[New structures of mTORC1: Focus on Rag GTPases]. / Une moisson de nouvelles structures de mTORC1 - Coup de projecteur sur les GTPases Rag.

mTORC1 is a central player in cell growth, a process that is tightly regulated by the availability of nutrients and that controls various aspects of metabolism in the normal cell and in severe diseases such as cancers. mTORC1 is a large multiprotein complex, composed of the kinase subunit mTOR, of Ragulator, which attaches mTOR to the lysosome membrane, of the atypical Rag GTPases and the small GTPase RheB, whose nucleotide states directly dictate its localization to the lysosome and its kinase activity, and of RAPTOR, an adaptor that assembles the complex. The activity of the Rag GTPases is further controlled by the GATOR1 and folliculin complexes, which regulate their GTP/GDP conversion. Here, we review recent structures of important components of the mTORC1 machinery, determined by cryo-electron microscopy for the most part, which allow to reconstitute the architecture of active mTORC1 at near atomic resolution. Notably, we discuss how these structures shed new light on the roles of Rag GTPases and their regulators in mTORC1 regulation, and the perspectives that they open towards understanding the inner workings of mTORC1 on the lysosomal membrane.

TITLE: Une moisson de nouvelles structures de mTORC1 - Coup de projecteur sur les GTPases Rag. ABSTRACT: mTORC1 est un acteur central de la croissance cellulaire, un processus étroitement régulé par la disponibilité de nutriments et qui contrôle diverses étapes du métabolisme dans la cellule normale et au cours de maladies, comme les cancers. mTORC1 est un complexe multiprotéique de grande taille constitué de nombreuses sous-unités, parmi lesquelles deux types de GTPases, Rag et RheB, contrôlent directement sa localisation membranaire et son activité kinase. Dans cette revue, nous faisons le point sur une moisson de structures récentes, déterminées pour la plupart par cryo-microscopie électronique, qui sont en passe de reconstituer le puzzle de l'architecture de mTORC1. Nous discutons ce que ces structures révèlent sur le rôle des GTPases, et ce que leur connaissance ouvre comme perspectives pour comprendre comment mTORC1 fonctionne à la membrane du lysosome.

Protein-membrane interactions in small GTPase signalling and pharmacology: perspectives from Arf GTPases studies.

Small GTPases, in association with their GEFs, GAPs and effectors, control major intracellular processes such as signal transduction, cytoskeletal dynamics and membrane trafficking. Accordingly, dysfunctions in their biochemical properties are associated with many diseases, including cancers, diabetes, infections, mental disorders and cardiac diseases, which makes them attractive targets for therapies. However, small GTPases signalling modules are not well-suited for classical inhibition strategies due to their mode of action that combines protein-protein and protein-membrane interactions. As a consequence, there is still no validated drug available on the market that target small GTPases, whether directly or through their regulators. Alternative inhibitory strategies are thus highly needed. Here we review recent studies that highlight the unique modalities of the interaction of small GTPases and their GEFs at the periphery of membranes, and discuss how they can be harnessed in drug discovery.

A Ca2+-regulated deAMPylation switch in human and bacterial FIC proteins.

FIC proteins regulate molecular processes from bacteria to humans by catalyzing post-translational modifications (PTM), the most frequent being the addition of AMP or AMPylation. In many AMPylating FIC proteins, a structurally conserved glutamate represses AMPylation and, in mammalian FICD, also supports deAMPylation of BiP/GRP78, a key chaperone of the unfolded protein response. Currently, a direct signal regulating these FIC proteins has not been identified. Here, we use X-ray crystallography and in vitro PTM assays to address this question. We discover that Enterococcus faecalis FIC (EfFIC) catalyzes both AMPylation and deAMPylation and that the glutamate implements a multi-position metal switch whereby Mg2+ and Ca2+ control AMPylation and deAMPylation differentially without a conformational change. Remarkably, Ca2+ concentration also tunes deAMPylation of BiP by human FICD. Our results suggest that the conserved glutamate is a signature of AMPylation/deAMPylation FIC bifunctionality and identify metal ions as diffusible signals that regulate such FIC proteins directly.

PH-domain-binding inhibitors of nucleotide exchange factor BRAG2 disrupt Arf GTPase signaling.

