ACS CHEMICAL BIOLOGY • VOL.1 NO.4 www.acschemicalbiology.org

Stressed Out CellsHumans aren’t the only ones who get “stressed out”. Cells get stressed out under a variety of circumstances, such as glucose depri­vation, altered glyco­sylation, or accumulation of unfolded proteins. Fortunately, the cell has intricate systems in place to deal with such stress, including the unfolded pro­tein response (UPR). The UPR initiates three signaling pathways, one of which is mediated by the endoplasmic reticulum (ER) transmembrane protein kinase and endoribonuclease inositol­requiring enzyme 1a (IRE1a). Members of the BCL­2 protein family, known for their intimate involvement in apoptosis, are also found in the ER, and various agents that induce apoptosis also induce the UPR. To further define the role of BCL­2 proteins in the cellular stress response, Hetz et al. (Science 2006, 312, 572–576) have examined the involvement of the proapoptotic BCL­2 proteins BAK and BAX in UPR signaling events.

The researchers created BAX and BAK double knockout (DKO) mice and explored the effects on UPR signaling pathways.

Comparison of DKO and wild­type cells using a variety of experiments including protein expression and

phosphorylation analysis, coimmunoprecipitation, protein mutagenesis, and small interfering RNA revealed that BAX and BAK modulate the IRE1a signaling pathway by affecting X­box­binding protein 1 (XBP­1) expression, a transcriptional activator of UPR­related genes that is activated by IRE1a. Furthermore, it was discovered that BAK expression at the ER membrane augmented IRE1a signaling, BAK and BAX interacted directly

with IRE1a through their BH3 and BH1 domains, and this interaction was enhanced in cells undergoing ER stress suggesting that BAX and BAK may stabilize the active form of IRE1a.

The authors propose that BCL­2 proteins may function not only as proapoptotic molecules but also as modulators of ER homeostasis that link stress signals to the apoptotic

circuitry in cells. This study provides a tangible connection between the UPR and proteins involved in apoptosis and will contribute to further deciphering of these two fundamental cellular processes. EG

Published online May 19, 2006 • 10.1021/cb600194v • © 2006 American Chemical Society

188

SpotlightArabidopsis Resists Hormone Stimulation

Reprinted with permission from AAAS

Plants and animals have mecha-nisms to detect the presence of a pathogen. In plants, a 22 amino acid peptide derived from the N-terminus

of flagellin (flg22) triggers a rapid down regulation of a subset of genes,

in part through a posttranscriptional mechanism. One such mechanism is RNA silencing, an mRNA degradation process mediated by short 20–24 nucleotide short interfering (siRNA) and microRNA (miRNA). These short RNAs bind to target mRNAs and mediate their cleavage. Until recently, it was not clear if miRNAs, which modulate develop-mental processes, were also involved in antibacterial resis-tance. Now, Navarro et al. (Science 2006, 312, 436–439) show that the flg22 induces the generation of a miRNA that targets mRNAs of receptors for auxin, a plant hormone, resulting in reduced susceptibility to bacterial infection.

To investigate the mechanism by which flg22 functions in bacterial resistance, transgenic

Arabidopsis plants expressing viral genes that suppress miRNA- and siRNA-mediated processes were treated with flg22. Analysis of the mRNA levels from treated and untreated plant cells showed that a subset of mRNAs TIR1, AFB2 and AFB3 (but not AFB1) were more prevalent in these transgenic plants. These F-box proteins are auxin receptors. The repres-sion of TIR1 and the AFB proteins coincides with increases in the level of miR393, a conserved miRNA. This miRNA-mediated repression leads to the down-regulation of auxin signaling pathways implicated in disease susceptibility. The miR393 pathway works in parallel with transcriptional repression of auxin receptors to ensure a rapid and robust immune response to the attaching bacteria. Precisely how auxin promotes disease susceptibility is not clear.

