Mahel Zeghouf

Regulation of Small GTPases by GEFs, GAPs, and GDIs

Small GTPases use GDP/GTP alternation to actuate a variety of functional switches that are pivotal for cell dynamics. The GTPase switch is turned on by GEFs, which stimulate dissociation of the tightly bound GDP, and turned off by GAPs, which accelerate the intrinsically sluggish hydrolysis of GTP. For Ras, Rho, and Rab GTPases, this switch incorporates a membrane/cytosol alternation regulated by GDIs and GDI-like proteins. The structures and core mechanisms of representative members of small GTPase regulators from most families have now been elucidated, illuminating their general traits combined with scores of unique features. Recent studies reveal that small GTPase regulators have themselves unexpectedly sophisticated regulatory mechanisms, by which they process cellular signals and build up specific cell responses. These mechanisms include multilayered autoinhibition with stepwise release, feedback loops mediated by the activated GTPase, feed-forward signaling flow between regulators and effectors, and a phosphorylation code for RhoGDIs. The flipside of these highly integrated functions is that they make small GTPase regulators susceptible to biochemical abnormalities that are directly correlated with diseases, notably a striking number of missense mutations in congenital diseases, and susceptible to bacterial mimics of GEFs, GAPs, and GDIs that take command of small GTPases in infections. This review presents an overview of the current knowledge of these many facets of small GTPase regulation.

Mapping the Cellular Response to Small Molecules Using Chemogenomic Fitness Signatures

Yeasty HIPHOP In order to identify how chemical compounds target genes and affect the physiology of the cell, tests of the perturbations that occur when treated with a range of pharmacological chemicals are required. By examining the haploinsufficiency profiling (HIP) and homozygous profiling (HOP) chemogenomic platforms, Lee et al. (p. 208) analyzed the response of yeast to thousands of different small molecules, with genetic, proteomic, and bioinformatic analyses. Over 300 compounds were identified that targeted 121 genes within 45 cellular response signature networks. These networks were used to extrapolate the likely effects of related chemicals, their impact upon genetic pathways, and to identify putative gene functions. Guilt by association helps identify the chemogenomic signatures of compounds targeting yeast genes. Genome-wide characterization of the in vivo cellular response to perturbation is fundamental to understanding how cells survive stress. Identifying the proteins and pathways perturbed by small molecules affects biology and medicine by revealing the mechanisms of drug action. We used a yeast chemogenomics platform that quantifies the requirement for each gene for resistance to a compound in vivo to profile 3250 small molecules in a systematic and unbiased manner. We identified 317 compounds that specifically perturb the function of 121 genes and characterized the mechanism of specific compounds. Global analysis revealed that the cellular response to small molecules is limited and described by a network of 45 major chemogenomic signatures. Our results provide a resource for the discovery of functional interactions among genes, chemicals, and biological processes.

Structure-based discovery of an inhibitor of Arf activation by Sec7 domains through targeting of protein-protein complexes

Small molecules that produce nonfunctional protein–protein complexes are an alternative to competitive inhibitors for the inhibition of protein functions. Here we target the activation of the small GTP-binding protein Arf1, a major regulator of membrane traffic, by the Sec7 catalytic domain of its guanine nucleotide exchange factor ARNO. The crystal structure of the Arf1-GDP/ARNO complex, which initiates the exchange reaction, was used to discover an inhibitor, LM11, using in silico screening of a flexible pocket near the Arf1/ARNO interface. Using fluorescence kinetics and anisotropy, NMR spectroscopy and mutagenesis, we show that LM11 acts following a noncompetitive mechanism in which the inhibitor targets both Arf1-GDP and the Arf1-GDP/ARNO complex and produces a nonfunctional Arf-GDP/ARNO complex whose affinity is similar to that of the native complex. In addition, LM11 recognizes features of both Arf and ARNO near the Arf/Sec7 interface, a characteristic reminiscent of the paradigm interfacial inhibitor Brefeldin A. We then show that LM11 is a cell-active inhibitor that impairs Arf-dependent trafficking structures at the Golgi. Furthermore, LM11 inhibits ARNO-dependent migration of Madin–Darby canine kidney (MDCK) cells, demonstrating that ARNO is a target of LM11 in cells. Remarkably, LM11 inhibits the activation of Arf1 but not Arf6 in vitro, pointing to a possible synergy between Arf1 and Arf6 activation by ARNO in cell migration. Our design method shows that flexible regions in protein–protein complexes provide drugable sites with the potential to develop novel tools for investigating and inhibiting signaling pathways.

