Inhibition of neuraminidase-1 sialidase activity by interfering peptides impairs insulin receptor activity in vitro and glucose homeostasis in vivo.

Neuraminidases (NEUs) also called sialidases are glycosidases which catalyze the removal of terminal sialic acid residues from glycoproteins, glycolipids, and oligosaccharides. Mammalian NEU-1 participates in regulation of cell surface receptors such as insulin receptor (IR), epithelial growth factor receptor, low-density lipoprotein receptor, and toll-like receptor 4. At the plasma membrane, NEU-1 can be associated with the elastin-binding protein and the carboxypeptidase protective protein/cathepsin A to constitute the elastin receptor complex. In this complex, NEU-1 is essential for elastogenesis, signal transduction through this receptor and for biological effects of the elastin-derived peptides on atherosclerosis, thrombosis, insulin resistance, nonalcoholic steatohepatitis, and cancers. This is why research teams are developing inhibitors targeting this sialidase. Previously, we developed interfering peptides to inhibit the dimerization and the activation of NEU-1. In this study, we investigated the effects of these peptides on IR activation in vitro and in vivo. Using cellular overexpression and endogenous expression models of NEU-1 and IR (COS-7 and HepG2 cells, respectively), we have shown that interfering peptides inhibit NEU-1 dimerization and sialidase activity which results in a reduction of IR phosphorylation. These results demonstrated that NEU-1 positively regulates IR phosphorylation and activation in our conditions. In vivo, biodistribution study showed that interfering peptides are well distributed in mice. Treatment of C57Bl/6 mice during 8 weeks with interfering peptides induces a hyperglycemic effect in our experimental conditions. Altogether, we report here that inhibition of NEU-1 sialidase activity by interfering peptides decreases IR activity in vitro and glucose homeostasis in vivo.

Sialic acids cleavage induced by elastin-derived peptides impairs the interaction between insulin and its receptor in adipocytes 3T3-L1.

The insulin receptor (IR) plays an important role in insulin signal transduction, the defect of which is believed to be the root cause of type 2 diabetes. In 3T3-L1 adipocytes as in other cell types, the mature IR is a heterotetrameric cell surface glycoprotein composed of two α subunits and two ß subunits. Our objective in our study, is to understand how the desialylation of N-glycan chains, induced by elastin-derived peptides, plays a major role in the function of the IR. Using the 3T3-L1 adipocyte line, we show that removal of the sialic acid from N-glycan chains (N893 and N908), induced by the elastin receptor complex (ERC) and elastin derived-peptides (EDPs), leads to a decrease in the autophosphorylation activity of the insulin receptor. We demonstrate by molecular dynamics approaches that the absence of sialic acids on one of these two sites is sufficient to generate local and general modifications of the structure of the IR. Biochemical approaches highlight a decrease in the interaction between insulin and its receptor when ERC sialidase activity is induced by EDPs. Therefore, desialylation by EDPs is synonymous with a decrease of IR sensitivity in adipocytes and could thus be a potential source of insulin resistance associated with diabetic conditions.

Neuraminidase-1: A Sialidase Involved in the Development of Cancers and Metabolic Diseases.

Sialidases or neuraminidases (NEU) are glycosidases which cleave terminal sialic acid residues from glycoproteins, glycolipids and oligosaccharides. Four types of mammalian sialidases, which are encoded by different genes, have been described with distinct substrate specificity and subcellular localization: NEU-1, NEU-2, NEU-3 and NEU-4. Among them, NEU-1 regulates many membrane receptors through desialylation which results in either the activation or inhibition of these receptors. At the plasma membrane, NEU-1 also associates with the elastin-binding protein and the carboxypeptidase protective protein/cathepsin A to form the elastin receptor complex. The activation of NEU-1 is required for elastogenesis and signal transduction through this receptor, and this is responsible for the biological effects that are mediated by the elastin-derived peptides (EDP) on obesity, insulin resistance and non-alcoholic fatty liver diseases. Furthermore, NEU-1 expression is upregulated in hepatocellular cancer at the mRNA and protein levels in patients, and this sialidase regulates the hepatocellular cancer cells' proliferation and migration. The implication of NEU-1 in other cancer types has also been shown notably in the development of pancreatic carcinoma and breast cancer. Altogether, these data indicate that NEU-1 plays a key role not only in metabolic disorders, but also in the development of several cancers which make NEU-1 a pharmacological target of high potential in these physiopathological contexts.

