Adult T-cell leukaemia/lymphoma’, Journal of Clinical Pathology. BMJ Publishing Group, 60(12), pp. 1373–1377. doi: 10.1136/jcp.2007.052456.

Maupin-Furlow, J. A. (2014) ‘Prokaryotic Ubiquitin-Like Protein Modification’, Annual Review of Microbiology, 68(1), pp. 155–175. doi: 10.1146/annurev-micro-091313-103447.

McEwen, D. G. (2005) ‘Puckered, a Drosophila MAPK phosphatase, ensures cell viability by antagonizing JNK-induced apoptosis’, Development, 132(17), pp. 3935–3946. doi: 10.1242/dev.01949.

McGurk, L., Berson, A. and Bonini, N. M. (2015) ‘Drosophila as an in vivo model for human neurodegenerative disease’, Genetics. Genetics, 201(2), pp. 377–402. doi: 10.1534/genetics.115.179457.

Menon, K. P., Carrillo, R. A. and Zinn, K. (2013) ‘Development and plasticity of the Drosophila larval neuromuscular junction’, Wiley Interdisciplinary Reviews: Developmental Biology. NIH Public Access, 2(5), pp. 647–670. doi: 10.1002/wdev.108.

Merbl, Y. et al. (2013) ‘Profiling of ubiquitin-like modifications reveals features of mitotic control’, Cell. Elsevier, 152(5), pp. 1160–1172. doi: 10.1016/j.cell.2013.02.007.

Meulmeester, E. and Melchior, F. (2008) ‘Cell biology: SUMO’, Nature. Springer Nature, 452(7188), pp. 709–711. doi: 10.1038/452709a.

Michalak, K., Orr, W. C. and Radyuk, S. N. (2008) ‘Drosophila peroxiredoxin 5 is the second gene in a dicistronic operon’, Biochemical and Biophysical Research Communications, 368(2), pp. 273–278. doi: 10.1016/j.bbrc.2008.01.052.

Miech, C. et al. (2008) ‘Presynaptic Local Signaling by a Canonical Wingless Pathway Regulates Development of the Drosophila Neuromuscular Junction’, Journal of Neuroscience, 28(43), pp. 10875–10884. doi: 10.1523/JNEUROSCI.0164-08.2008.

56

Miles, W. O., Dyson, N. J. and Walker, J. A. (2011) ‘Modeling tumor invasion and metastasis in Drosophila’, Disease Models & Mechanisms. The Company of Biologists Ltd, 4(6), pp. 753–761. doi: 10.1242/dmm.006908.

Milton, V. J. et al. (2011) ‘Oxidative stress induces overgrowth of the Drosophila neuromuscular junction’, Proceedings of the National Academy of Sciences. National Academy of Sciences, 108(42), pp. 17521–17526. doi: 10.1073/pnas.1014511108.

Minakhina, S. and Steward, R. (2006) ‘Nuclear factor-kappa B pathways in Drosophila’, Oncogene, 25(51), pp. 6749–6757. doi: 10.1038/sj.onc.1209940.

Miranda, H. V. et al. (2011) ‘E1- and ubiquitin-like proteins provide a direct link between protein conjugation and sulfur transfer in archaea’, Proceedings of the National Academy of Sciences, 108(11), pp. 4417–4422. doi: 10.1073/pnas.1018151108.

Misof, B. et al. (2014) ‘Phylogenomics resolves the timing and pattern of insect evolution’, Science. American Association for the Advancement of Science, 346(6210), pp. 763–767. doi: 10.1126/science.1257570.

Morgan, T. H. (1910) ‘Sex Limited Inheritance in Drosophila’, Science, 32(812), pp. 120–122. doi: 10.1126/science.32.812.120.

Morgan, T. H., Sturtevant, A. H. and Bridge, C. B. (1920) ‘The Evidence for the Linear Order of the Genes’, Source: Proceedings of the National Academy of Sciences of the United States of America, 6(4), pp. 162–164. doi: 10.1073/pnas.6.4.162.

Muller, H. J. (1918) ‘Genetic Variability, Twin Hybrids and Constant Hybrids, in a Case of Balanced Lethal Factors.’, Genetics, 3(5), pp. 422–99.

