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608 ACS CHEMICAL BIOLOGY • VOL.1 NO.10 www.acschemicalbiology.org

Published online November 17, 2006 • 10.1021/cb600447n CCC: $33.50

© 2006 by American Chemical Society

Plants Get Hormonal TooApparently, hormones play a role in how plants deal with stress too. The plant hormone abscisic acid (ABA) is involved in various physiological processes during the plant life cycle, including adapting to environmen-

tally stressful conditions such as dehydra-tion. Plants tweak their ABA levels in order to adjust to continually changing conditions, but the molecular mechanisms involved are not well understood. Now, Lee et al. (Cell 2006, 126, 1109-1120) demonstrate that the b-glucosidase AtBG1 is an important modulator of ABA levels and reveal regula-tory mechanisms behind AtBG1 activity.

The observation that stress conditions or exposure to exogenous ABA induces the expression of AtBG1 led to the discovery that loss of AtBG1 results in defects in responses

mediated by ABA. The use of wild-type and mutant proteins to investigate the activity of AtBG1 indicated that the enzyme specifically hydrolyzes the glucose ester of ABA (ABA-GE) to ABA. The presence of a pep-

tide sequence suspiciously similar to an endoplasmic reticulum (ER) retention signal suggested that AtBG1 resides in the ER. Indeed, ER-localized AtBG1 hydro-lyzes ABA-GE, which appears to be imported into the ER by a membrane-localized transporter. Further investigations demonstrated that increased ABA levels in response to dehydration are correlated with AtBG1 levels, an indication that AtBG1 is activated under these conditions. Clues from previous studies suggesting that multimerization of b-glucosidases results in increased activity led to the discovery that dehydration causes polymerization of AtBG1, which results in higher enzymatic activity. The authors also demonstrated that the ABA produced by AtBG1 contributes to both intracellular and extracellular ABA signaling. Taken together, these data suggest that, in addition to de novo synthesis, an alternative regula-tory mechanism for ABA exists. The activity of AtBG1 may facilitate rapid adjustment of ABA levels, which is required for adaptation to the ever-changing environ-ment in the daily life of a plant. EG

Membrane ManipulationPhosphoinositide phospholipids are important signaling components of the plasma membrane (PM). Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) by phospholipase C (PLC) results in the closing of KCNQ ion chan-nels, a family of potassium channels that regulates neuron excitability and is asso-ciated with certain inherited diseases, including epilepsy, cardiac ventricular arrhythmias, and deafness. However, it is not known whether depletion of PtdIns(4,5)P2 alone is sufficient to close KCNQ channels or whether other signal-ing events also contribute to this event. Using a chemical dimerizer strategy, Suh et al. (Science Express, published online Sept 21, 2006, DOI: 10.1126/sci-

ence.1131163) present a method for inves-tigating PtdIns(4,5)P2 depletion without activating the PLC pathway.

The chemical dimerizer strategy relies on the ability of the small molecule rapamycin to bring together two protein domains, FKBP (FK506 binding protein) and FRB (FKBP-rapamycin binding protein). Fusions of these proteins were generated to create a specific, non-invasive, inducible method for evaluat-ing the cellular consequences of PLC-inde-pendent depletion of PtdIns(4,5)P2. Inp54p, a phosphatase specific for the phosphate at the 5-position of PtdIns(4,5)P2, was fused to a fluorescent derivative of FKBP (CF-Inp) and transfected into cells along with a mem-brane-anchored derivative of FRB, called Lyn11-FRB. In addition, a fluorescent pleck-

strin homology domain from PLCδ1 was created as a PtdIns(4,5)P2/InsP3 biosen-sor. Addition of the rapamycin derivative iRap to cells transfected with these three constructs resulted in rapid translocation of CF-Inp to the PM, in situ depletion of PtdIns(4,5)P2, and concomitant irrevers-ible suppression of KCNQ current. Similar approaches were used to increase the levels of PtdIns(4,5)P2, which augmented the current, and to induce the synthesis of PtdIns(3,4,5)P3, which did not affect PtdIns(4,5)P2 levels or the amplitude of the KCNQ current. These results further define the role of PtdIns(4,5)P2 in KCNQ channel function and validate this method as a versatile approach for manipulating lipid composition of the PM. EG

Reprinted from Cell, 126, Lee, K. H., et al., Activation of glucosidase via stress-induced polymerization rapidly increases active pools of abscisic acid, 1109-1120, Copyright 2006, with permission from Elsevier.

