PROTEIN AGGREGATION AND DISEASES

IntroductionEvolution has guided the design of amino acid sequences such

that globular proteins reliably assume a specific functional

native state, precisely bringing together residues to form,

for example, catalytic sites in enzymes or specific binding

site architectures for protein complexation and signaling.

The ability of the protein to find and maintain the native

state is therefore dependent on an amino acid sequence that

gives rise to a structural ensemble that is

thermodynamically stable at the physiological pressures and

temperatures and solution conditions in the normal cellular

or extracellular environment. Destabilizing sequence

mutations,1 chemical modification,2 or changes in protein

concentration and solution environment of the protein3 can

shift the equilibrium from the native state in favor of

aggregates, that is, misfolded states with interchain

contacts made with other proteins. These aggregates range

from structurally amorphous collections of misfolded

proteins often found in inclusion bodies when proteins are

overexpresssed in bacterial hosts4 to fibrils with regular

and repeating structure associated with a number of human

diseases.1 In order to change deleterious aggregation

outcomes, it is of critical importance to develop an

understanding of the molecular driving forces for early and

late aggregation events, which in turn might be reversed to

prevent disease proteins from nucleating thermodynamically

stable aggregate assemblies or to break up inclusion bodies

to recover functional protein.

Though the gross morphology of

large fibril aggregates can be investigated with current

biochemical or protein structural experimental techniques,1,5

these are more limited in application to early aggregation

events involving small and likely disordered oligomers at

very dilute concentration. Molecular simulations currently

offer great promise of directly observing the entire

aggregation process in molecular detail. In this project

report, we present the recent studies which show how

judicious use of coarse-grained models, validated against

appropriate experimental observables, can characterize the

aggregation thermodynamics and kinetic pathways at a level

of detail and insight not possible with experiment alone. We

use these models to quantify molecular mechanistic

differences in aggregation outcomes for nondisease proteins

L and G and the A - peptide indicted in Alzheimer’s

disease.

What is protein aggregation…………?Protein aggregation is a biological phenomenon in which

miss-folded protein aggregate(i.e; accumulate and clump

togather) either intra or extracellularly. These protein

aggregates are often toixic. Protein aggregates have

beenimplicated in a wide variety of diseases known

amyloidoses, including Alzheimer’s, Parkinson’ andPrion

diseases.

In this account we present the development and application

of computational models for theinvestigation of diseases

protein aggregatibn as illustrated for the proteins L and G

and the Alzheimers’s A beta systems, Parkinsons, huntingtons

and Prion disease’s

Common factors in neurodegenerative phenomena:-

Here we list …… Deposition of fibrillarproteinacious material in the

intracellular matrix.

Mitrochondrial dysfunction,increased oxidative stress

andproduction ROS(many mitrocondria,a lot of energy)

Increased apoptosis

Decreased proteasomal activity (ubiquitin is present in

all thelesions, plaques Lewy bodies, huntingtin

aggregates etc)

Decresed autophagy and lysosomal degradation of protein

Excitotoxicity

Alteration in the intrigrity of the cell membrane:

implication for alterd levels of intracellular

cholesterol

Protein misfolding, aggregation andneurodegeneration:-

Deposition of fibrillarproteinacious materialin the intracellular or extracellular matrix

Amyloid: amyloid fibrils are filamentous, hydrophobicstructures, width ~10nm, length between 0.1-10 mM. Ribbon-like -sheet motifs are formed by --strands and -turns.These kind of fibrils are common to differentneurodegenerative diseases, from Alzheimer’s to Huntington’sdisease. Conformation-specific antibodies (raised againstspecific proteins which form a specific amyloid formation)can recognize only the -sheet -polymeric conformation butnot the monomeric-soluble conformation of the proteinsubstrate. Thus, the β-sheet conformation and amyloidformation may be linked to neurodegenerative diseases.

Fig 1: beta sheet conformation of protein

Ref: ( Ross CA, Poirier MA. Nat Med. 2004 Jul: 10 Suppl:S 10-7. Review)

How proteins aggregate and form Amyloidinsoluble Fibrils…………?

Several factors may induce protein aggregation:-

That’s are………..

1-Protein oxidation (a-synuclein in PD)/ 2-Metal chelation (Prion disease

and AD) - 3-Specific protein cleavage (AD) 4-Inefficient protein degradation of b-sheet proteins/ubiquitin accumulates within the les (PD, AD, ALS, Prion Disease, HD) 5-Change in intracellular pH (AD)

Different evidences in the different neurodegenerative diseases :-AD: plaques, soluble Ab, correlate with progression of the

disease. However, Ab oligomers seem to be the more toxic

species.

PD: inclusion bodies do not follow the progression of the

disease.

HD: aggregates may be present ONLY in surviving neurons.

