Membranes prime the RapGEF EPAC1 to transduce cAMP signaling.

EPAC1, a cAMP-activated GEF for Rap GTPases, is a major transducer of cAMP signaling and a therapeutic target in cardiac diseases. The recent discovery that cAMP is compartmentalized in membrane-proximal nanodomains challenged the current model of EPAC1 activation in the cytosol. Here, we discover that anionic membranes are a major component of EPAC1 activation. We find that anionic membranes activate EPAC1 independently of cAMP, increase its affinity for cAMP by two orders of magnitude, and synergize with cAMP to yield maximal GEF activity. In the cell cytosol, where cAMP concentration is low, EPAC1 must thus be primed by membranes to bind cAMP. Examination of the cell-active chemical CE3F4 in this framework further reveals that it targets only fully activated EPAC1. Together, our findings reformulate previous concepts of cAMP signaling through EPAC proteins, with important implications for drug discovery.

Novel 2,7-Diazaspiro[4,4]nonane Derivatives to Inhibit Mouse and Human Osteoclast Activities and Prevent Bone Loss in Ovariectomized Mice without Affecting Bone Formation.

Osteoporosis is currently treated with drugs targeting the differentiation or viability osteoclasts, the cells responsible for physiological and pathological bone resorption. Nevertheless, osteoporosis drugs that target only osteoclast activity are expected to preserve bone formation by osteoblasts in contrast to current treatments. We report here the design, synthesis, and biological characterization of a series of novel N-arylsufonamides featuring a diazaspiro[4,4]nonane nucleus to target the guanine nucleotide exchange activity of DOCK5, which is essential for bone resorption by osteoclasts. These compounds can inhibit both mouse and human osteoclast activity. In particular, 4-chlorobenzyl-4-hydroxy-2-phenyl-1-thia-2,7-diazaspiro[4,4]nonane 1,1-dioxide (compound E197) prevented pathological bone loss in mice. Most interestingly, treatment with E197 did not affect osteoclast and osteoblast numbers and hence did not impair bone formation. E197 could represent a lead molecule to develop new antiosteoporotic drugs targeting the mechanism of osteoclast adhesion onto the bone.

Allosteric inhibition of the guanine nucleotide exchange factor DOCK5 by a small molecule.

Rac small GTPases and their GEFs of the DOCK family are pivotal checkpoints in development, autoimmunity and bone homeostasis, and their abnormal regulation is associated to diverse pathologies. Small molecules that inhibit their activities are therefore needed to investigate their functions. Here, we characterized the mechanism of inhibition of human DOCK5 by C21, a small molecule that inhibits mouse Dock5 in cells and blocks bone degradation in mice models of osteoporosis. We showed that the catalytic DHR2 domain of DOCK5 has a high basal GEF activity in the absence of membranes which is not regulated by a simple feedback loop. C21 blocks this activity in a non-competitive manner and is specific for DOCK5. In contrast, another Dock inhibitor, CPYPP, inhibits both DOCK5 and an unrelated GEF, Trio. To gain insight into structural features of the inhibitory mechanism of C21, we used SAXS analysis of DOCK5DHR2 and crystallographic analysis of unbound Rac1-GDP. Together, these data suggest that C21 takes advantage of intramolecular dynamics of DOCK5 and Rac1 to remodel the complex into an unproductive conformation. Based on this allosteric mechanism, we propose that diversion of intramolecular dynamics is a potent mechanism for the inhibition of multidomain regulators of small GTPases.

Characterization of the activation of small GTPases by their GEFs on membranes using artificial membrane tethering.

Active, GTP-bound small GTPases need to be attached to membranes by post-translational lipid modifications in order to process and propagate information in cells. However, generating and manipulating lipidated GTPases has remained difficult, which has limited our quantitative understanding of their activation by guanine nucleotide exchange factors (GEFs) and their termination by GTPase-activating proteins. Here, we replaced the lipid modification by a histidine tag in 11 full-length, human small GTPases belonging to the Arf, Rho and Rab families, which allowed to tether them to nickel-lipid-containing membranes and characterize the kinetics of their activation by GEFs. Remarkably, this strategy uncovered large effects of membranes on the efficiency and/or specificity in all systems studied. Notably, it recapitulated the release of autoinhibition of Arf1, Arf3, Arf4, Arf5 and Arf6 GTPases by membranes and revealed that all isoforms are efficiently activated by two GEFs with different regulatory regimes, ARNO and Brag2. It demonstrated that membranes stimulate the GEF activity of Trio toward RhoG by ∼30 fold and Rac1 by ∼10 fold, and uncovered a previously unknown broader specificity toward RhoA and Cdc42 that was undetectable in solution. Finally, it demonstrated that the exceptional affinity of the bacterial RabGEF DrrA for the phosphoinositide PI(4)P delimits the activation of Rab1 to the immediate vicinity of the membrane-bound GEF. Our study thus validates the histidine-tag strategy as a potent and simple means to mimic small GTPase lipidation, which opens a variety of applications to uncover regulations brought about by membranes.

The αRep artificial repeat protein scaffold: a new tool for crystallization and live cell applications.