609www.acschemicalbiology.org VOL.1 NO.10 • ACS CHEMICAL BIOLOGY

An Axon Balancing ActAxons, the long projections of nerve cells, have complex regulatory mechanisms that enable their function as the transmission lines of the nervous system. Mapping the networks involved in axon regulation will help contribute to our knowledge of brain development and could lead to new therapeutic strategies for nerve regeneration. Srahna et al. (PLoS Biol. 2006, 4, 2076-2090) use the visual system of the Drosophila brain to investigate the signals that regulate axon extension and retraction, putting forth a model describ-ing the network that controls this remarkable process.

A candidate gene approach was employed to decipher the role of specific signaling pathways during axon extension and retraction in dorsal cluster neurons (DCNs), a group of ~40 neurons located in each brain hemisphere. Interest-ingly, all DCN axons initially extend toward the developing part of the fly optic lobe, termed the medulla, but as devel-opment continues only 11 or 12 of the 40 axons continue along defined paths, while the remaining ones retract. The authors found that blocking the Jun N-terminal kinase (JNK) pathway significantly decreased axon extension but that blocking fibroblast growth factor (FGF) receptor activity or the

ras-related C3 botulinum toxin substrate 1 (Rac1) GTPase promoted DCN axon extension. Further prob-ing revealed that signaling through the Wnt pathway via Wnt5 and the Wnt signal-

ing adaptor protein Dishevelled (Dsh) can attenuate Rac1 activity and thereby suppress Rac1 suppression of JNK. Exploration of how these pathways are intertwined indicated that JNK acts downstream of Rac1, which acts downstream of Dsh. It appears that a careful molecular balancing act between the Wnt and FGF signals governs the number of DCN axons that continue to extend versus the number that retract. Further investigation of the mechanisms that deter-mine the identity of those axons that continue to extend will help connect the complex wiring of neuronal networks. EG

CCA CinemaTransfer RNAs (tRNAs) are linked to their cognate amino

acids just after an invariant terminal nucleotide stretch,

CCA. This sequence is not encoded by the tRNA genes,

but rather, it is appended later by a specialized enzyme,

the CCA-adding RNA polymerase. Although such a tailing

event appears reminiscent of the eukaryotic messenger

RNAs and the poly-A tail, the story with tRNAs is far more

complex. This polymerase must specifically recognize

the shape of tRNAs, position the 3′ terminus, and then

sequentially add just three nucleotides onto the end. This

phenomenon occurs without any RNA or DNA template

guiding the reaction. Thus, a

puzzling question has long

remained: how does the enzyme

achieve its exquisite specificity?

Now, Tomita et al. (Nature 2006,

443, 956-960) add an impres-

sive collection of X-ray crystal

structures capturing an archaeal

CCA-adding enzyme at various

points in the reaction pathway.

The authors determined the structure of the protein in

complex with a number of small RNAs corresponding to

intermediates along the pathway. In addition, they care-

fully looked for structural changes that might occur from

the incoming nucleoside triphosphate. Using six different

high-resolution structures, the authors postulate on the

mechanism of the enzyme and even construct a movie

that incorporates the X-ray snapshots along the path.

The enzyme first stretches part of the tRNA to bring the 3′ terminus into the active site. After addition of one C, the

RNA snaps back by one nucleotide and repositions the

new terminus into the active site. Once another cytidine

triphosphate is provided, the enzyme changes conforma-

tion to a more closed state for addition of the next C to

the tRNA. This open–closed switch is similar to how DNA

polymerases clamp down on a template. Unlike tradi-

tional polymerases, the next addition of an A occurs in a

closed and locked structure that cannot translocate. The

enzyme clamps around the RNA helix and prevents further

additions. This study displays the dynamic workings of an

interesting and highly specialized polymerase found in all

three kingdoms of life. JU

Reprinted from PLoS Biol., 4, Srahna, M., et al., A signaling network for patterning of neuronal connectivity in the Drosophila brain, 2076-2090.

Reprinted by permission from Macmillan Publishers Ltd: Nature, Tomita, K, et al., 443, 956-960, copyright 2006.


Making and Breaking MoenomycinThe enzymes involved in bacterial cell wall

biosynthesis are excellent targets for antibiotic

development because of their critical role in

bacterial survival and the lack of analogous

enzymes in humans. Transglycosylases catalyze

formation of the glycan units of peptidoglycan,

the major component of the cell wall. The natural

product moenomycin A is the only known

natural product inhibitor of transglycosylases,

but its poor pharmacokinetic properties and

complex structure have prohibited its use as an

antibiotic in humans. Two recent papers (Adachi

et al., J. Am. Chem. Soc.