Dual Specificity of the Interfacial Inhibitor Brefeldin A for Arf Proteins and Sec7 Domains

Guanine nucleotide exchange factors (GEFs), which activate small GTP-binding proteins (SMG) by stimulating their GDP/GTP exchange, are emerging as candidate targets for the inhibition of cellular pathways involved in diseases. However, their specific inhibition by competitive inhibitors is challenging, because GEF and SMG families comprise highly similar members. Nature shows us an alternative strategy called interfacial inhibition, exemplified by Brefeldin A (BFA). BFA inhibits the activation of Arf1 by its GEFs in vivo by stabilizing an abortive complex between Arf-GDP and the catalytic Sec7 domain of some of its GEFs. Here we characterize the specificity of BFA toward wild-type (ARNO and BIG1) and mutant Sec7 domains and toward class I, II, and III Arfs. We find that BFA sensitivity of the exchange reaction depends on the nature of both the Sec7 domain and the Arf protein. A single Phe/Tyr substitution is sufficient to achieve BFA sensitivity of the Sec7 domain, which is supported by our characterization of brefeldin C (BFC), a BFA analog that cannot interact with the Tyr residue, and by free energy computations. We further show that Arf1 and Arf5, but not Arf6, are BFA-sensitive, despite their having every BFA-interacting residue in common. Analysis of Arf6 mutants points to the dynamics of the interswitch, which is involved in membrane-to-nucleotide signal propagation, as contributing to, although not sufficient for, BFA sensitivity. Altogether, our results reveal the Tyr/Phe substitution as a novel tool for monitoring BFA sensitivity of cellular ArfGEFs and document the exquisite and dual specificity that can be achieved by an interfacial inhibitor.

RPAP1, a novel human RNA polymerase II-associated protein affinity purified with recombinant wild-type and mutated polymerase subunits.

ABSTRACT We have programmed human cells to express physiological levels of recombinant RNA polymerase II (RNAPII) subunits carrying tandem affinity purification (TAP) tags. Double-affinity chromatography allowed for the simple and efficient isolation of a complex containing all 12 RNAPII subunits, the general transcription factors TFIIB and TFIIF, the RNAPII phosphatase Fcp1, and a novel 153-kDa polypeptide of unknown function that we named RNAPII-associated protein 1 (RPAP1). The TAP-tagged RNAPII complex is functionally active both in vitro and in vivo. A role for RPAP1 in RNAPII transcription was established by shutting off the synthesis of Ydr527wp, a Saccharomyces cerevisiae protein homologous to RPAP1, and demonstrating that changes in global gene expression were similar to those caused by the loss of the yeast RNAPII subunit Rpb11. We also used TAP-tagged Rpb2 with mutations in fork loop 1 and switch 3, two structural elements located strategically within the active center, to start addressing the roles of these elements in the interaction of the enzyme with the template DNA during the transcription reaction.