The Elastin Receptor Complex: An Emerging Therapeutic Target Against Age-Related Vascular Diseases.

The incidence of cardiovascular diseases is increasing worldwide with the growing aging of the population. Biological aging has major influence on the vascular tree and is associated with critical changes in the morphology and function of the arterial wall together with an extensive remodeling of the vascular extracellular matrix. Elastic fibers fragmentation and release of elastin degradation products, also known as elastin-derived peptides (EDPs), are typical hallmarks of aged conduit arteries. Along with the direct consequences of elastin fragmentation on the mechanical properties of arteries, the release of EDPs has been shown to modulate the development and/or progression of diverse vascular and metabolic diseases including atherosclerosis, thrombosis, type 2 diabetes and nonalcoholic steatohepatitis. Most of the biological effects mediated by these bioactive peptides are due to a peculiar membrane receptor called elastin receptor complex (ERC). This heterotrimeric receptor contains a peripheral protein called elastin-binding protein, the protective protein/cathepsin A, and a transmembrane sialidase, the neuraminidase-1 (NEU1). In this review, after an introductive part on the consequences of aging on the vasculature and the release of EDPs, we describe the composition of the ERC, the signaling pathways triggered by this receptor, and the current pharmacological strategies targeting ERC activation. Finally, we present and discuss new regulatory functions that have emerged over the last few years for the ERC through desialylation of membrane glycoproteins by NEU1, and its potential implication in receptor transactivation.

Apelin expression deficiency in mice contributes to vascular stiffening by extracellular matrix remodeling of the aortic wall.

Numerous recent studies have shown that in the continuum of cardiovascular diseases, the measurement of arterial stiffness has powerful predictive value in cardiovascular risk and mortality and that this value is independent of other conventional risk factors, such as age, cholesterol levels, diabetes, smoking, or average blood pressure. Vascular stiffening is often the main cause of arterial hypertension (AHT), which is common in the presence of obesity. However, the mechanisms leading to vascular stiffening, as well as preventive factors, remain unclear. The aim of the present study was to investigate the consequences of apelin deficiency on the vascular stiffening and wall remodeling of aorta in mice. This factor freed by visceral adipose tissue, is known for its homeostasic role in lipid and vascular metabolisms, or again in inflammation. We compared the level of metabolic markers, inflammation of white adipose tissue (WAT), and aortic wall remodeling from functional and structural approaches in apelin-deficient and wild-type (WT) mice. Apelin-deficient mice were generated by knockout of the apelin gene (APL-KO). From 8 mice by groups, aortic stiffness was analyzed by pulse wave velocity measurements and by characterizations of collagen and elastic fibers. Mann-Whitney statistical test determined the significant data (p < 5%) between groups. The APL-KO mice developed inflammation, which was associated with significant remodeling of visceral WAT, such as neutrophil elastase and cathepsin S expressions. In vitro, cathepsin S activity was detected in conditioned medium prepared from adipose tissue of the APL-KO mice, and cathepsin S activity induced high fragmentations of elastic fiber of wild-type aorta, suggesting that the WAT secretome could play a major role in vascular stiffening. In vivo, remodeling of the extracellular matrix (ECM), such as collagen accumulation and elastolysis, was observed in the aortic walls of the APL-KO mice, with the latter associated with high cathepsin S activity. In addition, pulse wave velocity (PWV) and AHT were increased in the APL-KO mice. The latter could explain aortic wall remodeling in the APL-KO mice. The absence of apelin expression, particularly in WAT, modified the adipocyte secretome and facilitated remodeling of the ECM of the aortic wall. Thus, elastolysis of elastic fibers and collagen accumulation contributed to vascular stiffening and AHT. Therefore, apelin expression could be a major element to preserve vascular homeostasis.

Identification and Evaluation of New Potential Inhibitors of Human Neuraminidase 1 Extracted from Olyra latifolia L.: A Preliminary Study.