Murphy, F. V. et al. (2004) ‘The role of modifications in codon discrimination by tRNALysUUU’, Nature Structural and Molecular Biology, 11(12), pp. 1186–1191. doi: 10.1038/nsmb861.

Nakai, Y. et al. (2004) ‘Yeast Nfs1p Is Involved in Thio-modification of Both Mitochondrial and Cytoplasmic tRNAs’, Journal of Biological Chemistry. American Society for Biochemistry and Molecular Biology, 279(13), pp. 12363–12368. doi: 10.1074/jbc.M312448200.

Nakai, Y. et al. (2012) ‘Arabidopsis molybdopterin biosynthesis protein Cnx5 collaborates with the ubiquitin-like protein urm11 in the thio-modification of tRNA’, Journal of Biological Chemistry. American Society for Biochemistry and Molecular Biology, 287(36), pp. 30874–30884. doi: 10.1074/jbc.M112.350090.

Nakai, Y., Nakai, M. and Hayashi, H. (2008) ‘Thio-modification of yeast cytosolic tRNA requires a ubiquitin-related system that resembles bacterial sulfur transfer systems’, Journal of Biological Chemistry, 283(41), pp. 27469–27476. doi: 10.1074/jbc.M804043200.

Napetschnig, J. and Wu, H. (2013) ‘Molecular Basis of NF-κB Signaling’, Annual Review of Biophysics. Annual Reviews, 42(1), pp. 443–468. doi: 10.1146/annurev-biophys-083012-130338.

Nasr, R. et al. (2003) ‘Arsenic / interferon specifically reverses 2 distinct gene networks critical for the survival of HTLV-1 – infected leukemic cells’, Networks, 101(11), pp. 4576–4582. doi: 10.1182/blood-2002-09-2986.Supported.

Nasr, R. et al. (2006) ‘Tax ubiquitylation and sumoylation control critical cytoplasmic and nuclear steps of NF-κB activation’, Blood, 107(10), pp. 4021–4029. doi: 10.1182/blood-2005-09-3572.

Nathan, C. and Ding, A. (2010) ‘Nonresolving Inflammation’, Cell, pp. 871–882. doi:

57

10.1016/j.cell.2010.02.029.

Neuveut, C. et al. (1998) ‘Human T-cell leukemia virus type 1 Tax and cell cycle progression: role of cyclin D-cdk and p110Rb.’, Molecular and cellular biology, 18(6), pp. 3620–32.

Ng, P. W. P. et al. (2001) ‘Genome-wide expression changes induced by HTLV-1 Tax: Evidence for MLK-3 mixed lineage kinase involvement in Tax-mediated NF-κB activation’, Oncogene, 20(33), pp. 4484–4496. doi: 10.1038/sj.onc.1204513.

Noma, A., Sakaguchi, Y. and Suzuki, T. (2009) ‘Mechanistic characterization of the sulfur-relay system for eukaryotic 2-thiouridine biogenesis at tRNA wobble positions’, Nucleic Acids Research, 37(4), pp. 1335–1352. doi: 10.1093/nar/gkn1023.

Noselli, S. and Agnès, F. (1999) ‘Roles of the JNK signaling pathway in Drosophila morphogenesis’, Current Opinion in Genetics and Development, 9(4), pp. 466–472. doi: 10.1016/S0959-437X(99)80071-9.

Novack, D. V. (2011) ‘Role of NF-kappaB in the skeleton’, Cell Research, 21(1), pp. 169–182. doi: 10.1038/cr.2010.159.

Nüsslein-volhard, C. and Wieschaus, E. (1980) ‘Mutations affecting segment number and polarity in drosophila’, Nature. Nature Publishing Group, 287(5785), pp. 795–801. doi: 10.1038/287795a0.

Ohsugi, T. and Koito, A. (2007) ‘Human T cell leukemia virus type I is resistant to the antiviral effects of APOBEC3’, Journal of Virological Methods, 139(1), pp. 93–96. doi: 10.1016/j.jviromet.2006.08.016.