2006, 128, 14,012-14,013, and Taylor

et al., J. Am. Chem.

Soc. published online

Nov 4, 2006, DOI:

10.1021/ja065907x)

now describe flexible synthetic approaches to

moenomycin and its analogues and report the

inhibitory activity of moenomycin and a deriva-

tive against purified transglycosylases.

Moenomycin A is composed of a pentasac-

charide attached to a 2-O-moenocinyl glycer-

ate chain via a phosphodiester linkage. The

total synthesis of moenomycin A, described in

the paper by Taylor et al., was designed to be

efficient while facilitating generation of moeno-

mycin analogues. The authors constructed the

pentasaccharide unit by first synthesizing two

disaccharide fragments, linking them together,

and then attaching the fifth ring in the last

steps. Efficient stereoselective glycosylations

were accomplished with the sulfoxide glyco-

sylation reaction, and the reaction conditions

were tweaked depending on the donor–acceptor

reactivity profiles. Inverse addition and appropri-

ate use of scavengers proved essential in order

to suppress certain side reactions in some of the

glycosylations.

The synthesis of the 2-O-moenocinyl glycerate

piece and the generation of the phosphodiester

linkage are described in the paper by Adachi et al.

2-O-Moenocinyl glycerate was created through

conversion of the allyl alcohol functionality in

moenocinol to an allyl ether, followed by protect-

ing group shuffling and esterification. Conversion

of the anomeric hydroxyl of the pentasaccharide to

an H-phosphonate ester followed by reaction with

2-O-moenocinyl glycerate in the presence of 1-

adamantanecarbonyl chloride, mild oxidation, and

global deprotection afforded moenomycin A.

A method for degrading and reconstructing

moenomycin was also developed to facilitate

manipulation of the reducing end of the com-

pound. This approach enables creation of moeno-

mycin analogues with modified lipid chains

without the need to synthesize the compounds

from scratch. Successful implementation of this

method required developing degradation condi-

tions that left the pentasaccharide unit intact. The

inherent lability of the allyl ether functionality that

connects the pentasaccharide to the lipid chain

enabled the researchers to come up with degrada-

tion conditions that were selective for the glycidyl

ether linkage while leaving the glycosidic bonds

untouched.

With these methods, moenomycin was suc-

cessfully ripped apart and subsequently put

(continued on page 611)

OHO

OH

O NHHOO

HONHAc

OHO

NHAc

OOO

HOO

O

OHO

OHO

HOOH H2N

O

OP

O

CO2H

O

OHO

O

OH

O

NH2

Reprinted with permission from Adachi, M., et al., J. Am. Chem. Soc., 128, 14012–14013. Copyright 2006 American Chemical Society.


The receptor tyrosine kinase human

epidermal growth factor receptor 2

(HER2) is a key component of a complex

signaling network that regulates impor-

tant cellular processes such as migra-

tion and proliferation. Overexpression

of HER2 is notoriously associated with

breast and other cancers, and drugs

that selectively target HER2 have dem-

onstrated effective anticancer activity in

patients. In an effort to map the signal-

ing network of HER2, Wolf-Yadlin et al.

(Mol. Syst. Biol., published online Oct 3,

2006, DOI: 10.1038/msb4100094) use

quantitative mass spectrometry, biologi-

cal response data, and computational

analysis to correlate phosphorylation

patterns with cell proliferation or with

migration.

The cellular state of tyrosine phos-

phorylation was examined across 16

dimensions: four time points, two cell

lines (one that did and one that did not

overexpress HER2), and treatment with

Phosphorylation Phenotyping?either epidermal growth factor (EGF) or

heregulin (HRG), growth factors that dif-

ferentially stimulate HER2 heterodimers.

Astonishingly, 332 phosphorylated pep-

tides from 175 proteins were identified,

122 of which had not previously been

described. Using the self-organizing

map clustering algorithm, which enables

the identification of clusters of tyrosine-

phosphorylated peptides with similar

temporal dynamics, the authors readily

identified four clusters that reveal con-

nectivity in the data. In order to correlate

this signaling data with a phenotypic

effect, they also measured cell migration

and proliferation under the same condi-

tions. In general, HER2 overexpressing

cells exhibited enhanced cell migration.