Sialidases, also called neuraminidases, are involved in several human pathologies such as neurodegenerative disorders, cancers, as well as infectious and cardiovascular diseases. Several studies have shown that neuraminidases, such as neuraminidase 1 (NEU-1), may be promising pharmacological targets. Therefore, the discovery of new selective inhibitors of NEU-1 are necessary to better understand the biological functions of this sialidase. In the present study, we describe the isolation and characterization of nine known compounds from Olyra latifolia L. leaves. This plant, known to have several therapeutic properties, belongs to the family of Poaceae and is found in the neotropics and in tropical Africa and Madagascar. Among the purified compounds, feddeiketone B, 2,3-dihydroxy-1-(4-hydroxy-3,5-diméthoxyphényl)-l-propanone, and syringylglycerol were shown to present structural analogy with DANA, and their effects on membrane NEU-1 sialidase activity were evaluated. Our results show that they possess inhibitory effects against NEU-1-mediated sialidase activity at the plasma membrane. In conclusion, we identified new natural bioactive molecules extracted from Olyra latifolia as inhibitors of human NEU-1 of strong interest to elucidate the biological functions of this sialidase and to target this protein involved in several pathophysiological contexts.

Adverse Effects of Oseltamivir Phosphate Therapy on the Liver of LDLR-/- Mice Without Any Benefit on Atherosclerosis and Thrombosis.

ABSTRACT: Desialylation, governed by sialidases or neuraminidases, is strongly implicated in a wide range of human disorders, and accumulative data show that inhibition of neuraminidases, such as neuraminidases 1 sialidase, may be useful for managing atherosclerosis. Several studies have reported promising effects of oseltamivir phosphate, a widely used anti-influenza sialidase inhibitor, on human cancer cells, inflammation, and insulin resistance. In this study, we evaluated the effects of oseltamivir phosphate on atherosclerosis and thrombosis and potential liver toxicity in LDLR-/- mice fed with high-fat diet. Our results showed that oseltamivir phosphate significantly decreased plasma levels of LDL cholesterol and elastin fragmentation in aorta. However, no effect was observed on both atherosclerotic plaque size in aortic roots and chemically induced thrombosis in carotid arteries. Importantly, oseltamivir phosphate administration had adverse effects on the liver of mice and significantly increased messenger RNA expression levels of F4/80, interleukin-1ß, transforming growth factor-ß1, matrix metalloproteinase-12, and collagen. Taken together, our findings suggest that oseltamivir phosphate has limited benefits on atherosclerosis and carotid thrombosis and may lead to adverse side effects on the liver with increased inflammation and fibrosis.

Transmembrane Peptides as Inhibitors of Protein-Protein Interactions: An Efficient Strategy to Target Cancer Cells?

Cellular functions are regulated by extracellular signals such as hormones, neurotransmitters, matrix ligands, and other chemical or physical stimuli. Ligand binding on its transmembrane receptor induced cell signaling and the recruitment of several interacting partners to the plasma membrane. Nowadays, it is well-established that the transmembrane domain is not only an anchor of these receptors to the membrane, but it also plays a key role in receptor dimerization and activation. Indeed, interactions between transmembrane helices are associated with specific biological activity of the proteins as cell migration, proliferation, or differentiation. Overexpression or constitutive dimerization (due notably to mutations) of these transmembrane receptors are involved in several physiopathological contexts as cancers. The transmembrane domain of tyrosine kinase receptors as ErbB family proteins (implicated in several cancers as HER2 in breast cancer) or other receptors as Neuropilins has been described these last years as a target to inhibit their dimerization/activation using several strategies. In this review, we will focus on the strategy which consists in using peptides to disturb in a specific manner the interactions between transmembrane domains and the signaling pathways (induced by ligand binding) of these receptors involved in cancer. This approach can be extended to inhibit other transmembrane protein dimerization as neuraminidase-1 (the catalytic subunit of elastin receptor complex), Discoidin Domain Receptor 1 (a tyrosine kinase receptor activated by type I collagen) or G-protein coupled receptors (GPCRs) which are involved in cancer processes.

Transmembrane Peptides as a New Strategy to Inhibit Neuraminidase-1 Activation.

Sialidases, or neuraminidases, are involved in several human disorders such as neurodegenerative, infectious and cardiovascular diseases, and cancers. Accumulative data have shown that inhibition of neuraminidases, such as NEU1 sialidase, may be a promising pharmacological target, and selective inhibitors of NEU1 are therefore needed to better understand the biological functions of this sialidase. In the present study, we designed interfering peptides (IntPep) that target a transmembrane dimerization interface previously identified in human NEU1 that controls its membrane dimerization and sialidase activity. Two complementary strategies were used to deliver the IntPep into cells, either flanked to a TAT sequence or non-tagged for solubilization in detergent micelles. Combined with molecular dynamics simulations and heteronuclear nuclear magnetic resonance (NMR) studies in membrane-mimicking environments, our results show that these IntPep are able to interact with the dimerization interface of human NEU1, to disrupt membrane NEU1 dimerization and to strongly decrease its sialidase activity at the plasma membrane. In conclusion, we report here new selective inhibitors of human NEU1 of strong interest to elucidate the biological functions of this sialidase.