These data show for the first time that miRNAs also contribute to antibacterial resistance in plants. It will be

interesting to determine if other stress-induced processes also utilize miRNAs to control cellular responses. EJ

Image reprinted with permission from AAAS

www.acschemicalbiology.org VOL.1 NO.4 • ACS CHEMICAL BIOLOGY

(Poly)Combing through the Stem Cell Genome

Spotlight

189

Mycoplasma pneumoniae is responsible for community­acquired pneumonia and various other airway disorders. This unique species of bacterium is characterized by the lack of a cellular wall, making them resistant to antibiotics that disrupt cell wall synthesis such as penicillins. In addition, like viruses, they are dependent on host cells for virulence. These unusual characteristics have posed unique challenges in the investigation and treatment of M. pneumoniae infections, and many of the molecular mechanisms involved in their pathology remain a mystery. Now, Kannan and Baseman (PNAS 2006, 103, 6724–6729) report the identification of a virulence factor seemingly responsible for respiratory injury associated with M. pneumoniae.

In the search for agents responsible for M. pneumoniae virulence, affinity chromatography using a human lung protein led to the identification of a 68 kDa protein desig­nated community­acquired respiratory distress syndrome toxin (CARDS TX). Analysis of the primary sequence revealed that CARDS TX shares sequence similarities to pertus­sis toxin (PTX), hinting that, like PTX, CARDS TX may be an ADP­ribosylating toxin (ADPRT). Indeed, experiments

demonstrated that CARDS TX possesses ADP­ribosyltransferase (ART) activity, modify­ing an overlapping but distinct set of proteins than that of PTX. Treat­ment of mammalian cell cultures and baboon tracheal rings with the toxin revealed that CARDS TX causes

the characteristic cytoplasmic vacuolization observed in infected specimens. Notably, infected individuals seroconvert to CARDS TX, providing further evidence that CARDS TX is the virulent factor in M. pneumoniae. The authors propose that intimate contact between the mycoplasma and the host cell in the early stages of infection could enable release of CARDS TX into target cells, leading to ADP ribosylation, vacuolization, and eventual cytotoxicity. Identification of this mycoplasma­associated toxin provides an explanation for the pathology of M. pneumoniae and will facilitate diagnostic, preventative, and treatment strategies for M. pneumoniae infections. EG

The extraordinary ability of stem cells to differentiate into most cell types has stim­ulated much research spanning the basic biology behind their metamorphosis to their tantalizing clinical potential in regen­erative medicine. Recently, intriguing con­nections have been made between stem cell regulation and chromatin structure. Now, four studies tackle genome­wide analyses of chromatin structure to provide insight into the role of chromatin in stem cell regulation.

Chromatin, the structural building block of chromosomes, is made up largely of chromosomal DNA and proteins called histones. Posttranslational modification of histones, such as methylation or acetyl­ation of specific residues, modulates chromatin structure, and these modifi­cations are part of the gene regulation machinery. The polycomb group (PcG) of proteins are chromatin­binding proteins

and are integral pieces of this machinery. PcG proteins form two major complexes, termed polycomb repressive complexes (PRCs). PRC2 binds to sites of transcrip­tional repression and modifies chromatin

structure through epigenetic changes, or “heritable” changes that do not involve alteration of DNA sequence, by catalyzing histone H3 lysine­27 (H3K27) methylation. Trimethylation of H3K27

(H3K27me3) in turn provides a landscape that recruits PRC1, which facilitates chromatin reorganization, including oligo­merization and condensation, reinforcing transcriptional repression. Investigation

into the identity and function of the specific genes that PRCs target will help elucidate mecha­nisms of gene regulation and define the pathways involved in embryonic development.

To this end, Tolhuis et al. (Nature Genetics, published online April 20, 2006, doi:10.1038/ng1792), Boyer et al. (Nature, pub­lished online April 19, 2006,

doi:10.1038/ng04733), and Lee et al. (Cell 2006, 125, 301–313) have mapped binding patterns of PcG proteins in Drosophila melanogaster, mouse, and

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(continued on page 190)

ACS CHEMICAL BIOLOGY • VOL.1 NO.4 www.acschemicalbiology.org

Spotlight

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(Poly)Combing through the Stem Cell Genome, continued

human embryonic stem (ES) cells, respec­tively, and Bernstein et al. (Cell 2006, 125, 315–326) have mapped histone methylation patterns in mouse ES cells. In all three species, PcG proteins strate­gically bind to sites of transcriptional repression of developmentally important genes, including genes that regulate early and late developmental processes in D. melanogaster, such as ectoderm development and eye morphogenesis, and genes involved in mammalian cell differentiation, cell­fate commitment, organogenesis, hematopoiesis, and neurogenesis. In addition, there is a strong correlation between PcG binding and sites of H3K27me3 that substan­tially decreases upon cell differentiation. This cross­species, genome­wide data demonstrates that polycomb complexes target developmental genes and are a critical component of the gene silencing

machinery. This implicates chromatin structure as an important regulator in ES cell pluripotency.

