Structural evidence of a phosphoinositide-binding site in the Rgd1-RhoGAP domain.

Phosphoinositide lipids recruit proteins to the plasma membrane involved in the regulation of cytoskeleton organization and in signalling pathways that control cell polarity and growth. Among those, Rgd1p is a yeast GTPase-activating protein (GAP) specific for Rho3p and Rho4p GTPases, which control actin polymerization and stress signalling pathways. Phosphoinositides not only bind Rgd1p, but also stimulate its GAP activity on the membrane-anchored form of Rho4p. Both F-BAR (F-BAR FCH, and BAR) and RhoGAP domains of Rgd1p are involved in lipid interactions. In the Rgd1p-F-BAR domain, a phosphoinositide-binding site has been recently characterized. We report here the X-ray structure of the Rgd1p-RhoGAP domain, identify by NMR spectroscopy and confirm by docking simulations, a new but cryptic phosphoinositide-binding site, comprising contiguous A1, A1' and B helices. The addition of helix A1', unusual among RhoGAP domains, seems to be crucial for lipid interactions. Such a site was totally unexpected inside a RhoGAP domain, as it was not predicted from either the protein sequence or its three-dimensional structure. Phosphoinositide-binding sites in RhoGAP domains have been reported to correspond to polybasic regions, which are located at the unstructured flexible termini of proteins. Solid-state NMR spectroscopy experiments confirm the membrane interaction of the Rgd1p-RhoGAP domain upon the addition of PtdIns(4,5)P2 and indicate a slight membrane destabilization in the presence of the two partners.

The Saccharomyces cerevisiae RhoGAP Rgd1 is phosphorylated by the Aurora B like kinase Ipl1.

Polarized growth of the yeast Saccharomyces cerevisiae depends on different biological processes and requires several signaling pathways. Signaling is mediated through a set of proteins, which include Rho3p and Rho4p GTPases. Although these two proteins are involved in the control of distinct aspects of polarized growth in yeast, they have a common regulator: the Rgd1 RhoGAP protein. Here we demonstrate that Rgd1p is phosphorylated by the Aurora B like kinase Ipl1 and we observe that loss of Ipl1 function leads to a new Rgd1p distribution in a small part of the cell population.

Secretory pathway-dependent localization of the Saccharomyces cerevisiae Rho GTPase-activating protein Rgd1p at growth sites.

Establishment and maintenance of cell polarity in eukaryotes depends upon the regulation of Rho GTPases. In Saccharomyces cerevisiae, the Rho GTPase activating protein (RhoGAP) Rgd1p stimulates the GTPase activities of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively. Consistent with the distribution of Rho3p and Rho4p, Rgd1p is found mostly in areas of polarized growth during cell cycle progression. Rgd1p was mislocalized in mutants specifically altered for Golgi apparatus-based phosphatidylinositol 4-P [PtdIns(4)P] synthesis and for PtdIns(4,5)P(2) production at the plasma membrane. Analysis of Rgd1p distribution in different membrane-trafficking mutants suggested that Rgd1p was delivered to growth sites via the secretory pathway. Rgd1p may associate with post-Golgi vesicles by binding to PtdIns(4)P and then be transported by secretory vesicles to the plasma membrane. In agreement, we show that Rgd1p coimmunoprecipitated and localized with markers specific to secretory vesicles and cofractionated with a plasma membrane marker. Moreover, in vivo imaging revealed that Rgd1p was transported in an anterograde manner from the mother cell to the daughter cell in a vectoral manner. Our data indicate that secretory vesicles are involved in the delivery of RhoGAP Rgd1p to the bud tip and bud neck.

Characterization of a Family of Scorpion Toxins Modulating Ca2+-Activated Cl- Current in Vascular Myocytes.

The pharmacology of calcium-activated chloride current is not well developed. Peptides from scorpion venom present potent pharmacological actions on ionic conductance used to characterize the function of channels but can also be helpful to develop organic pharmacological tools. Using electrophysiological recording coupled with calcium measurement, we tested the potent effect of peptides extracted from Leuirus quinquestratus quinquestratus venom on the calcium-activated chloride current expressed in smooth muscle cells freshly dissociated from rat portal veins. We identified one peptide which selectively inhibited the chloride conductance without effects on either calcium signaling or calcium and potassium currents expressed in this cell type. The synthetic peptide had the same affinity, but the chemical modification of the amino acid sequence altered the efficiency to inhibit the calcium-activated chloride conductance.