Role of elastin peptides and elastin receptor complex in metabolic and cardiovascular diseases.

The Cardiovascular Continuum describes a sequence of events from cardiovascular risk factors to end-stage heart disease. It includes conventional pathologies affecting cardiovascular functions such as hypertension, atherosclerosis or thrombosis and was traditionally considered from the metabolic point of view. This Cardiovascular Continuum, originally described by Dzau and Braunwald, was extended by O'Rourke to consider also the crucial role played by degradation of elastic fibers, occurring during aging, in the appearance of vascular stiffness, another deleterious risk factor of the continuum. However, the involvement of the elastin degradation products, named elastin-derived peptides, to the Cardiovascular Continuum progression has not been considered before. Data from our laboratory and others clearly showed that these bioactive peptides are central regulators of this continuum, thereby amplifying appearance and evolution of cardiovascular risk factors such as diabetes or hypertension, of vascular alterations such as atherothrombosis and calcification, but also nonalcoholic fatty liver disease and nonalcoholic steatohepatitis. The Elastin Receptor Complex has been shown to be a crucial actor in these processes. We propose here the participation of these elastin-derived peptides and of the Elastin Receptor Complex in these events, and introduce a revisited Cardiovascular Continuum based on their involvement, for which elastin-based pharmacological strategies could have a strong impact in the future.

Receptor for advanced glycation end products: a key molecule in the genesis of chronic kidney disease vascular calcification and a potential modulator of sodium phosphate co-transporter PIT-1 expression.

BACKGROUND: Chronic kidney disease (CKD) is associated with increased cardiovascular mortality, frequent vascular calcification (VC) and accumulation of uraemic toxins. Advanced glycation end products and S100 proteins interact with the receptor for advanced glycation end products (RAGE). In the present work, we aimed to investigate the role(s) of RAGE in the CKD-VC process. METHODS: Apoe-/- or Apoe-/-Ager (RAGE)-/- male mice were assigned to CKD or sham-operated groups. A high-phosphate diet was given to a subgroup of Apoe-/-and Apoe-/-Ager-/- CKD mice. Primary cultures of Ager+/+ and Ager-/- vascular smooth muscle cells (VSMCs) were established and stimulated with either vehicle, inorganic phosphate (Pi) or RAGE ligands (S100A12; 20 µM). RESULTS: After 12 weeks of CKD we observed a significant increase in RAGE ligand (AGE and S100 proteins) concentrations in the serum of CKD Apoe-/- mice. Ager messenger RNA (mRNA) levels were 4-fold higher in CKD vessels of Apoe-/- mice. CKD Apoe-/- but not CKD Apoe-/- or Ager-/- mice displayed a marked increase in the VC surface area. Similar trends were found in the high-phosphate diet condition. mRNA levels of Runx2 significantly increased in the Apoe-/- CKD group. In vitro, stimulation of Ager+/+VSMCs with Pi or S100A12 induced mineralization and osteoblast transformation, and this was inhibited by phosphonoformic acid (Pi co-transporters inhibitor) and Ager deletion. In vivo and in vitro RAGE was necessary for regulation of the expression of Pit-1, at least in part through production of reactive oxygen species. CONCLUSION: RAGE, through the modulation of Pit-1 expression, is a key molecule in the genesis of VC.

Identification of CD36 as a new interaction partner of membrane NEU1: potential implication in the pro-atherogenic effects of the elastin receptor complex.

In addition to its critical role in lysosomes for catabolism of sialoglycoconjugates, NEU1 is expressed at the plasma membrane and regulates a myriad of receptors by desialylation, playing a key role in many pathophysiological processes. Here, we developed a proteomic approach dedicated to the purification and identification by LC-MS/MS of plasma membrane NEU1 interaction partners in human macrophages. Already known interaction partners were identified as well as several new candidates such as the class B scavenger receptor CD36. Interaction between NEU1 and CD36 was confirmed by complementary approaches. We showed that elastin-derived peptides (EDP) desialylate CD36 and that this effect was blocked by the V14 peptide, which blocks the interaction between bioactive EDP and the elastin receptor complex (ERC). Importantly, EDP also increased the uptake of oxidized LDL by macrophages that is blocked by both the V14 peptide and the sialidase inhibitor 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (DANA). These results demonstrate, for the first time, that binding of EDP to the ERC indirectly modulates CD36 sialylation level and regulates oxidized LDL uptake through this sialidase. These effects could contribute to the previously reported proatherogenic role of EDP and add a new dimension in the regulation of biological processes through NEU1.