Remarkably, a unique characteristic of ES cell chromatin appears to allow for the reversal of gene silencing upon appropriate developmental signals. While H3K27 methylation is a transcriptional repression signal, methylation of H3 lysine 4 (H3K4) signifies transcriptional activation. Upon analysis of histone methylation patterns across the mouse ES cell genome, Bernstein et al. identified a specific pattern in the vicinity of many developmental genes, termed “bivalent domains” that consists of large regions of H3K27 methylation harboring smaller regions of H3K4 methylation. The impli­cated genes are largely silenced in ES cells, consistent with the H3K27 mark and with the findings of the other studies. The authors propose that the coincident H3K4

methylation keeps these critical genes poised for activation at later develop­mental stages. Interestingly, strong corre­lations between genome sequence and histone methylation were also observed, highlighting the importance of DNA sequence in defining the initial epigenetic state, which is then altered as embryonic development progresses.

Taken together, these studies point to a model of ES cell regulation whereby PcG proteins and histone methylation function to control transcription of developmen­tally relevant genes, silencing them until developmental cues call for their activa­tion. Further elucidation of the molecular details of this intricate regulation system will enhance our understanding of myriad developmental processes, including stem cell differentiation, embryonic develop­ment, tissue homeostasis, aging, and oncogenesis. EG

Natural products are an incredibly valuable resource for discovery of potential new medicines and molecular tools for biological exploration. One major hurdle in natural products research is the tedious isolation and structure determination processes that are often required. Clarkson et al. (J. Nat. Prod. 2006, 69, 527–530) have devel-oped a new technique, high-performance liquid chromatography–solid-phase extraction–nuclear magnetic resonance (HPLC–SPE–NMR) that enables the rapid structure determination of constituents of natural product extracts.

The researchers used HPLC–SPE–NMR to analyze the petroleum ether root extract from Harpagophytum procumbens, a plant native to South Africa whose large roots are used as an anti-inflammatory agent and to stimulate digestion. From 11 major and minor HPLC peaks analyzed, two novel structures were identified. Structure deter-mination was facilitated by several key features of the HPLC–SPE–NMR technique. First, extensive 2D NMR data can be obtained from all HPLC peaks without interruption of the mobile phase flow, as in direct, stopped-flow HPLC–NMR. In addition, multiple SPE trappings can be conducted, dramatically improving signal-to-noise ratios

in the NMR analysis. Finally, the entire analysis can be performed under inert conditions, facilitating structure elucidation of air- and moisture-sensitive compounds. The novel compounds were found to contain an uncommon diterpene skeleton, named chinane, with an unusually placed isopropyl group on the terpene ring system. On the basis of comparison of their NMR data with that of other diterpenes, the authors propose that the chinane structure may exist in other known compounds whose structures have been misinterpreted. This new technique could revolutionize the structure elucidation process in natural products research and revitalize industrial drug discovery programs based on natural products by providing accelerated access to chemical diversity of biological sources. EG

Extracting Extract Structures

www.acschemicalbiology.org VOL.1 NO.4 • ACS CHEMICAL BIOLOGY

Some marine mollusks secrete small molecules that inhibit biological processes such as protein translation. Mechanistic studies of translation have benefited significantly from the use of these ligands,

many of which target specific steps in protein synthesis. Furthermore, several inhibitors of translation have been used as anticancer drugs and are in clinical trials. Recent studies showed that chlorinated lissoclimides from the marine mollusk Pleurobranchus forskalii affect translation. Now, Robert et al. (RNA 2006, 12, 717–724) find that two members of this family of molecules, chlorolissoclimide and dichlorolisso­climide, are potent inhibitors of eukaryotic translation elongation.