Evidence for specific interaction between the RhoGAP domain from the yeast Rgd1 protein and phosphoinositides.

The Rho GTPase activating protein Rgd1 increases the GTPase activity of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively, in the budding yeast Saccharomyces cerevisiae. Rgd1p is a member of the F-BAR family conserved in eukaryotes; indeed, in addition to the C-terminal RhoGAP domain Rgd1p possesses an F-BAR domain at its N-terminus. Phosphoinositides discriminate between the GTPase activities of Rho3p and Rho4p through Rgd1p and specifically stimulate the RhoGAP activity of Rgd1p on Rho4p. Determining specific interactions and resolving the structure of Rgd1p should provide insight into the functioning of this family of protein. We report the preparation of highly pure and functional RhoGAP domain of Rgd1 RhoGAP domain using a high yield expression procedure. By gel filtration and circular dichroïsm we provide the first evidences for a specific interaction between a RhoGAP domain (the RhoGAP domain of Rgd1p) and phosphoinositides.

The transport of high amounts of vascular endothelial growth factor by blood platelets underlines their potential contribution in systemic sclerosis angiogenesis.

OBJECTIVES: Altered angiogenesis is a characteristic feature in SSc and remains ill-understood. VEGF is believed to play a central role. Serum VEGF is elevated in SSc patients but questions remain concerning the source of circulating VEGF. Here we investigated platelet activation and the role of platelets as a source of VEGF and other angiogenic mediators in this disease. METHODS: A cohort of 40 patients with SSc was included. Age- and sex-matched healthy subjects and subjects presenting a primary RP were included as controls. Platelets were isolated, activated with thrombin and the secretion of VEGF, platelet derived growth factor, homodimeric form BB (PDGF-BB), TGF-beta1 and angiopoietins-1 and -2 measured. Plasma concentrations of these mediators and the functionality of platelet-derived VEGF were also studied. Platelet activation was assayed by measuring plasma beta-thromboglobulin and expression of P-selectin on platelets. The effect of iloprost on VEGF secretion by platelets was studied. RESULTS: Platelets from SSc patients, in contrast to controls, secreted large amounts of VEGF when activated, but not PDGF-BB, TGF-beta1 or angiopoietins. Increased expression of membrane P-selectin confirmed platelet activation in the patients. Iloprost inhibited VEGF secretion by platelets both in vivo and in vitro, through inhibition of platelet activation. CONCLUSIONS: Platelets transport high levels of VEGF in SSc. They may contribute to circulating VEGF because of ongoing activation in the course of the disease. If activated at the contact of injured endothelium, platelets may be important in the altered angiogenesis associated with the disease through the secretion of high levels of VEGF.

Assignment of 1H, 13C and 15N resonances and secondary structure of the Rgd1-RhoGAP domain.

The protein Rgd1 is involved in the regulation of cytoskeleton formation and in signalling pathways that control cell polarity and growth in Saccharomyces cerevisiae. Rgd1p is composed of a F-BAR domain required for membrane binding and a RhoGAP domain responsible for activating Rho3p and Rho4p, two GTPases respectively involved in bud growth and cytokinesis. Rgd1p is recruited to the membrane through interactions with phosphoinositide lipids, which bind the two isolated domains and stimulate the RhoGAP activity on Rho4p. As previously shown by crystallography, the membrane-binding F-BAR domain contains a conserved inositol phosphate binding site, which explains the preferential binding of phosphoinositides. In contrast, RhoGAP domains are not expected to bind lipids. In order to unravel this puzzling feature, we solved the three-dimensional structure of the isolated protein and found a cryptic phosphoinositide binding site involving non conserved residues (Martinez et al. 2017). The assignment of the resonances and secondary structure of Rgd1-RhoGAP (aa 450-666) is presented here.

Mapacalcine protects mouse neurons against hypoxia by blocking cell calcium overload.