Pan, Y. et al. (2009) ‘Differential roles of the fan-shaped body and the ellipsoid body in Drosophila visual pattern memory’, Learning & Memory, 16(5), pp. 289–295. doi: 10.1101/lm.1331809.

Pandey, U. B. and Nichols, C. D. (2011) ‘Human Disease Models in Drosophila melanogaster and the Role of the Fly in Therapeutic Drug Discovery’, Pharmacological Reviews. American Society for Pharmacology and Experimental Therapeutics, 63(2), pp. 411–436. doi: 10.1124/pr.110.003293.

Pedrioli, P. G. A., Leidel, S. and Hofmann, K. (2008) ‘Urm1 at the crossroad of modifications. “Protein Modifications: Beyond the usual suspects” review series’, EMBO Reports, 9(12), pp. 1196–1202. doi: 10.1038/embor.2008.209.

Peng, J. et al. (2004) ‘The herbicide paraquat induces dopaminergic nigral apoptosis through sustained activation of the JNK pathway’, Journal of Biological Chemistry, 279(31), pp. 32626–32632. doi: 10.1074/jbc.M404596200.

Petroski, M. D. and Deshaies, R. J. (2005) ‘Function and regulation of cullin-RING ubiquitin ligases’, Nature Reviews Molecular Cell Biology, 6(1), pp. 9–20. doi: 10.1038/nrm1547.

Petroski, M. D., Salvesen, G. S. and Wolf, D. A. (2011) ‘Urm1 couples sulfur transfer to ubiquitin-like protein function in oxidative stress: Fig. 1.’, Proceedings of the National Academy of Sciences, 108(5), pp. 1749–1750. doi: 10.1073/pnas.1019043108.

Phelps, S. S. et al. (2004) ‘Modified nucleotides in tRNALys and tRNAVal are important for translocation’, Journal of Molecular Biology, 338(3), pp. 439–444. doi: 10.1016/j.jmb.2004.02.070.

Phizicky, E. M. and Hopper, A. K. (2010) ‘tRNA biology charges to the front’, Genes and Development, 24(17), pp. 1832–1860. doi: 10.1101/gad.1956510.

58

Piper, M. D. W. et al. (2011) ‘Dietary restriction and aging: A unifying perspective’, Cell Metabolism, 14(2), pp. 154–160. doi: 10.1016/j.cmet.2011.06.013.

Piper, M. D. W. and Partridge, L. (2017) ‘Drosophila as a model for ageing’, Biochimica et Biophysica Acta - Molecular Basis of Disease. doi: 10.1016/j.bbadis.2017.09.016.

Potter, C. J., Turenchalk, G. S. and Xu, T. (2000) ‘Drosophila in cancer research. An expanding role.’, Trends in genetics : TIG. Elsevier Current Trends, 16(1996), pp. 33–39. doi: 10.1016/S0168-9525(99)01878-8.

Prabakaran, S. et al. (2012) ‘Post-translational modification: Nature’s escape from genetic imprisonment and the basis for dynamic information encoding’, Wiley Interdisciplinary Reviews: Systems Biology and Medicine, 4(6), pp. 565–583. doi: 10.1002/wsbm.1185.

Radyuk, S. N. et al. (2009) ‘Peroxiredoxin 5 confers protection against oxidative stress and apoptosis and also promotes longevity in Drosophila.’, The Biochemical journal, 419(2), pp. 437–445. doi: 10.1042/BJ20082003.

Rafatpanah, H. et al. (2011) ‘High prevalence of HTLV-I infection in Mashhad, Northeast Iran: A population-based seroepidemiology survey’, Journal of Clinical Virology. Elsevier, 52(3), pp. 172–176. doi: 10.1016/j.jcv.2011.07.004.

Raghu, K. et al. (2013) ‘Paraquat poisoning: A case report and review of literature.’, Journal of family & community medicine. Wolters Kluwer -- Medknow Publications, 20(3), pp. 198–200. doi: 10.4103/2230-8229.122023.

Ranjan, N. et al. (2011) ‘Solution structure and activation mechanism of ubiquitin-like small archaeal modifier proteins’, Journal of Molecular Biology, 405(4), pp. 1040–1055. doi: 10.1016/j.jmb.2010.11.040.