Chlorolissoclimide and dichlorolisso­climide are toxic bicyclic diterpene alkaloids and are members of a larger family of diterpenoids called labdanes. Investigation into the mechanism of inhibition of these compounds revealed

that they inhibit the elongation phase of translation, as opposed to preventing translation initiation. Furthermore, unlike other translation elonga­tion inhibitors such as phyllanthoside and nagilactone C, which disrupt polyribosomes, chlorolissoclimide and dichlorolissoclimide block translation elongation by stalling the ribosome on the mRNA. The authors note that there is a structural similarity between the lissoclimides and cycloheximide, a translation inhibitor that also inhibits trans­location by interfering with the release of ribo­somes from polysomes.

Diterpenoids from the labdane class have been isolated from a variety of plants and animals, and they have been found to possess inhibitory activity against a wide range of eurkayotic organisms, including Trypanosoma cruzi, algae, and cancer cells. The authors propose that, although more research is required into the mechanisms of action of these compounds, diverse species may use inhibition of protein synthesis as a defense mechanism against predators. EG

Spotlight

191

R1 OH

Cl

NHO

O

OH

Reprinted with permission from the RNA Society

Elongate No MoreG Protein Hot SpotsHeterotrimeric guanine nucleotide-binding proteins (G pro-

teins) mediate diverse physiological processes, and small

molecule modulators of these

proteins have broad therapeutic

potential. G protein coupled

receptor activation results in the

release of G protein βγ subunits

that then participate in inter-

actions with multiple down-

stream effector molecules. Many

of these effector interactions

occur at a hotspot for protein-

protein interactions on the

β-subunit surface. Bonacci et al.

(Science, 2006, 312, 443–446)

use molecular modeling to

discover small molecules that interact with this surface and

demonstrate that these compounds can differentially affect

G protein function.

Virtual docking of a structurally diverse small molecule

library to the interaction hotspot led to the identification

of several molecules, including M119 and M201, that

bound to distinct subsurfaces of the hotspot. While M119

inhibited Gβγ-dependent phospholipase C β3 (PLC β3) and

phosphoinositide 3-kinase activation, M201 potentiated

these interactions. In addition, cellular assays demonstrated

that M119 attenuated peptide-induced, G protein-mediated

calcium increases while M201 had no effect. Moreover, in

vivo studies showed that M119 increased the sensitivity of

mice to morphine, an expected effect from a compound that

blocks PLC β3 activity. Given the diversity of interactions

in which G proteins participate, the ability to differentially

manipulate G protein function with small molecules is a

powerful strategy. EG

Reprinted with permission from Biochemistry, 2005, 44, 10593–10604.

Spotlight

192 ACS CHEMICAL BIOLOGY • VOL.1 NO.4 www.acschemicalbiology.org

Go with the FlowFlow cytometry is a powerful tool for analysis of many cellular properties, including cell surface markers, DNA content, calcium flux, apoptosis, and intracellular protein concentra-tions. The wide range of applica-tions make this a tempting method for large-scale investigations such as drug screening and cellular profiling, but increasing the capacity of the technol-ogy has been hampered by corresponding increases in reagent expense, inadequate sample throughput, and variability in sample labeling. Now, Krutzik and Nolan (Nature Methods 2006, 3, 361–368) present a cell-based multiplexing approach, termed fluor-escent cell barcoding (FCB), that overcomes these limitations and enables a wide range of high-throughput flow cytometry applications.

Flow cytometry permits multiparameter analysis in complex cell populations. For example, subpopulations of white blood cells in a heterogeneous sample can each be analyzed for the presence of multiple cell surface markers. FCB takes this multi-parameter capacity to an even higher level by exploiting the ability of flow cytometers to discriminate between samples labeled with different intensities of the same fluoro-phore. FCB combines the use of multiple fluorophores at multiple intensities per fluorophore so that each sample acquires a unique fluorescent signature, or barcode. For example, use of two fluorophores at six

different intensities each results in 36 unique

fluorescent barcodes. The uniquely bar-coded samples can

then be run together as one sample in the evalua-

tion of the characteristics of interest (for example, the presence of specific

phosphorylated proteins), resulting in signi-ficant reductions in reagent cost, sample variability, and time on the instrument. Deconvolution of the data after the samples are run enables analysis of the original samples based on their FCB signature.