Stroke is one of a major cause of death and adult disability. Despite intense researches, treatment for stroke remains reduced to fibrinolysis, a technique useful for less than 10% of patients. Finding molecules able to treat or at least to decrease the deleterious consequences of stroke is an urgent need. Here, we showed that mapacalcine, a homodimeric peptide purified from the marine sponge Cliona vastifica, is able to protect mouse cortical neurons against hypoxia. We have also identified a subtype of L-type calcium channel as a target for mapacalcine and we showed that the channel has to be open for mapacalcine binding. The two main L-type subunits at the brain level are CaV1.3 and CaV1.2 subunits but mapacalcine was unable to block these calcium channels.Mapacalcine did not interfere with N-, P/Q- and R-type calcium channels. The protective effect was studied by measuring internal calcium level variation triggered by Oxygen Glucose Deprivation protocol, which mimics stroke, or glutamate stimulation. We showed that NMDA/AMPA receptors are not involved in the mapacalcine protection. The protective effect was confirmed by measuring the cell survival rate after Oxygen Glucose Deprivation condition. Our data indicate that mapacalcine is a promising molecule for stroke treatment.

Overexpression and role of the ATPase and putative DNA helicase RuvB-like 2 in human hepatocellular carcinoma.

UNLABELLED: Using a proteomic analysis of human hepatocellular carcinoma (HCC), we identified the overexpression in 4 tumors of RuvB-like 2 (RUVBL2), an ATPase and putative DNA helicase known to interact with beta-catenin and cellular v-myc myelocytomatosis viral oncogene homolog (c-myc). RUVBL2 expression was further analyzed in tumors with quantitative reverse-transcription polymerase chain reaction analysis and immunohistochemistry; in addition, RUVBL2 expression in a HuH7 cell line was silenced by small interfering RNA or increased with a lentiviral vector. RUVBL2 messenger RNA overexpression was confirmed in 72 of 96 HCC cases, and it was associated with poorly differentiated tumors (P = 0.02) and a poor prognosis (P = 0.02) but not with beta-catenin mutations or c-myc levels. Although RUVBL2 was strictly nuclear in normal hepatocytes, tumoral hepatocytes exhibited additional cytoplasmic staining. There was no mutation in the coding sequence of RUVBL2 in 10 sequenced cases. Silencing RUVBL2 in HuH7 HCC cells reduced cell growth (P < 0.001) and increased apoptosis, as shown by DNA fragmentation (P < 0.001) and caspase 3 activity (P < 0.005). This was associated with an increased expression of several proapoptotic genes and with an increased conformational activation of Bak-1 and Bax. On the other hand, HuH7 cells with an overexpression of RUVBL2 grew better in soft agar (P < 0.03), had increased resistance to C2 ceramide-induced apoptosis (P < 0.001), and gave rise to significantly larger tumors when injected into immunodeficient Rag2/gammac mice (P = 0.016). CONCLUSION: RUVBL2 is overexpressed in a large majority of HCCs. RUVBL2 overexpression enhances tumorigenicity, and RUVBL2 is required for tumor cell viability. These results argue for a major role of RUVBL2 in liver carcinogenesis.

Mapacalcine specifically blocks hypoxia-induced calcium influx in rat hepatocytes.

Post ischaemic cell calcium invasion has been described as one of the main causes of graft failure. Protective effects of calcium antagonists have been investigated but are not convincing and their mechanisms of action remain unclear. In this work we tested the protective effect of a new calcium inhibitor described to block a calcium current insensitive to all known calcium blockers. Specific mapacalcine receptors were first characterized on rat hepatocytes membranes using the 125I-labeled mapacalcine. 45Ca fluxes were then measured on cultured hepatocytes submitted (or not) to an hypoxic period. The action of mapacalcine was investigated on the ischaemia-induced calcium influx. We demonstrate here that: (a) there are specific receptors for mapacalcine in rat hepatocytes; (b) Mapacalcine is able to specifically block ischaemia-induced calcium influx with an IC50 of 0.3 micro m and does not significantly interact with the basal calcium flux. Our work demonstrates that the mapacalcine receptor is a cellular structure directly involved in the phenomenon of postischaemic cell invasion by calcium. Specific block of ischaemia-induced Ca2+ influx by mapacalcine suggests that the development of a panel of pharmacological drugs acting on this receptor could lead to the discovery of therapeutic agents able to protect cells against one of the events responsible for organ failure after transplantation or simply after an ischaemic period. Moreover, identification of the cellular protein which binds mapacalcine may become an important step in the research of mechanisms involved in postischaemic cell invasion by calcium.