Reiter, L. T. et al. (2001) ‘A systematic analysis of human disease-associated gene sequences in Drosophila melanogaster’, Genome Research, 11(6), pp. 1114–1125. doi: 10.1101/gr.169101.

Ren, J. et al. (2006) ‘FAT10 plays a role in the regulation of chromosomal stability’, Journal of Biological Chemistry, 281(16), pp. 11413–11421. doi: 10.1074/jbc.M507218200.

Riesgo-Escovar, J. R. et al. (1996) ‘The Drosophila jun-N-terminal kinase is required for cell morphogenesis but not for DJun-dependent cell fate specification in the eye’, Genes and Development, 10(21), pp. 2759–2768. doi: 10.1101/gad.10.21.2759.

Rizzi, M. and Schindelin, H. (2002) ‘Structural biology of enzymes involved in NAD and molybdenum cofactor biosynthesis’, Current Opinion in Structural Biology. Elsevier Current Trends, 12(6), pp. 709–720. doi: 10.1016/S0959-440X(02)00385-8.

Rodríguez, J. A. (2014) ‘Interplay between nuclear transport and ubiquitin/SUMO modifications in the regulation of cancer-related proteins’, Seminars in Cancer Biology, 27, pp. 11–19. doi: 10.1016/j.semcancer.2014.03.005.

Roos, J. et al. (2000) ‘Drosophila Futsch regulates synaptic microtubule organization and is necessary for synaptic growth’, Neuron, 26(2), pp. 371–382. doi: 10.1016/S0896-6273(00)81170-8.

Roucoux, D. F. and Murphy, E. L. (2004) ‘The epidemiology and disease outcomes of human T-lymphotropic virus type II’, AIDS Reviews, pp. 144–154.

Rudolph, M. J. et al. (2001) ‘Crystal structure of molybdopterin synthase and its evolutionary relationship to ubiquitin activation’, Nature Structural Biology, 8(1), pp. 42–46. doi: 10.1038/83034.

59

Ruiz-Canada, C. et al. (2004) ‘New synaptic bouton formation is disrupted by misregulation of microtubule stability in aPKC mutants’, Neuron, 42(4), pp. 567–580. doi: 10.1016/S0896-6273(04)00255-7.

Ryder, E. and Russell, S. (2003) ‘Transposable elements as tools for genomics and genetics in Drosophila’, Briefings in Functional Genomics and Proteomics, 2(1), pp. 57–71. doi: 10.1093/bfgp/2.1.57.

Saitoh, M. et al. (1998) ‘Mammalian thioredoxin is a direct inhibitor of apoptosis signal-regulating kinase (ASK) 1’, EMBO Journal, 17(9), pp. 2596–2606. doi: 10.1093/emboj/17.9.2596.

Sanyal, S. et al. (2002) ‘Ap-1 functions upstream of creb to control synaptic plasticity in drosophila’, Nature. Nature Publishing Group, 416(6883), pp. 870–874. doi: 10.1038/416870a.

Sanyal, S. et al. (2003) ‘Evidence for cell autonomous AP1 function in regulation of Drosophila motor-neuron plasticity.’, BMC neuroscience, 4(1), p. 20. doi: 10.1186/1471-2202-4-20.

Schlieker, C. D. et al. (2008) ‘A functional proteomics approach links the ubiquitin-related modifier Urm1 to a tRNA modification pathway.’, Proceedings of the National Academy of Sciences of the United States of America, 105(47), pp. 18255–60. doi: 10.1073/pnas.0808756105.

Schmitz, J. et al. (2008) ‘The sulfurtransferase activity of Uba4 presents a link between ubiquitin-like protein conjugation and activation of sulfur carrier proteins’, Biochemistry. American Chemical Society, 47(24), pp. 6479–6489. doi: 10.1021/bi800477u.

Schwartz, D. C. and Hochstrasser, M. (2003) ‘A superfamily of protein tags: Ubiquitin, SUMO and related modifiers’, Trends in Biochemical Sciences. Elsevier Current Trends, 28(6), pp. 321–328. doi: 10.1016/S0968-0004(03)00113-0.