Fig 2 : Example for gain and loss of function mechanism

leading toneuronal dysfunction and cell death. (red-AD- blue

prion diseases.)

Representation of structural components of protein structures:

Primary Secondary Tertiary Quternary structure Structure structure structure

Fig 3 :Schematic representation of the structural components ofprotein . The order of the amino acids determines the primarystructure of the protein which can fold into secondary elementsalpha-helix, beta-sheet or random coils. The distribution ofsecondary elements ascertains tertiary structure that isstabilized by hydrogen bonds or ionic interactions. Finaly thequartenary structure describe the connection of poly peptidesubunits in multimeric proteins.

Ref: (Eszter Herczenik and Martijn F.B. G. Gebbink)

Diferent states of protein……….

Protein folding: a folding funnel to change the structure

and the energy of proteins.

Only folded, native proteins are functionally active.

Unfolded states: characterized by higher degree of

conformational entropy and free energy than native states.

This leads to “unstableness” of proteins when in the

unfolded state. As the folding funnel proceeds,

conformational entropy decreases as proteins have lower

number of conformational states, as well as the free energy

decreases. The minimum of the energy level of a protein is

reached when it’s in its native/folded state

Thermodynamics of proteinfolding/misfolding

Fig 4 : Energy state of protein folding under physiological andmisfolding condition. The shape of the graph shows energy stateof the protein conformations moving towards its native ormisfolded condition through multiple inter or intramolecularcontact arrangements.

(“The energy of the different conformations decreases withthe development of organized, native-like proteins”.)

Steps that lead to formation of aggregates:

Unfolding……

Nucleation: when proteins attach REVERSIBLY to a growing

core.

Aggregation: when proteins attach IRREVERSIBLY to the core

forming larger aggregates. Aggregation can be triggered by

hydrophobic residues in the sequence of the protein and by

b-sheet structure. Amyloid is one of the forms of protein

aggregates that occurs in nature, is very stable but its

formation can be still reversible. This is not true,

unfortunately, for most of the protein aggregates that are

responsible of neurodegenerative diseases.

Summary of protein folding diseases :

(Ref: Eszter Herczenik Martijn F. B. G. Gebbink)

Protein aggregation and neurodegenerative disease:Neurodegenerative diseases such as Alzheimer's disease (AD),

Parkinson's disease (PD), Huntington's disease (HD),

amyotrophic lateral sclerosis (ALS) and prion diseases are

increasingly being realized to have common cellular and

molecular mechanisms including protein aggregation and

inclusion body formation. The aggregates usually consist of

fibers containing misfolded protein with a -sheet

conformation, termed amyloid. There is partial but not

perfect overlap among the cells in which abnormal proteins

are deposited and the cells that degenerate. The most likely

explanation is that inclusions and other visible protein

aggregates represent an end stage of a molecular cascade of

several steps, and that earlier steps in the cascade may be

more directly tied to pathogenesis than the inclusions

themselves. For several diseases, genetic variants assist in

explaining the pathogenesis of the more common sporadic

forms and developing mouse and other models. There is now

increased understanding of the pathways involved in protein

aggregation, and some recent clues have emerged as to the

molecular mechanisms of cellular toxicity. These are leading

to approaches toward rational therapeutics.

Deposition of fibrillar proteinacious

material in Alzhimers disease:

Alzheimer's disease and tauopathies. AD is a late-onset

dementing illness, with progressive loss of memory, task

performance, speech, and recognition of people and objects.

There is degeneration of neurons (particularly in the basal

forebrain and hippocampus), but at least as important for

pathogenesis may be synaptic pathology and altered neuronal

connections AD involves two major kinds of protein

aggregates. Extracellular aggregates known as neuritic

plaques have as their major constituent the A peptide,

which is derived from proteolytic processing of the amyloid

precursor protein (APP). The A -containing aggregates have

-sheet structure and Congo red and thioflavin-T reactivity

characteristic of amyloid. There are also intracellular

aggregates of the microtubule-associated protein tau, called

neurofibrillary tangles. The pathogenesis of AD has been

greatly clarified by the identification of genetic mutations

responsible for rare familial forms of the disease. These

mutations are in APP itself and also in the

presenilins, which are involved with the cleavage of APP.

Fig 5: Plaques

Tangles Phospho-Tau

(Ref: Bossy-Wetzel E.et al. Nat Med.2004 Jul; 10 Suppl:S2-9. Review )

Deposition of fibrillar proteinacious material in Parkinsons disease: Parkinson's disease. PD is characterized by resting tremor,

rigidity, slow movements and other features such as postural

and autonomic instability. It is caused by degeneration of

dopaminergic neurons in the substantia nigra of the midbrain

and other monoaminergic neurons in the brain stem . The

discovery of several genes in which mutations cause early-

onset forms of PD has greatly accelerated research progress.

