Plants Get Hormonal Too
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.
Apparently, 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 environmentally stressful conditions such as dehydration. 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 regulatory 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 hydrolyzes 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 regulatory mechanism for ABA exists. The activity of AtBG1 may facilitate rapid adjustment of ABA levels, which is required for adaptation to the ever-changing environment in the daily life of a plant. EG
Membrane Manipulation Phosphoinositide 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 channels, a family of potassium channels that regulates neuron excitability and is associated 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 signaling 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 investigating 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 (FKBPrapamycin binding protein). Fusions of these proteins were generated to create a specific, non-invasive, inducible method for evaluating the cellular consequences of PLC-independent 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 membrane-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 biosensor. 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 irreversible 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
Published online November 17, 2006 • 10.1021/cb600447n CCC: $33.50 © 2006 by American Chemical Society
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An Axon Balancing Act
CCA Cinema
Axons, 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 describing 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. Interestingly, all DCN axons initially extend toward the developing part of the fly optic lobe, termed the medulla, but as development 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 probing revealed that signaling through the Wnt pathway via Wnt5 and the Wnt signaling 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 determine the identity of those axons that continue to extend will help connect the complex wiring of neuronal networks. EG
Transfer 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 impressive collection of X-ray crystal structures capturing an archaeal CCA-adding enzyme at various Reprinted by permission from Macmillan Publishers Ltd: Nature, Tomita, K, et al., 443, 956-960, copyright 2006. 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 carefully 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 conformation 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 traditional 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.
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Making and Breaking Moenomycin
O
The 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 H N HO OH et al., J. Am. Chem. Soc. O O O O O 2006, 128, 14,012NH OH O O O O O 14,013, and Taylor HO O NHAc O NHAc et al., J. Am. Chem. P O O OH Soc. published online CO H 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 derivative against purified transglycosylases. Moenomycin A is composed of a pentasaccharide attached to a 2-O-moenocinyl glycerate 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 moenomycin 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 glyco2
HO OH HO
HO O NH O O HO HO OH
2
2
Reprinted with permission from Adachi, M., et al., J. Am. Chem. Soc., 128, 14012–14013. Copyright 2006 American Chemical Society.
sylation reaction, and the reaction conditions were tweaked depending on the donor–acceptor reactivity profiles. Inverse addition and appropriate 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 protecting 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 1adamantanecarbonyl 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 compound. This approach enables creation of moenomycin analogues with modified lipid chains without the need to synthesize the compounds from scratch. Successful implementation of this method required developing degradation conditions 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 degradation conditions that were selective for the glycidyl ether linkage while leaving the glycosidic bonds untouched. With these methods, moenomycin was successfully ripped apart and subsequently put (continued on page 611)
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Phosphorylation Phenotyping? The receptor tyrosine kinase human
either epidermal growth factor (EGF) or
epidermal growth factor receptor 2
heregulin (HRG), growth factors that dif-
(HER2) is a key component of a complex ferentially stimulate HER2 heterodimers.
ERK2|T/Y|185/187 P38 A|Y|182 paxillin|S/Y|84/88 PTRF|Y|308 c1 PZR|Y|263 EGFR|Y|1173 SHC|Y|239 SHC|Y|317 C18 orf 11|Y|297 ERK1|Y|204 ERK2|Y|187 STAT3-1|Y|705 STAT3-2|Y|704 Ack|Y|857 EGFR|Y|1068 EGFR|Y|1148 SHIP-2|Y|986 SHC|Y/Y|239/240 An A2|Y|23 An A2|Y|29 TfR|Y|20 Caveolin 1|Y|14 Dsc3a|Y|818 SCF38 m2|Y|20 Rin1|Y|36
FAK|Y|397 FAK|Y|576 IGF1R|Y|1161 RPTPa|Y|798 GIT1|Y|545 IGF1R|Y|1165 paxillin|Y|118 PI3KR2|Y|464 RAI3|Y|347
signaling network that regulates impor-
Astonishingly, 332 phosphorylated pep-
tant cellular processes such as migra-
tides from 175 proteins were identified,
tion and proliferation. Overexpression
122 of which had not previously been
of HER2 is notoriously associated with
described. Using the self-organizing
breast and other cancers, and drugs
map clustering algorithm, which enables
that selectively target HER2 have dem-
the identification of clusters of tyrosine-
onstrated effective anticancer activity in
phosphorylated peptides with similar
patients. In an effort to map the signal-
temporal dynamics, the authors readily
ing network of HER2, Wolf-Yadlin et al.
identified four clusters that reveal con-
growth factors promote cell migration.