Schwarz, G. and Mendel, R. R. (2006) ‘Molybdenum Cofactor Biosynthesis and Molybdenum Enzymes’, Annual Review of Plant Biology. Annual Reviews, 57(1), pp. 623–647. doi: 10.1146/annurev.arplant.57.032905.105437.

Shaulian, E. and Karin, M. (2001) ‘AP-1 in cell proliferation and survival’, Oncogene. Nature Publishing Group, 20(19 REV. ISS. 2), pp. 2390–2400. doi: 10.1038/sj.onc.1204383.

Shembade, N. et al. (2007) ‘The human T-cell leukemia virus type 1 Tax oncoprotein requires the ubiquitin-conjugating enzyme Ubc13 for NF-kappaB activation.’, Journal of virology, 81(24), pp. 13735–42. doi: 10.1128/JVI.01790-07.

Shembade, N. (2010) ‘Role of post-translational modifications of HTLV-1 Tax in NF-κB activation’, World Journal of Biological Chemistry. Baishideng Publishing Group Inc, 1(1), p. 13. doi: 10.4331/wjbc.v1.i1.13.

Shen, W. and Ganetzky, B. (2009) ‘Autophagy promotes synapse development in Drosophila’, Journal of Cell Biology, 187(1), pp. 71–79. doi: 10.1083/jcb.200907109.

Shields, S. B. and Piper, R. C. (2011) ‘How ubiquitin functions with ESCRTs’, Traffic, 12(10), pp. 1306–1317. doi: 10.1111/j.1600-0854.2011.01242.x.

Shigi, N. (2012) ‘Posttranslational modification of cellular proteins by a ubiquitin-like protein in bacteria’, Journal of Biological Chemistry, 287(21), pp. 17568–17577. doi: 10.1074/jbc.M112.359844.

Shimoyama, M. (1991) ‘Diagnostic criteria and classification of clinical subtypes of adult T‐cell leukaemia‐lymphoma: A REPORT FROM THE LYMPHOMA STUDY GROUP

60

(1984–87)’, British Journal of Haematology, 79(3), pp. 428–437. doi: 10.1111/j.1365-2141.1991.tb08051.x.

Shirinian, M. et al. (2013) ‘Tax-1 and tax-2 similarities and differences: Focus on post-translational modifications and NF-κB activation’, Frontiers in Microbiology. Frontiers, 4(AUG), p. 231. doi: 10.3389/fmicb.2013.00231.

Shirinian, M. et al. (2015) ‘A Transgenic Drosophila melanogaster Model To Study Human T-Lymphotropic Virus Oncoprotein Tax-1-Driven Transformation In Vivo’, J Virol. American Society for Microbiology (ASM), 89(15), pp. 8092–8095. doi: 10.1128/JVI.00918-15.

Singh, S. et al. (2005) ‘Three-dimensional structure of the AAH26994.1 protein from Mus musculus, a putative eukaryotic Urm1.’, Protein science : a publication of the Protein Society. Wiley-Blackwell, 14(8), pp. 2095–2102. doi: 10.1110/ps.051577605.

Sittipunt, C. (2005) ‘Paraquat poisoning.’, Respiratory care, 50(3), pp. 383–5. doi: 10.1016/S0140-6736(05)74910-2.

Sluss, H. K. et al. (1996) ‘A JNK signal transduction pathway that mediates morphogenesis and an immune response in Drosophila.’, Genes & Development, 10(21), pp. 2745–2758. doi: 10.1101/gad.10.21.2745.

Spradling, A. C. and Rubin, G. M. (1982) ‘Transposition of cloned P elements into Drosophila germ line chromosomes.’, Science, 218(4570), pp. 341–347. doi: 10.1126/science.6289435.

Stronach, B. (2005) ‘Dissecting JNK signaling, one KKKinase at a time’, Developmental Dynamics, 232(3), pp. 575–584. doi: 10.1002/dvdy.20283.

Stronach, B. E. and Perrimon, N. (1999) ‘Stress signaling in Drosophila.’, Oncogene, 18(45), pp. 6172–82. doi: 10.1038/sj.onc.1203125.