Peripheral membrane proteins orchestrate many physiological and pathological processes, making regulation of their activities by small molecules highly desirable. However, they are often refractory to classical competitive inhibition. Here, we demonstrate that potent and selective inhibition of peripheral membrane proteins can be achieved by small molecules that target protein-membrane interactions by a noncompetitive mechanism. We show that the small molecule Bragsin inhibits BRAG2-mediated Arf GTPase activation in vitro in a manner that requires a membrane. In cells, Bragsin affects the trans-Golgi network in a BRAG2- and Arf-dependent manner. The crystal structure of the BRAG2-Bragsin complex and structure-activity relationship analysis reveal that Bragsin binds at the interface between the PH domain of BRAG2 and the lipid bilayer to render BRAG2 unable to activate lipidated Arf. Finally, Bragsin affects tumorsphere formation in breast cancer cell lines. Bragsin thus pioneers a novel class of drugs that function by altering protein-membrane interactions without disruption.

FIC proteins: from bacteria to humans and back again.

During the last decade, FIC proteins have emerged as a large family comprised of a variety of bacterial enzymes and a single member in animals. The air de famille of FIC proteins stems from a domain of conserved structure, which catalyzes the post-translational modification of proteins (PTM) by a phosphate-containing compound. In bacteria, examples of FIC proteins include the toxin component of toxin/antitoxin modules, such as Doc-Phd and VbhT-VbhA, toxins secreted by pathogenic bacteria to divert host cell processes, such as VopS, IbpA and AnkX, and a vast majority of proteins of unknown functions. FIC proteins catalyze primarily the transfer of AMP (AMPylation), but they are not restricted to this PTM and also carry out other modifications, for example by phosphocholine or phosphate. In a recent twist, animal FICD/HYPE was shown to catalyze both AMPylation and de-AMPylation of the endoplasmic reticulum BIP chaperone to regulate the unfolded protein response. FICD shares structural features with some bacterial FIC proteins, raising the possibility that bacteria also encode such dual activities. In this review, we discuss how structural, biochemical and cellular approaches have fertilized each other to understand the mechanism, regulation and function of FIC proteins from bacterial pathogens to humans.

Multiple interactions between an Arf/GEF complex and charged lipids determine activation kinetics on the membrane.

Lipidated small GTPases and their regulators need to bind to membranes to propagate actions in the cell, but an integrated understanding of how the lipid bilayer exerts its effect has remained elusive. Here we focused on ADP ribosylation factor (Arf) GTPases, which orchestrate a variety of regulatory functions in lipid and membrane trafficking, and their activation by the guanine-nucleotide exchange factor (GEF) Brag2, which controls integrin endocytosis and cell adhesion and is impaired in cancer and developmental diseases. Biochemical and structural data are available that showed the exceptional efficiency of Arf activation by Brag2 on membranes. We determined the high-resolution crystal structure of unbound Brag2 containing the GEF (Sec7) and membrane-binding (pleckstrin homology) domains, revealing that it has a constitutively active conformation. We used this structure to analyze the interaction of uncomplexed Brag2 and of the myristoylated Arf1/Brag2 complex with a phosphatidylinositol bisphosphate (PIP2) -containing lipid bilayer, using coarse-grained molecular dynamics. These simulations revealed that the system forms a close-packed, oriented interaction with the membrane, in which multiple PIP2 lipids bind the canonical lipid-binding site and unique peripheral sites of the PH domain, the Arf GTPase and, unexpectedly, the Sec7 domain. We cross-validated these predictions by reconstituting the binding and kinetics of Arf and Brag2 in artificial membranes. Our coarse-grained structural model thus suggests that the high efficiency of Brag2 requires interaction with multiple lipids and a well-defined orientation on the membrane, resulting in a local PIP2 enrichment, which has the potential to signal toward the Arf pathway.

Identification of cancer-associated missense mutations in hace1 that impair cell growth control and Rac1 ubiquitylation.