The feasibility of using the FCB platform in high-throughput applications was demon-strated with two large-scale assays. First, a drug screening assay was performed in which three FCB markers were used to barcode 96 samples (analogous to use of a 96-well plate), and 70 small molecule inhibitors were screened for inhibition of cytokine-induced protein phosphorylation events. Data analysis rapidly revealed compound selectivity for specific signaling pathways. Second, a cellular profiling assay was run where a heterogeneous population of mouse spleno-cytes was analyzed for differential response to cytokine stimulation, and cell-type specific effects were promptly discerned. Technical advancements in this powerful technology, including innovative fluorescent markers and more sophisticated data analysis methods, will further contribute to its utility in high-throughput applications. EG

Reprinted with

permission from Nature Methods

Spotlight

193www.acschemicalbiology.org VOL.1 NO.4 • ACS CHEMICAL BIOLOGY

As if creating effective inhibitors for potential drug targets isn’t hard enough, appropriate formulation and bioavailability are two additional major hurdles in crossing the drug discovery finish line. One method over these hurdles is conjugation of the potential drug to a molecular transporter that yields a water­soluble yet membrane­permeable entity. Once inside the cell, however, the compound likely needs to be released from the transporter so that it can perform its intended biological activity. Jones et al. (JACS, pub­lished online April 25, 2006, doi:10.1021/ja0586283) have created a controllable releasable linker system that allows for liberation of the mole­cule after cell entry, and they have developed a cellular assay that enables measurement of conjugate uptake and release of the molecule.

The conjugate design incorporates specific features that make it an attractive drug delivery system. First, the linker that connects the potential drug, or cargo, to the transporter (a D­octaarginine

molecule that has previously been shown to effectively transport small molecules into cells and tissue) contains two key functional groups, a carbonate and a disulfide. Upon entry of the conjugate into the reducing environment of the cell’s cytoplasm, the disulfide bond is cleaved. The exposed free thiol reacts with the carbon­ate, resulting in liberation of the cargo. Furthermore, conjugate stability can be increased from hours to days simply by increasing the linker length, pro­viding a tunable system. To test the effectiveness of the conju­gate, luciferin was employed as the cargo, and luciferase­transfected cells were treated with the conjugate. Conjugate exposure resulted in an initial increase in luminescence fol­lowed by a gradual decay over the course of several minutes, and luminescence was dem­onstrated to be dependent on intracellular release of luciferin. This releasable luciferin conju­gate enables the exploration of other transporters and linkers for evaluation of delivery, release, and target interaction of potential drugs. EG

Nucleic Acids, GRCJune 4–9, 2006 Newport, RI

Nucleic Acid Enzyme, FASEBJune 10–15, 2006Saxton River, VT

Single Molecule Approaches to Biology, GRCJune 18–23, 2006 New London, NH

Biological Methylation, FASEBJune 24–29, 2006Saxton River, VT

Ubiquitin & Cellular Regulation, FASEBJuly 22–27, 2006 Saxton River, VT

Natural Products, GRCJuly 23–28, 2006 Tilton, NH

UPCOMING CONFERENCES

Spotlight written by Eva Gordon and Evelyn Jabri

Illuminating the Drug Delivery PathwayThe design of proteins whose functions can be manipulated under specific conditions is an innovative strategy for the development of macro-molecules with novel sensor capabilities. Ha et al. (J. Mol. Biol., 2006, 357, 1058–1062) have created a fusion protein that can be induced either to be catalytically active or to bind DNA, depending on the conditions.

The fusion protein, termed BG, consists of a target protein, the catalytically active bacterial ribonuclease barnase (Bn), and an ‘inserted’ protein, the DNA binding domain of GCN4. The GCN4 sequence is strategically integrated at a surface-exposed loop in Bn. In the absence of the GCN4 ligand, a DNA oligonucleotide called AP-1, the Bn domain of the chimera is more stable than the GCN4 sequence. Bn is folded and catalytically active, while GCN4 is largely disordered. In con-trast, in the presence of AP-1, the GCN4 portion is more stable than the Bn region and a thermodynamic ‘tug-of-war’ ensues, wherein folding of the ligand binding domain of GCN4 effectively splits Bn in two and abolishes enzymatic activity.

BG is a model for the design of additional chimeric proteins that possess novel sensor capabilities. For example, a potential therapeutic agent could be designed by inserting a different binding domain into Bn. The enzymatic activity of the new chimera would be controlled by binding of a specific ligand, which may be present only in certain cell types. EG

Reprinted with permission from the Journal of Molecular Biology

Protein Tug-of-War