Sturtevant, A. H. (1913) ‘The linear arrangement of six sex-linked factors in Drosophila, as shown by their mode of association’, Journal of Experimental Zoology, 14(1), pp. 43–59. doi: 10.1002/jez.1400140104.

Sue, G. R., Ho, Z. C. and Kim, K. (2005) ‘Peroxiredoxins: A historical overview and speculative preview of novel mechanisms and emerging concepts in cell signaling’, Free Radical Biology and Medicine, 38(12), pp. 1543–1552. doi: 10.1016/j.freeradbiomed.2005.02.026.

Suganuma, T. et al. (2008) ‘ATAC is a double histone acetyltransferase complex that stimulates nucleosome sliding’, Nature Structural and Molecular Biology. Nature Publishing Group, 15(4), pp. 364–372. doi: 10.1038/nsmb.1397.

Suganuma, T. et al. (2010) ‘The ATAC Acetyltransferase Complex Coordinates MAP Kinases to Regulate JNK Target Genes’, Cell. Cell Press, 142(5), pp. 726–736. doi: 10.1016/j.cell.2010.07.045.

Sun, L. et al. (2008) ‘Molecular identification and functional characterization of a Drosophila dual-specificity phosphatase DMKP-4 which is involved in PGN-induced activation of the JNK pathway’, Cellular Signalling, 20(7), pp. 1329–1337. doi: 10.1016/j.cellsig.2008.03.003.

Sun, S.-C. and Yamaoka, S. (2005) ‘Activation of NF-κB by HTLV-I and implications for cell transformation’, Oncogene, 24(39), pp. 5952–5964. doi: 10.1038/sj.onc.1208969.

Sun, T. Q. et al. (2001) ‘PAR-1 is a Dishevelled-associated kinase and a positive regulator of Wnt signalling’, Nature Cell Biology, 3(7), pp. 628–636. doi: 10.1038/35083016.

61

Suntres, Z. E. (2002) ‘Role of antioxidants in paraquat toxicity’, Toxicology, 180(1), pp. 65–77. doi: 10.1016/S0300-483X(02)00382-7.

Suzuki, T. et al. (1996) ‘HTLV-1 Tax protein interacts with cyclin-dependent kinase inhibitor p16INK4A and counteracts its inhibitory activity towards CDK4.’, The EMBO journal, 15(7), pp. 1607–14.

Swatek, K. N. and Komander, D. (2016) ‘Ubiquitin modifications’, Cell Research, 26(4), pp. 399–422. doi: 10.1038/cr.2016.39.

Sykiotis, G. P. and Bohmann, D. (2010) ‘Stress-Activated Cap’n’collar Transcription Factors in Aging and Human Disease’, Science Signaling, 3(112), p. re3-re3. doi: 10.1126/scisignal.3112re3.

Takatsuki, K. (2005) ‘Discovery of adult T-cell leukemia.’, Retrovirology. BioMed Central, 2, p. 16. doi: 10.1186/1742-4690-2-16.

Taylor, C. T. and Pouyssegur, J. (2007) ‘Oxygen, hypoxia, and stress’, Annals of the New York Academy of Sciences. Blackwell Publishing Inc, 1113(1), pp. 87–94. doi: 10.1196/annals.1391.004.

Teo, H., Veprintsev, D. B. and Williams, R. L. (2004) ‘Structural insights into endosomal sorting complex required for transport (ESCRT-I) recognition of ubiquitinated proteins’, Journal of Biological Chemistry, 279(27), pp. 28689–28696. doi: 10.1074/jbc.M400023200.

Toohey, J. I. and Cooper, A. J. L. (2014) ‘Thiosulfoxide (Sulfane) sulfur: New chemistry and new regulatory roles in biology’, Molecules. NIH Public Access, 19(8), pp. 12789–12813. doi: 10.3390/molecules190812789.

Vassin, H., Vielmetter, J. and Campos-Ortega, J. A. (1985) ‘Genetic interactions in early neurogenesis of Drosophila melanogaster’, J.Neurogenet., 2(0167–7063 (Print)), pp. 291–308. doi: 10.3109/01677068509102325.