The E3 ubiquitin ligase HACE1 is a potent tumor suppressor that controls cell proliferation and ubiquitylates the small GTPase Rac1 to target it to proteasomal degradation. Whether and how the activity of HACE1 is regulated by the N-terminal ankyrin (ANK) and the middle (MID) domains is ill defined. Here, we identified in the version 64 of the Catalogue of Somatic Mutations in Cancer (COSMIC) 13 missense mutations of hace1 located outside the HECT domain, and found that all lead to defective control of cell proliferation. In addition, several mutations located in the ankyrin domain displayed a dramatic reduction in Rac1 ubiquitylation associated with a decrease of colony formation in soft agar. 3D structure modelling of the 7 ankyrin-repeats coupled to functional analysis identified a surface epitope centered on one of the mutated residue, Gly-175, which is critical for controlling Rac1 binding and ubiquitylation. We also identified a role for the MID domain in conferring the specificity of association of HACE1 to the active form of Rac1. Our study of the functional interplay between HACE1 and Rac1 in cancer thus sheds a new light on the molecular mechanism of Rac1 ubiquitylation by HACE1 and the impact of its cancer-associated mutations in cell proliferation.

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.

Mapping the cellular response to small molecules using chemogenomic fitness signatures.

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.

Inhibitory signalling to the Arp2/3 complex steers cell migration.

Cell migration requires the generation of branched actin networks that power the protrusion of the plasma membrane in lamellipodia. The actin-related proteins 2 and 3 (Arp2/3) complex is the molecular machine that nucleates these branched actin networks. This machine is activated at the leading edge of migrating cells by Wiskott-Aldrich syndrome protein (WASP)-family verprolin-homologous protein (WAVE, also known as SCAR). The WAVE complex is itself directly activated by the small GTPase Rac, which induces lamellipodia. However, how cells regulate the directionality of migration is poorly understood. Here we identify a new protein, Arpin, that inhibits the Arp2/3 complex in vitro, and show that Rac signalling recruits and activates Arpin at the lamellipodial tip, like WAVE. Consistently, after depletion of the inhibitory Arpin, lamellipodia protrude faster and cells migrate faster. A major role of this inhibitory circuit, however, is to control directional persistence of migration. Indeed, Arpin depletion in both mammalian cells and Dictyostelium discoideum amoeba resulted in straighter trajectories, whereas Arpin microinjection in fish keratocytes, one of the most persistent systems of cell migration, induced these cells to turn. The coexistence of the Rac-Arpin-Arp2/3 inhibitory circuit with the Rac-WAVE-Arp2/3 activatory circuit can account for this conserved role of Arpin in steering cell migration.

Integrated conformational and lipid-sensing regulation of endosomal ArfGEF BRAG2.

The mechanisms whereby guanine nucleotide exchange factors (GEFs) coordinate their subcellular targeting to their activation of small GTPases remain poorly understood. Here we analyzed how membranes control the efficiency of human BRAG2, an ArfGEF involved in receptor endocytosis, Wnt signaling, and tumor invasion. The crystal structure of an Arf1-BRAG2 complex that mimics a membrane-bound intermediate revealed an atypical PH domain that is constitutively anchored to the catalytic Sec7 domain and interacts with Arf. Combined with the quantitative analysis of BRAG2 exchange activity reconstituted on membranes, we find that this PH domain potentiates nucleotide exchange by about 2,000-fold by cumulative conformational and membrane-targeting contributions. Furthermore, it restricts BRAG2 activity to negatively charged membranes without phosphoinositide specificity, using a positively charged surface peripheral to but excluding the canonical lipid-binding pocket. This suggests a model of BRAG2 regulation along the early endosomal pathway that expands the repertoire of GEF regulatory mechanisms. Notably, it departs from the auto-inhibitory and feedback loop paradigm emerging from studies of SOS and cytohesins. It also uncovers a novel mechanism of unspecific lipid-sensing by PH domains that may allow sustained binding to maturating membranes.

Structure of the Legionella effector AnkX reveals the mechanism of phosphocholine transfer by the FIC domain.

The FIC motif and the eukaryotic-like ankyrin repeats are found in many bacterial type IV effectors, yet little is known about how these domains enable bacteria to modulate host cell functions. Bacterial FIC domains typically bind ATP and transfer adenosine monophosphate moiety onto target proteins. The ankyrin repeat-containing protein AnkX encoded by the intracellular pathogen Legionella pneumophila is unique in that its FIC domain binds to CDP-choline and transfers a phosphocholine residue onto proteins in the Rab1 GTPase family. By determining the structures of unbound AnkX and AnkX with bound CDP-choline, CMP/phosphocholine and CMP, we demonstrate that the orientation of substrate binding in relation to the catalytic FIC motif enables this protein to function as a phosphocholinating enzyme rather than a nucleotidyl transferase. Additionally, the structure reveals that the ankyrin repeats mediate scaffolding interactions that resemble those found in protein-protein interactions, but are unprecedented in intramolecular interactions. Together with phosphocholination experiments, our structures unify a general phosphoryl transferase mechanism common to all FIC enzymes that should be conserved from bacteria to human.