Moreover, phosphorylation patterns of

cells stimulated with EGF versus HRG

pointed to the network connections

behind the increased migratory ability of

HER2 overexpressing cells and eluci-

dated distinct pathways by which these

ERK2|T/Y|185/187P38 A|Y|182paxillin|S/Y|84/88PTRF|Y|308PZR|Y|263EGFR|Y|1173SHC|Y|239SHC|Y|317C18 orf 11|Y|297ERK1|Y|204ERK2|Y|187STAT3-1|Y|705STAT3-2|Y|704Ack|Y|857EGFR|Y|1068EGFR|Y|1148SHIP-2|Y|986SHC|Y/Y|239/240An A2|Y|23An A2|Y|29TfR|Y|20Caveolin 1|Y|14Dsc3a|Y|818SCF38 m2|Y|20Rin1|Y|36

BCAR3|Y|267p130Cas|Y|327p130Cas|Y|387

EphA2|Y|588SHB|Y|355EphA2|Y|772EphA2|Y/Y|588/594LDLR|Y|845SHP-2|Y|62

FAK|Y|397FAK|Y|576IGF1R|Y|1161RPTPa|Y|798GIT1|Y|545IGF1R|Y|1165paxillin|Y|118PI3KR2|Y|464RAI3|Y|347

c1

c2 c3

c4

growth factors promote cell migration.

In contrast, HER2 overexpression had

a minimal effect on cell proliferation;

rather, EGF treatment emerged as the

primary driver of cell growth. A model

using partial-least-squares regression

was constructed to quantitatively

correlate phosphorylation patterns

with cell migration or proliferation,

establishing a powerful approach for

exploring the relationship between

protein phosphorylation and cellular

processes. EG

back together. In addition, a moenomycin derivative

containing a 10-carbon neryl chain in place of the

much longer natural polyprenyl unit in moenomycin

was also constructed. The compounds were tested for

their ability to inhibit purified transglycosylases from

two clinically relevant bacterial species, Staphylococ-

cus aureus and Enterococcus faecalis. Notably, both

compounds inhibited the purified proteins compa-

rably, but moenomycin was a much more potent

Making and Breaking Moenomycin, continued from page 610

inhibitor of bacterial growth, an indication of the

importance of the lipid chain in the context of a

biological system.

These studies provide access to new synthetic

methods for creating moenomycin analogues,

facilitating investigations into the mechanism

of inhibition of moenomycin, the biological role

of transglycosylases, and the development of

moenomycin-based antibiotics. EG

Reprinted by permission from Macmillan Publishers Ltd: Mol. Syst. Biol., advance online publication, Oct 3, 2006, DOI: 10.1038/msb4100094.


Getting Connected!Establishing connections among physiological and

pathological processes and genetic and small-molecule

perturbations can lead to unanticipated links that could

help decipher the incredibly complex web that defines a

biological state. In an attempt to establish a systematic

method for exploring these relationships, Lamb et al.

(Science 2006, 313, 1929-1935) present the Connectivity

Map, a resource in which gene-expression profiles of cells

exposed to small molecules are assembled into a public

database for which data-mining tools are available to

detect noteworthy relationships among the profiles.

Data from the expression profiles of breast cancer cells

exposed to 164 distinct bioactive small molecules were

used to create a first-generation Connectivity Map. A

query signature, or list of genes whose expression is cor-

related with a biological state of interest, could then be

scanned in the Connectivity Map in the search for promi-

nent relationships. A range of query signatures from both

Linking Lipids to LifeSome pathogens exert their destructive

behavior by producing pore-forming toxins,

which essentially poke holes through

cell membranes and potentially lead to

cell death. The molecular processes that

govern this pathway, however, are not well

understood. Gurcel et al. (Cell 2006, 126,

1135-1145) explore the cellular response

to aerolysin, a pore-forming toxin produced

by certain bacteria, and demonstrate

evidence for a chain of events that helps

explain the cell’s ability to repair its mem-

brane and survive.

The authors initially observed that

exposure of mammalian cells to aerolysin

resulted in activation of the sterol regula-

tory element binding proteins (SREBPs),

transcription factors that regulate choles-

Reference database(profiles)

Connections

Query Output

Biological stateof interest(signature)

Up

Down

Strongpositive

Positive

Negative

Weakpositive

Null Strongnegative

internal and external studies were collected and evalu-

ated. The data included the effects on gene expression of

small molecules, such as histone deacetylase inhibitors,

estrogens, and phenothiazines, and of disease states, such

as diet-induced obesity, Alzheimer’s disease, and dexa-

methasone resistance in acute lymphoblastic leukemia.