Van der Veen, A. G. et al. (2011) ‘Role of the ubiquitin-like protein Urm1 as a noncanonical lysine-directed protein modifier’, Proceedings of the National Academy of Sciences. National Academy of Sciences, 108(5), pp. 1763–1770. doi: 10.1073/pnas.1014402108.

van der Veen, A. G. and Ploegh, H. L. (2012) ‘Ubiquitin-Like Proteins’, Annual Review of Biochemistry, 81(1), pp. 323–357. doi: 10.1146/annurev-biochem-093010-153308.

Vijay-Kumar, S. et al. (1985) ‘Three-dimensional structure of ubiquitin at 2.8 A resolution.’, Proceedings of the National Academy of Sciences of the United States of America, 82(11), pp. 3582–3585. doi: VL - 82.

Vonhoff, F. and Keshishian, H. (2017) ‘Activity-Dependent Synaptic Refinement: New Insights from Drosophila’, Frontiers in Systems Neuroscience. Frontiers, 11, p. 23. doi: 10.3389/fnsys.2017.00023.

Wan, H. I. et al. (2000) ‘Highwire regulates synaptic growth in Drosophila’, Neuron, 26(2), pp. 313–329. doi: 10.1016/S0896-6273(00)81166-6.

Wang, F. et al. (2011) ‘The dual role of ubiquitin-like protein Urm1 as a protein modifier and sulfur carrier’, Protein and Cell, 2(8), pp. 612–619. doi: 10.1007/s13238-011-1074-6.

Wang, M. C., Bohmann, D. and Jasper, H. (2003) ‘JNK signaling confers tolerance to oxidative stress and extends lifespan in Drosophila’, Developmental Cell. Elsevier, 5(5), pp. 811–816. doi: 10.1016/S1534-5807(03)00323-X.

Wang, M. C., Bohmann, D. and Jasper, H. (2005) ‘JNK extends life span and limits growth by antagonizing cellular and organism-wide responses to insulin signaling’, Cell, 121(1),

62

pp. 115–125. doi: 10.1016/j.cell.2005.02.030.

Wang, X., Yan, Q. and Guan, M. X. (2007) ‘Deletion of the MTO2 gene related to tRNA modification causes a failure in mitochondrial RNA metabolism in the yeast Saccharomyces cerevisiae’, FEBS Letters, 581(22), pp. 4228–4234. doi: 10.1016/j.febslet.2007.07.067.

Wang, Y. L. et al. (2008) ‘Human ATAC is a GCN5/PCAF-containing acetylase complex with a novel NC2-like histone fold module that interacts with the TATA-binding protein’, Journal of Biological Chemistry. American Society for Biochemistry and Molecular Biology, 283(49), pp. 33808–33815. doi: 10.1074/jbc.M806936200.

Watanabe, T. (2017) ‘Adult T-cell Leukemia: Molecular basis for clonal expansion and transformation of HTLV-1-infected T cells’, Blood, 129(9), pp. 1071–1081. doi: 10.1182/blood-2016-09-692574.

Wei, H. C. et al. (2003) ‘The Sac1 Lipid Phosphatase Regulates Cell Shape Change and the JNK Cascade during Dorsal Closure in Drosophila’, Current Biology, 13(21), pp. 1882–1887. doi: 10.1016/j.cub.2003.09.056.

Weston, C. R. and Davis, R. J. (2007) ‘The JNK signal transduction pathway’, Current Opinion in Cell Biology. Elsevier Current Trends, 19(2), pp. 142–149. doi: 10.1016/j.ceb.2007.02.001.

Wilkinson, K. D. (2000) ‘Ubiquitination and deubiquitination: Targeting of proteins for degradation by the proteasome’, Seminars in Cell & Developmental Biology, 11(3), pp. 141–148. doi: 10.1006/scdb.2000.0164.

Wilkinson, K. D. (2005) ‘The discovery of ubiquitin-dependent proteolysis’, Proceedings of the National Academy of Sciences, 102(43), pp. 15280–15282. doi: 10.1073/pnas.0504842102.