Point mutations or increased gene dosage of the -synuclein

gene cause autosomal dominant PD via a gain-of-function

mechanism. Recessive early-onset PD can be caused by

mutations in the genes encoding parkin, DJ-1 or PINK1,

presumably by a loss-of-function mechanism. The pathological

hallmark of adult-onset PD is the Lewy body, an inclusion

body found in the cytoplasm of neurons, often near the

nucleus. Lewy bodies are densest in the substantial nigra

but can also be present in monoaminergic, cerebral cortical

and other neurons. There are also aggregates in neurites,

which are referred to as Lewy neurites. A major constituent

of Lewy bodies is aggregated -synuclein protein. Lewy

bodies can also be labeled for ubiquitin, a synuclein

interactor termed synphilin-1, proteasome proteins, and

cytoskeletal and other proteins.

Lewy bodies Lewy bodies

Lewy bodies lewy neurite

(H&E) ubiquitin (α-

Synuclein) (α-synuclein)

Fig 6 :Ref: (Bossy-wetzel E.et al.. Nat Med. 2004 Jul:s2-9 Revew )

Deposition of fibrillar proteinacious material in Hungtons disease:

Huntington's disease. HD is a progressive neurodegenerative

disorder caused by expansion of a CAG repeat coding for

polyglutamine in the N terminus of the huntingtin protein.

Because it is caused by a mutation in a single gene, HD has

emerged as a model for studying neurodegenerative disease

pathogenesis. There is a remarkable threshold effect, in

that polyglutamine stretches of 36 in huntingtin cause

disease, whereas 35 do not. Within the expanded range,

longer repeats cause earlier onset. There is a striking

correlation between the threshold for aggregation in vitro and

the threshold for disease in humans, consistent with the

idea that aggregation is related to pathogenesis. Inclusions

containing huntingtin are present in regions of the brain

that degenerate. However, the neurons with inclusions do not

correspond exactly to the neurons that degenerate. For

instance, inclusions are present in the striatum, which is

most affected, but they are more enriched in populations of

large interneurons, which are spared, than in medium spiny

projection neurons, which are selectively lost. There is a

good correlation, however, between the length of the CAG

repeat and the density of inclusions.

Huntingtin aggregates can be labeled with antibodies to the

N terminus of huntingtin or antibodies to ubiquitin, a

marker for misfolded proteins, and a signal for degradation

by the proteasome. Proteasomes may have difficulty digesting

them, however, leading to their accumulation. The aggregates

contain fibers and appear to have -sheet structure

characteristic of amyloid, although there is controversy

about whether they bind dyes that intercalate into - SHEETS ,

as is a characteristic of amyloid. Other proteins, such as

Creb binding protein (CBP; discussed later) containing

polyglutamine may be recruited into huntingtin aggregates.

Fig 7 :

Ref: (Ross CA, Poirier MA. Nat Med. 2004 Jul;10 Suppl:S10-7. Review)

Deposition of fibrillar proteinacious

material in Creutzfeldt –Jakobs

disease(Prions disease):

Neurodegenerative diseases caused by prions can be sporadic

or can be acquired either by environmental transmission or

via genetic mutations Environmental pathways include eating

prion particles derived from infected brain tissue or

surgical implantation via infected instruments. Prion

disease can also be caused by point mutations in the prion

gene, leading to alterations of the prion protein. Pathology

can include amyloid plaques that appear similar to those of

AD and that can be labeled with prion antibodies. Prion

disease is a prototypical protein conformation disease, in

that highly sophisticated studies have shown that it is

caused by abnormal protein structure and not an infective

viral agent. Mechanisms of prion disease have been

illuminated by the discovery of prionlike protein

conformational changes in yeast. In all cases, disease is

caused by abnormally folded prion proteins. Prion

aggregation can take place both extracellularly and

intracellularly.

Alzheimers Prions

Fig 8 : Both Alzheimer’s and Prion diseases are characterized bythe deposition of pathological proteins in the brain, often inthe form of plaques. The brown colour is indicative ofimmunostained cortical deposit of A-beta peptide and of the prpsc

protein in brains of patients suffering from alzheimer’s disease(A) and prion disease (B) respectively

Deposition of fibrillar proteinacious

material in Cataract disease: A cataract is a clouding that develops in the crystallineiens of the eye or in its envelope (lens capsule), varying

in degree from slight to complete opacity and obstructing

the passage of light. Early in the development of age-

related cataract, the power of the lens may be increased,

causing near-sightedness (myopia), and the gradual yellowing

and opacification of the lens may reduce the perception of

blue colors. Cataracts typically progress slowly to cause

vision loss and are potentially blinding if untreated. The

condition usually affects both eyes, but almost always one

eye is affected earlier than the other

Fig 9: Cataract disease in human eye

Role of Water in ProteinAggregation andAmyloid Polymorphism:

Protein aggregation leading to amyloid fibril formation is

linked to a number of neurodegenerative diseases although in

some instances their formation is also beneficial.