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.

Ras superfamily GEFs and GAPs: validated and tractable targets for cancer therapy?

There is now considerable and increasing evidence for a causal role for aberrant activity of the Ras superfamily of small GTPases in human cancers. These GTPases function as GDP-GTP-regulated binary switches that control many fundamental cellular processes. A common mechanism of GTPase deregulation in cancer is the deregulated expression and/or activity of their regulatory proteins, guanine nucleotide exchange factors (GEFs) that promote formation of the active GTP-bound state and GTPase-activating proteins (GAPs) that return the GTPase to its GDP-bound inactive state. In this Review, we assess the association of GEFs and GAPs with cancer and their druggability for cancer therapeutics.

Modulation of cell adhesion and migration by the histone methyltransferase subunit mDpy-30 and its interacting proteins.

We have previously shown that a subset of mDpy-30, an accessory subunit of the nuclear histone H3 lysine 4 methyltransferase (H3K4MT) complex, also localizes at the trans-Golgi network (TGN), where its recruitment is mediated by the TGN-localized ARF guanine nucleotide exchange factor (ArfGEF) BIG1. Depletion of mDpy-30 inhibits the endosome-to-TGN transport of internalized CIMPR receptors and concurrently promotes their accumulation at the cell protrusion. These observations suggest mDpy-30 may play a novel role at the crossroads of endosomal trafficking, nuclear transcription and adhesion/migration. Here we provide novel mechanistic and functional insight into this association. First, we demonstrate a direct interaction between mDpy-30 and BIG1 and locate the binding region in the N-terminus of BIG1. Second, we provide evidence that the depletion or overexpression of mDpy-30 enhances or inhibits cellular adhesion/migration of glioma cells in vitro, respectively. A similar increase in cell adhesion/migration is observed in cells with reduced levels of BIG1 or other H3K4MT subunits. Third, knockdown of mDpy-30, BIG1, or the RbBP5 H3K4MT subunit increases the targeting of beta1 integrin to cell protrusions, and suppression of H3K4MT activity by depleting mDpy-30 or RbBP5 leads to increased protein and mRNA levels of beta1 integrin. Moreover, stimulation of cell adhesion/migration via mDpy-30 knockdown is abolished after treating cells with a function-blocking antibody to beta1 integrin. Taken together, these data indicate that mDpy-30 and its interacting proteins function as a novel class of cellular adhesion/migration modulators partially by affecting the subcellular distribution of endosomal compartments as well as the expression of key adhesion/migration proteins such as beta1 integrin.

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.

Unique GMP-binding site in Mycobacterium tuberculosis guanosine monophosphate kinase.

Bacterial nucleoside monophosphate (NMP) kinases, which convert NMPs to nucleoside diphosphates (NDP), are investigated as potential antibacterial targets against pathogenic bacteria. Herein, we report the biochemical and structural characterization of GMP kinase from Mycobacterium tuberculosis (GMPKMt). GMPKMt is a monomer with an unusual specificity for ATP as a phosphate donor, a lower catalytic efficiency compared with eukaryotic GMPKs, and it carries two redox-sensitive cysteines in the central CORE domain. These properties were analyzed in the light of the high-resolution crystal structures of unbound, GMP-bound, and GDP-bound GMPKMt. The latter structure was obtained in both an oxidized form, in which the cysteines form a disulfide bridge, and a reduced form which is expected to correspond to the physiological enzyme. GMPKMt has a modular domain structure as most NMP kinases. However, it departs from eukaryotic GMPKs by the unusual conformation of its CORE domain, and by its partially open LID and GMP-binding domains which are the same in the apo-, GMP-bound, and GDP-bound forms. GMPKMt also features a unique GMP binding site which is less close-packed than that of mammalian GMPKs, and in which the replacement of a critical tyrosine by a serine removes a catalytic interaction. In contrast, the specificity of GMPKMt for ATP may be a general feature of GMPKs because of an invariant structural motif that recognizes the adenine base. Altogether, differences in domain dynamics and GMP binding between GMPKMt and mammalian GMPKs should reveal clues for the design of GMPKMt-specific inhibitors.