Aptamer-Derived Peptides as Potent Inhibitors of the Oncogenic RhoGEF Tgat

Guanine nucleotide exchange factors (GEFs) activate the Rho GTPases by accelerating their GDP/GTP exchange rate. Some RhoGEFs have been isolated based on their oncogenic potency, and strategies to inhibit their activity are therefore actively being sought. In this study we devise a peptide inhibitor screening strategy to target the GEF activity of Tgat, an oncogenic isoform of the RhoGEF Trio, based on random mutations of the Trio inhibitor TRIP alpha, which we previously isolated using a peptide aptamer screen. This identifies one peptide, TRIP(E32G), which specifically inhibits Tgat GEF activity in vitro and significantly reduces Tgat-induced RhoA activation and foci formation. Furthermore, subcutaneous injection of cells expressing Tgat and TRIP(E32G) into nude mice reduces the formation of Tgat-induced tumors. Our approach thus demonstrates that peptide aptamers are potent inhibitors that can be used to interfere with RhoGEF functions in vivo.

Chronicles of the GTPase switch

The GTPase switch is a versatile molecular device used by many proteins, such as the small GTPases, to regulate an astounding number of functions. Although the basics of the guanine nucleotide cycle are now well established, the next challenge is to reach an integrated view of how these proteins use it to orchestrate signaling pathways.

LG186: An Inhibitor of GBF1 Function that Causes Golgi Disassembly in Human and Canine Cells

Brefeldin A‐mediated inhibition of ADP ribosylation factor (Arf) GTPases and their guanine nucleotide exchange factors, Arf‐GEFs, has been a cornerstone of membrane trafficking research for many years. Brefeldin A (BFA) is relatively non‐selective inhibiting at least three targets in human cells, Golgi brefeldin A resistance factor 1 (GBF1), brefeldin A inhibited guanine nucleotide exchange factor 1 (BIG1) and brefeldin A inhibited guanine nucleotide exchange factor 2 (BIG2). Here, we show that the previously described compound Exo2 acts through inhibition of Arf‐GEF function, but causes other phenotypic changes that are not GBF1 related. We describe the engineering of Exo2 to produce LG186, a more selective, reversible inhibitor of Arf‐GEF function. Using multiple‐cell‐based assays and GBF1 mutants, our data are most consistent with LG186 acting by selective inhibition of GBF1. Unlike other Arf‐GEF and reported GBF1 inhibitors including BFA, Exo2 and Golgicide A, LG186 induces disassembly of the Golgi stack in both human and canine cells.

Fam65b Is a New Transcriptional Target of FOXO1 That Regulates RhoA Signaling for T Lymphocyte Migration

Forkhead box O (FOXO) transcription factors favor both T cell quiescence and trafficking through their control of the expression of genes involved in cell cycle progression, adhesion, and homing. In this article, we report that the product of the fam65b gene is a new transcriptional target of FOXO1 that regulates RhoA activity. We show that family with sequence similarity 65 member b (Fam65b) binds the small GTPase RhoA via a noncanonical domain and represses its activity by decreasing its GTP loading. As a consequence, Fam65b negatively regulates chemokine-induced responses, such as adhesion, morphological polarization, and migration. These results show the existence of a new functional link between FOXO1 and RhoA pathways, through which the FOXO1 target Fam65b tonically dampens chemokine-induced migration by repressing RhoA activity.

Pharmacological inhibition of Dock5 prevents osteolysis by affecting osteoclast podosome organization while preserving bone formation

Osteoporosis is caused by excessive activity of bone-degrading osteoclasts over bone-forming osteoblast. Standard antiosteolytic treatments inhibit bone resorption by inducing osteoclast loss, with the adverse effect of hindering also bone formation. Formation of the osteoclast sealing zone requires Dock5, a guanine nucleotide exchange factor for the small GTPase Rac, and C21, a chemical inhibitor of Dock5, decreases bone resorption by cultured osteoclasts. Here we show that C21 directly inhibits the exchange activity of Dock5 and disrupts osteoclast podosome organization. Remarkably, C21 administration protects mice against bone degradation in models recapitulating major osteolytic diseases: menopause, rheumatoid arthritis and bone metastasis. Furthermore, C21 administration does not affect bone formation and is not toxic. Our results validate the pharmacological inhibition of Dock5 as a novel therapeutic route for fighting osteolytic diseases while preserving bone formation.