(Mol. Syst. Biol., published online Oct 3,
nectivity in the data. In order to correlate
In contrast, HER2 overexpression had
2006, DOI: 10.1038/msb4100094) use
this signaling data with a phenotypic
a minimal effect on cell proliferation;
c4
c2
EphA2|Y|588 c3 SHB|Y|355 EphA2|Y|772 EphA2|Y/Y|588/594 LDLR|Y|845 SHP-2|Y|62
Reprinted by permission from Macmillan Publishers Ltd: Mol. Syst. Biol., advance online publication, Oct 3, 2006, DOI: 10.1038/msb4100094.
quantitative mass spectrometry, biologi- effect, they also measured cell migration
rather, EGF treatment emerged as the
cal response data, and computational
and proliferation under the same condi-
primary driver of cell growth. A model
analysis to correlate phosphorylation
tions. In general, HER2 overexpressing
using partial-least-squares regression
patterns with cell proliferation or with
cells exhibited enhanced cell migration.
was constructed to quantitatively
migration.
Moreover, phosphorylation patterns of
correlate phosphorylation patterns
cells stimulated with EGF versus HRG
with cell migration or proliferation,
phorylation was examined across 16
pointed to the network connections
establishing a powerful approach for
dimensions: four time points, two cell
behind the increased migratory ability of
exploring the relationship between
lines (one that did and one that did not
HER2 overexpressing cells and eluci-
protein phosphorylation and cellular
overexpress HER2), and treatment with
dated distinct pathways by which these
processes. EG
The cellular state of tyrosine phos-
BCAR3|Y|267 p130Cas|Y|327 p130Cas|Y|387
Making and Breaking Moenomycin, continued from page 610 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, Staphylococcus aureus and Enterococcus faecalis. Notably, both compounds inhibited the purified proteins comparably, but moenomycin was a much more potent
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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
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Reference database (profiles)
Biological state of interest (signature)
Connections Positive
Up
Output
Query Down
Negative
Getting Connected!
Strong Weak Null positive positive
Strong negative
From Lamb, J., et al., Science, 2006, Sept 29, 2006 DOI: 10.1126/science.1132939. Reprinted with permission from AAAS.
Establishing connections among physiological and
internal and external studies were collected and evalu-
pathological processes and genetic and small-molecule
ated. The data included the effects on gene expression of
perturbations can lead to unanticipated links that could
small molecules, such as histone deacetylase inhibitors,
help decipher the incredibly complex web that defines a
estrogens, and phenothiazines, and of disease states, such
biological state. In an attempt to establish a systematic
as diet-induced obesity, Alzheimer’s disease, and dexa-
method for exploring these relationships, Lamb et al.
methasone resistance in acute lymphoblastic leukemia.
(Science 2006, 313, 1929-1935) present the Connectivity
Remarkably, the Connectivity Map revealed both positive
Map, a resource in which gene-expression profiles of cells
and negative connectivity relationships that correctly
exposed to small molecules are assembled into a public
predicted several known relationships, pointed to the pre-
database for which data-mining tools are available to
viously unknown mechanism of action of a small molecule,
detect noteworthy relationships among the profiles.
and identified several molecules with potential therapeutic
Data from the expression profiles of breast cancer cells
utility. On the basis of these encouraging preliminary
exposed to 164 distinct bioactive small molecules were
results, the authors propose that an expanded Connectivity
used to create a first-generation Connectivity Map. A
Map should be generated as a community resource project.
query signature, or list of genes whose expression is cor-
Depending on the Map’s utility, the exciting prospect of
related with a biological state of interest, could then be
further expansion toward the ultimate goal of creating a
scanned in the Connectivity Map in the search for promi-
comprehensive description of all biological states in the
nent relationships. A range of query signatures from both
context of genomic signatures could be realized. EG
Linking Lipids to Life Some 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 membrane and survive. The authors initially observed that exposure of mammalian cells to aerolysin resulted in activation of the sterol regulatory element binding proteins (SREBPs), transcription factors that regulate choles612
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terol and fatty acid biosynthesis. Further investigations revealed that activation of the SREBPs was caused by loss of potassium through the toxin pores. Interestingly, they noted that potassium efflux had previously been linked to caspase-1 activation, 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 multiprotein complexes called inflammasomes, and they further demonstrated that aerolysin exposure triggers formation of inflammasomes. Moreover, it was demonstrated that prevention of caspase-1 or inflammasome activation blocked aerolysin-induced SREBP-2 activation and that caspase-1 activation induced SREBP activation through
a well-established pathway involving the escort protein SCAP (SREBP cleavage-activating protein) and two transmembrane proteases, S1P and S2P. These data undeniably 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 blocking 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 w w w. a c s c h e m i ca l biology.org
Quality of Protein Microarrays The assembly and deciphering of protein interaction networks promise to reveal valuable information about how organisms function. The accuracy of commonly used methods 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, 13,66813,669) 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 microarray 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 correct affinities of the interactions. A strength of microarrays is their ability to control protein concentrations, so obtaining this type of
Reprinted with permission from Gordus, A., and MacBeath, G., J. Am. Chem. Soc., 128, 13668-13669. Copyright 2006 American Chemical Society.
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
UPCOMING CONFERENCES American Society for Cell Biology Annual Meeting December 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 Meeting March 3-7, 2007 Baltimore, MD
Glycobiology, GRC March 4-9, 2007 Ventura, CA
2007 ACS Spring National Meeting March 25-29, 2007 Chicago, IL
Spotlights written by Eva Gordon and Jason Underwood
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