Remarkably, the Connectivity Map revealed both positive

and negative connectivity relationships that correctly

predicted several known relationships, pointed to the pre-

viously unknown mechanism of action of a small molecule,

and identified several molecules with potential therapeutic

utility. On the basis of these encouraging preliminary

results, the authors propose that an expanded Connectivity

Map should be generated as a community resource project.

Depending on the Map’s utility, the exciting prospect of

further expansion toward the ultimate goal of creating a

comprehensive description of all biological states in the

context of genomic signatures could be realized. EG

terol and fatty acid biosynthesis. Further

investigations revealed that activation of

the SREBPs was caused by loss of potas-

sium through the toxin pores. Interestingly,

they noted that potassium efflux had

previously been linked to caspase-1 activa-

tion, and indeed, caspase-1 was activated

in response to aerolysin exposure. It was

also known that activation of caspase-1 is

dependent on the assembly of large multi-

protein complexes called inflammasomes,

and they further demonstrated that aeroly-

sin exposure triggers formation of inflam-

masomes. Moreover, it was demonstrated

that prevention of caspase-1 or inflamma-

some activation blocked aerolysin-induced

SREBP-2 activation and that caspase-1 acti-

vation induced SREBP activation through

a well-established pathway involving the

escort protein SCAP (SREBP cleavage-acti-

vating protein) and two transmembrane

proteases, S1P and S2P. These data unde-

niably link caspase-1 and SREBP activation

to a common pathway and indicate that

caspase-1 activation is upstream of SREBP

activation. Finally, they showed that block-

ing the caspase-1 or SREBP pathways after

exposure of primary cells to aerolysin or

infection of cells with aerolysin-producing

bacteria increases cell death, an indication

that activation of these pathways promotes

cell survival. Taken together, these results

connect intracellular ion levels, caspases,

SREBPs, and lipid metabolism as part of

the survival mechanism that cells employ

to fight pore-forming toxins. EG

From Lamb, J., et al., Science, 2006, Sept 29, 2006 DOI: 10.1126/science.1132939. Reprinted with permission from AAAS.


Spotlights written by Eva Gordon and Jason Underwood

UPCOMING CONFERENCESAmerican Society for Cell Biology Annual MeetingDecember 9-13, 2006

San Diego, CA

Metals in Biology, GRC

January 28-February 2, 2007

Ventura, CA

MicroRNAs and siRNAs: Biological Functions and Mechanisms January 28-February 2, 2007

Keystone, CO

Biophysical Society Annual MeetingMarch 3-7, 2007

Baltimore, MD

Glycobiology, GRCMarch 4-9, 2007

Ventura, CA

2007 ACS Spring National MeetingMarch 25-29, 2007

Chicago, IL

Quality of Protein MicroarraysThe assembly and deciphering of protein

interaction networks promise to reveal valu-

able information about how organisms func-

tion. The accuracy of commonly used meth-

ods for accessing protein–protein interaction

data suffers from difficulties in normalizing

the behavior of proteins that by nature vary

widely in their physical properties. Gordus

et al. ( J. Am. Chem. Soc. 2006, 128, 13,668-13,669) propose a method for minimizing

the effects of variations in concentration,

surface density, and activity of proteins used

in microarrays.

To maximize the chances of working

with structures that behave similarly, the

authors chose protein domains, rather than

whole proteins, to systematically investigate

protein–protein interactions in a microar-

ray format. Seven Src homology 2 domains

labeled with a fluorescent tag were printed on

a microarray surface. The fraction of surface

area covered by each protein was evaluated

and was found to vary considerably. Next,

they used a labeled phosphopeptide known

to interact with five of the domains to evalu-

ate the amount of active protein on the surface;

it was found to vary substantially, to the extent

that spot intensity did not accurately reflect

interaction affinity. However, when saturation

binding curves were obtained and normalized

with respect to the amount of active protein

on the surface, the data did manifest the cor-

rect affinities of the interactions. A strength

of microarrays is their ability to control pro-

tein concentrations, so obtaining this type of

quantitative information should dramatically

improve the quality and quantitative integrity

of protein microarray data. The authors suggest

that because the dependencies of concentration

and activity of proteins also affect data obtained

in other protein interaction assays, such as yeast

two-hybrid systems and affinity purification of

protein complexes, more diligent efforts should

be made to obtain quantitative information

when defining protein interaction networks. EG

Reprinted with permission from Gordus, A., and MacBeath, G., J. Am. Chem. Soc., 128, 13668-13669. Copyright 2006 American Chemical Society.

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