Understanding how misfolded proteins polymerize into ordered

fibrils, which universallyhave a characteristic cross β-

structure, may be important in our ability to intervene and

prevent their formation. The physical basis of protein

aggregation involving a cascade of events that drive a

monomer to a fibrillar structure is complicated because of

interplay of a number of energy and time scales governing

amyloid formation. In addition, a number of other factors,

such as protein concentration, sequence of proteins, and

environmental conditions (pH, presence of osmolytes,

temperature) affect various kinetic steps in distinct ways,

thus making it difficult to describe even in vitro protein

aggregation in molecular terms. Despite these complexities,

significant advances have been made, especially in getting

structures of peptide amyloids and models for amyloid

fibrils on Aβ and fungal prion proteins. The availability of

structures have made it possible to undertake molecular

dynamics simulations, which have given insights into the

role water plays in oligomer formation as well as assembly

and growth of amyloid fibrils.

It has long been appreciated that water plays a major

role in the self-assembly of proteins5 in ensuring that

hydrophobic residues are (predominantly) sequestered in

protein interior. In contrast, the effects of water on

protein aggregation are poorly understood. Indeed, almost

all studies (experimental and computer simulations) on

amlyoid assembly mechanisms have been largely analyzed using

a protein centric perspective. The situation is further

exacerbated by experimental difficulties in directly

monitoring water activity during the growth process. Here,

we provide a perspective on the role water plays in protein

aggregation by synthesizing results from molecular dynamics

(MD) simulation studies. Briefly our goals are the

following: (i) Describe how water-mediated interactions

affect the energy landscape of monomers and drive oligomer

formation in Aβ peptides. (ii) The key role water plays in

late stages of fibril growth is described by large

variations in the sequence-dependent mechanism of self-

assembly to β-sheetrich amyloids. (iii) We use results of

recentMD simulations and concepts in protein crystallization

to provide scenarios for the role water plays in polymorphic

amyloid structures.

Fig 10 : Water absorb in protein

Water in protein aggregation and Amyloid polymorphis

Figure 11 : Schematic of protein aggregation mechanisms leadingto polymorphic fibrils. On the left are solvated peptides. Waterin the hydrationlayer is in red and the bulk water is in blue.Even isolated monomers sample aggregation-prone conformations,N*, which are coated with varyingnumbers of water molecules. Thepeptides with N* conformations aggregate to form disorderedprotein-rich droplets. A major driving force for aggregation isthe release of water molecules in the hydration layer into thebulk, which facilitates fibril formation being entropicallyfavorable. Thestructured protein aggregates nucleate from theprotein-rich droplet to form protofilaments, which further self-assemble to form a variety of matureamyloid fibrils. In some ofthe polymorphic structures, discrete numbers of water moleculesare confined in the fibril.

Conclusions:

Most of the neurodegenerative diseases are characterized byintracellular or extracellular deposition of insolublematerial. Whether this is a cause or a consequence of the diseases is

not known yet.

It is speculated that the early species in this process might be most toxic,

by being involved in abnormal interactions with other cellular proteins.

However, the fact that all these diseases are characterized

by the same common factors, and the observation that

inherited forms of these diseases cause a massive increase

in the production of b-sheet related proteins lead to

hypothesize that these b-sheet proteins and the subsequent

formation of the insoluble lesions may be upstream the

cascade of events that lead to neurodegeneration.

Department of Chemistry University of Kalyani

Kalyani, Nadia

Project report submitted for partial fulfillment of M.Sc . degree (PHYSICAL CHEMISTRY SPECIAL)

By

CHINMAY GHOSH MARCH 2012

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ACKNOWLEDGEMENT

Let me take the opportunity toexpress my deepest sense ofgratitude to my supervisor Prof.Nilashis Nandi for his patientguidance and teaching through courseof my project paper.

My sincerely gratitude goes toour H.O.D. Prof. Mitali Sarkar andall the faculties to give me theirvaluable suggestion and to completemy project paper.

I am also very thankful toSindrila Duttabanik , Md. Najib Alam, Kumarjyoti Roy (Resarch ScholarsDepartment of Chemistry, K.U.) fortheir valuable time and continuoushelp throughout course of myassigned paper.

Finally I gratefully thank tomy parents for inspiring me.

CHINMAY GHOSH

CONTENTS

1 - Acknowledgement

2 - Introduction

3 - What is proteinaggregation…?

4 - Protein missfolding,aggregation and Neurodegeneration.

5 - How protein aggregates andform amyloid Insoluble fibrils…..?

6 - Steps that lead to formationof aggregate. 7 - Protein aggregation and Neurodegenerative disease

8 - Role of water in proteinaggregation and Amyloid polymorphism.

9 - Conclusion

10 - References