Wu, C. (2005) ‘Highwire Function at the Drosophila Neuromuscular Junction: Spatial, Structural, and Temporal Requirements’, Journal of Neuroscience, 25(42), pp. 9557–9566. doi: 10.1523/JNEUROSCI.2532-05.2005.

Wu, K. et al. (2004) ‘Protein Profile of Tax-associated Complexes’, Journal of Biological Chemistry, 279(1), pp. 495–508. doi: 10.1074/jbc.M310069200.

Wu, S. et al. (2016) ‘Arsenic induced complete remission in a refractory t-all patient with a distinct t-cell clonal evolution without molecular complete remission: A case report’, Oncology Letters. Spandidos Publications, 11(6), pp. 4123–4130. doi: 10.3892/ol.2016.4529.

Wu, X. and Sun, S. C. (2007) ‘Retroviral oncoprotein Tax deregulates NF-κB by activating Tak1 and mediating the physical association of Tak1-IKK’, EMBO Reports, 8(5), pp. 510–515. doi: 10.1038/sj.embor.7400931.

Wullaert, A., Bonnet, M. C. and Pasparakis, M. (2011) ‘NF-κB in the regulation of epithelial homeostasis and inflammation’, Cell Research, 21(1), pp. 146–158. doi: 10.1038/cr.2010.175.

Xiao, G. et al. (2001) ‘Retroviral oncoprotein tax induces processing of NF-κB2/p100 in T cells: Evidence for the involvement of IKKα’, EMBO Journal, 20(23), pp. 6805–6815. doi: 10.1093/emboj/20.23.6805.

Xiao, G., Harhaj, E. W. and Sun, S. C. (2000) ‘Domain-specific interaction with the IkappaB kinase (IKK) regulatory subunit IKKgamma is an essential step in tax-mediated activation of IKK’, Journal of Biological Chemistry, 275(44), pp. 34060–34067. doi: 10.1074/jbc.M002970200.

63

Yamanaka, N., Rewitz, K. F. and O’Connor, M. B. (2013) ‘Ecdysone Control of Developmental Transitions: Lessons from Drosophila Research’, Annual Review of Entomology. NIH Public Access, 58(1), pp. 497–516. doi: 10.1146/annurev-ento-120811-153608.

Yaniv, S. P. and Schuldiner, O. (2016) ‘A fly’s view of neuronal remodeling’, Wiley Interdisciplinary Reviews: Developmental Biology, 5(5), pp. 618–635. doi: 10.1002/wdev.241.

Yarian, C. et al. (2002) ‘Accurate translation of the genetic code depends on tRNA modified nucleosides’, Journal of Biological Chemistry, 277(19), pp. 16391–16395. doi: 10.1074/jbc.M200253200.

Yu, Z. et al. (2013) ‘Highly efficient genome modifications mediated by CRISPR/Cas9 in Drosophila’, Genetics, 195(1), pp. 289–291. doi: 10.1534/genetics.113.153825.

Zaffran, S., Robrini, N. and Bertrand, N. (2014) Retinoids and Cardiac Development, Journal of Developmental Biology. Sinauer Associates. doi: 10.3390/jdb2010050.

Zaher, T. E. et al. (2007) ‘Hyperoxia-induced signal transduction pathways in pulmonary

epithelial cells☆’, Free Radical Biology and Medicine. NIH Public Access, 42(7), pp. 897–908. doi: 10.1016/j.freeradbiomed.2007.01.021.

Zhang, B., Wang, Y. and Su, Y. (2009) ‘Peroxiredoxins, a novel target in cancer radiotherapy’, Cancer Letters. Elsevier, 286(2), pp. 154–160. doi: 10.1016/j.canlet.2009.04.043.

Zhang, K. X. et al. (2016) ‘Rapid Changes in the Translatome during the Conversion of Growth Cones to Synaptic Terminals’, Cell Reports, 14(5), pp. 1258–1271. doi: 10.1016/j.celrep.2015.12.102.

Zhang, Y. et al. (2007) ‘PAR-1 Kinase Phosphorylates Dlg and Regulates Its Postsynaptic Targeting at the Drosophila Neuromuscular Junction’, Neuron. NIH Public Access, 53(2), pp. 201–215. doi: 10.1016/j.neuron.2006.12.016.