Calorimetric and crystallographic analysis of the oligomeric structure of Escherichia coli GMP kinase.

Guanosine monophosphate kinases (GMPKs), which catalyze the phosphorylation of GMP and dGMP to their diphosphate form, have been characterized as monomeric enzymes in eukaryotes and prokaryotes. Here, we report that GMPK from Escherichia coli (ecGMPK) assembles in solution and in the crystal as several different oligomers. Thermodynamic analysis of ecGMPK using differential scanning calorimetry shows that the enzyme is in equilibrium between a dimer and higher order oligomers, whose relative amounts depend on protein concentration, ionic strength, and the presence of ATP. Crystallographic structures of ecGMPK in the apo, GMP and GDP-bound forms were solved at 3.2A, 2.9A and 2.4A resolution, respectively. ecGMPK forms a hexamer with D3 symmetry in all crystal forms, in which the two nucleotide-binding domains are able to undergo closure comparable to that of monomeric GMPKs. The 2-fold and 3-fold interfaces involve a 20-residue C-terminal extension and a sequence signature, respectively, that are missing from monomeric eukaryotic GMPKs, explaining why ecGMPK forms oligomers. These signatures are found in GMPKs from proteobacteria, some of which are human pathogens. GMPKs from these bacteria are thus likely to form the same quaternary structures. The shift of the thermodynamic equilibrium towards the dimer at low ecGMPK concentration together with the observation that inter-subunit interactions partially occlude the ATP-binding site in the hexameric structure suggest that the dimer may be the active species at physiological enzyme concentration.

RhoGDIs revisited: novel roles in Rho regulation.

Small GTP-binding proteins of the Rho/Rac/Cdc42 family combine their GDP/GTP cycle, regulated by guanine nucleotide-exchange factors and GTPase-activating proteins, to a cytosol/membrane cycle, regulated by guanine nucleotide dissociation inhibitors (rhoGDIs). RhoGDIs are endowed with dual functions in the cytosol where they form soluble complexes with geranylgeranylated GDP-bound Rho proteins and at membrane interfaces where they monitor the delivery and extraction of Rho proteins to/from their site of action. They have little diversity compared with other Rho protein regulators and therefore have been regarded mostly as housekeeping regulators that distribute Rho proteins equally to any membranes. Recently, acquired data show that rhoGDIs, by interacting with candidate receptors/displacement factors or by phosphorylation, may in fact have active contributions to targeting Rho proteins to specific subcellular membranes and signaling pathways. In addition, the GDP/GTP and membrane/cytosol cycles can be uncoupled in certain cases, with Rho proteins either escaping the membrane/cytosol cycle or being regulated by rhoGDIs in their GTP-bound form. Here, we survey recent structure-function relationships and cellular studies on rhoGDIs and revisit their classical housekeeping role into novel and more specific functions. We also review their involvement in diseases.

Novel Rap1 dominant-negative mutants interfere selectively with C3G and Epac.

Rap1 is a Ras-related GTPase that is principally involved in integrin- and E-cadherin-mediated adhesion. Rap1 is transiently activated in response to many incoming signals via a large family of guanine nucleotide exchange factors (GEFs). The lack of potent Rap1 dominant-negative mutants has limited our ability to decipher Rap1-dependent pathways; we have therefore developed a procedure to generate such mutants consisting in the oligonucleotide-mediated mutagenesis of residues 14-19, selection of mutants presenting an enhanced interaction with Epac2 by yeast two-hybrid screening and counter-screening for mutants still interacting with Rap effectors. In detail analysis of their interaction capacity with various Rap-GEFs in the yeast two-hybrid system revealed that mutants of residues 15 and 16 interacted with Epacs, C3G and CalDAG-GEFI, whereas mutants of position 17 had selectively lost their ability to bind CalDAG-GEFI as well as, for some, C3G. In cellular models where Rap1 is activated via endogenous GEFs, the Rap1[S17A] mutant inhibits both the cAMP-Epac and EGF-C3G pathways, whereas Rap1[G15D] selectively interferes with the latter. Finally, Rap1[S17A] is able to act as a bona fide dominant-negative mutant in vivo since it phenocopies the eye-reducing and lethal effects of D-Rap1 deficiency in Drosophila, effects that are overcome by the overexpression of D-Epac or D-Rap1.