We have designed a new family of artificial proteins, named αRep, based on HEAT (acronym for Huntingtin, elongation factor 3 (EF3), protein pphosphatase 2A (PP2A), yeast kinase Tor1) repeat proteins containing an α-helical repeated motif. The sequence of the repeated motifs, first identified in a thermostable archae protein was optimized using a consensus design strategy and used for the construction of a library of artificial proteins. All proteins from this library share the same general fold but differ both in the number of repeats and in five highly randomized amino acid positions within each repeat. The randomized side chains altogether provide a hypervariable surface on αRep variants. Sequences from this library are efficiently expressed as soluble, folded and very stable proteins. αRep binders with high affinity for various protein targets were selected by phage display. Low micromolar to nanomolar dissociation constants between partners were measured and the structures of several complexes (specific αRep/protein target) were solved by X-ray crystallography. Using GFP as a model target, it was demonstrated that αReps can be used as bait in pull-down experiments. αReps can be expressed in eukaryotic cells and specifically interact with their target addressed to different cell compartments.

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.

Amphipol-mediated screening of molecular orthoses specific for membrane protein targets.

Specific, tight-binding protein partners are valuable helpers to facilitate membrane protein (MP) crystallization, because they can i) stabilize the protein, ii) reduce its conformational heterogeneity, and iii) increase the polar surface from which well-ordered crystals can grow. The design and production of a new family of synthetic scaffolds (dubbed αReps, for "artificial alpha repeat protein") have been recently described. The stabilization and immobilization of MPs in a functional state are an absolute prerequisite for the screening of binders that recognize specifically their native conformation. We present here a general procedure for the selection of αReps specific of any MP. It relies on the use of biotinylated amphipols, which act as a universal "Velcro" to stabilize, and immobilize MP targets onto streptavidin-coated solid supports, thus doing away with the need to tag the protein itself.

Ctenocephalides felis an in vitro potential vector for five Bartonella species.

The blood-sucking arthropod Ctenocephalides felis has been confirmed as a vector for Bartonella henselae and is a suspected vector for Bartonella clarridgeiae, Bartonella quintana and Bartonella koehlerae in Bartonella transmission to mammals. To understand the absence of other Bartonella species in the cat flea, we have developed an artificial flea-feeding method with blood infected successively with five different Bartonella species. The results demonstrated the ability of these five Bartonella species to persist in C. felis suggesting an ability of fleas to be a potential vector for several Bartonella species. In addition, we demonstrated a regurgitation of Bartonella DNA in uninfected blood used to feed C. felis thus suggesting a potential horizontal transmission of Bartonella through C. felis saliva. On the contrary, no vertical transmission was detected in these artificial conditions.

Heme binding proteins of Bartonella henselae are required when undergoing oxidative stress during cell and flea invasion.

Bartonella are hemotropic bacteria responsible for emerging zoonoses. These heme auxotroph alphaproteobacteria must import heme for their growth, since they cannot synthesize it. To import exogenous heme, Bartonella genomes encode for a complete heme uptake system enabling transportation of this compound into the cytoplasm and degrading it to release iron. In addition, these bacteria encode for four or five outer membrane heme binding proteins (Hbps). The structural genes of these highly homologous proteins are expressed differently depending on oxygen, temperature and heme concentrations. These proteins were hypothesized as being involved in various cellular processes according to their ability to bind heme and their regulation profile. In this report, we investigated the roles of the four Hbps of Bartonella henselae, responsible for cat scratch disease. We show that Hbps can bind heme in vitro. They are able to enhance the efficiency of heme uptake when co-expressed with a heme transporter in Escherichia coli. Using B. henselae Hbp knockdown mutants, we show that these proteins are involved in defense against the oxidative stress, colonization of human endothelial cell and survival in the flea.

Stoichiometry of the MexA-OprM binding, as investigated by blue native gel electrophoresis.

Multidrug resistance has become a serious concern in the treatment of bacterial infections. A prominent role is ascribed to the active efflux of xenobiotics out of the bacteria by a tripartite protein machinery. The mechanism of drug extrusion is rather well understood, thanks to the X-ray structures obtained for the Escherichia coli TolC/AcrA/AcrB model system and the related Pseudomonas aeruginosa OprM/MexA/MexB. However, many questions remain unresolved, in particular the stoichiometry of the efflux pump assembly. On the basis of blue native polyacrylamide gel electrophoresis (BN-PAGE) (Wittig et al., Nat. Protoc. 2006, 1, 418-428), we analyzed the binding stoichiometry of both palmitylated and non-palmitylated MexA with the cognate partner OprM trimer at different ratios and detergent conditions. We found that ß-octyl glucopyranoside (ß-OG) detergent was not suitable for this technique. Then we proved that MexA has to be palmitylated in order to stabilized the complex formation with OprM. Finally, we provided evidence for a two by two (2, 4, 6, or upper) binding of palmitylated MexA per trimer of OprM.

Novel mechanism of outer membrane targeting of proteins in Gram-negative bacteria.

SUMMARY: In Gram-negative bacteria, all the proteins destined for the outer membrane are synthesized with a signal sequence that is cleaved, either by the signal peptidase LepB for integral outer membrane proteins or by LspA for lipoproteins, when they cross the cytoplasmic membrane. The Dickeya dadantii protein PnlH does not possess a cleavable signal sequence but is anchored in the outer membrane by an N-terminal targeting signal. Addition of the 41 N-terminal amino acids of PnlH is sufficient for anchoring various hybrid proteins in the outer membrane. This targeting signal presents some of the characteristics of a Tat (twin arginine translocation) signal sequence but without an obvious cleavage site. We found that the Tat translocation pathway is required for the targeting process. This new mechanism of outer membrane protein targeting is probably widespread as PnlH was also addressed to the outer membrane when expressed in Escherichia coli. As PnlH was not detected as a substrate by Tat signal sequence prediction programmes, this would suggest that there may be many other unknown Tat-dependent outer membrane proteins.