Production of Elastin-Derived Peptides Contributes to the Development of Nonalcoholic Steatohepatitis.

Affecting more than 30% of the Western population, nonalcoholic fatty liver disease (NAFLD) is the most common liver disease and can lead to multiple complications, including nonalcoholic steatohepatitis (NASH), cancer, hypertension, and atherosclerosis. Insulin resistance and obesity are described as potential causes of NAFLD. However, we surmised that factors such as extracellular matrix remodeling of large blood vessels, skin, or lungs may also participate in the progression of liver diseases. We studied the effects of elastin-derived peptides (EDPs), biomarkers of aging, on NAFLD progression. We evaluated the consequences of EDP accumulation in mice and of elastin receptor complex (ERC) activation on lipid storage in hepatocytes, inflammation, and fibrosis development. The accumulation of EDPs induces hepatic lipogenesis (i.e., SREBP1c and ACC), inflammation (i.e., Kupffer cells, IL-1ß, and TGF-ß), and fibrosis (collagen and elastin expression). These effects are induced by inhibition of the LKB1-AMPK pathway by ERC activation. In addition, pharmacological inhibitors of EDPs demonstrate that this EDP-driven lipogenesis and fibrosis relies on engagement of the ERC. Our data reveal a major role of EDPs in the development of NASH, and they provide new clues for understanding the relationship between NAFLD and vascular aging.

Systematic protein-protein interaction mapping for clinically relevant human GPCRs.

G-protein-coupled receptors (GPCRs) are the largest family of integral membrane receptors with key roles in regulating signaling pathways targeted by therapeutics, but are difficult to study using existing proteomics technologies due to their complex biochemical features. To obtain a global view of GPCR-mediated signaling and to identify novel components of their pathways, we used a modified membrane yeast two-hybrid (MYTH) approach and identified interacting partners for 48 selected full-length human ligand-unoccupied GPCRs in their native membrane environment. The resulting GPCR interactome connects 686 proteins by 987 unique interactions, including 299 membrane proteins involved in a diverse range of cellular functions. To demonstrate the biological relevance of the GPCR interactome, we validated novel interactions of the GPR37, serotonin 5-HT4d, and adenosine ADORA2A receptors. Our data represent the first large-scale interactome mapping for human GPCRs and provide a valuable resource for the analysis of signaling pathways involving this druggable family of integral membrane proteins.

The Elastin Receptor Complex: A Unique Matricellular Receptor with High Anti-tumoral Potential.

Elastin, one of the longest-lived proteins, confers elasticity to tissues with high mechanical constraints. During aging or pathophysiological conditions such as cancer progression, this insoluble polymer of tropoelastin undergoes an important degradation leading to the release of bioactive elastin-derived peptides (EDPs), named elastokines. EDP exhibit several biological functions able to drive tumor development by regulating cell proliferation, invasion, survival, angiogenesis, and matrix metalloproteinase expression in various tumor and stromal cells. Although, several receptors have been suggested to bind elastokines (αvß3 and αvß5 integrins, galectin-3), their main receptor remains the elastin receptor complex (ERC). This heterotrimer comprises a peripheral subunit, named elastin binding protein (EBP), associated to the protective protein/cathepsin A (PPCA). The latter is bound to a membrane-associated protein called Neuraminidase-1 (Neu-1). The pro-tumoral effects of elastokines have been linked to their binding onto EBP. Additionally, Neu-1 sialidase activity is essential for their signal transduction. Consistently, EDP-EBP interaction and Neu-1 activity emerge as original anti-tumoral targets. Interestingly, besides its direct involvement in cancer progression, the ERC also regulates diabetes outcome and thrombosis, an important risk factor for cancer development and a vascular process highly increased in patients suffering from cancer. In this review, we will describe ERC and elastokines involvement in cancer development suggesting that this unique receptor would be a promising therapeutic target. We will also discuss the pharmacological concepts aiming at blocking its pro-tumoral activities. Finally, its emerging role in cancer-associated complications and pathologies such as diabetes and thrombotic events will be also considered.

Elastin-derived peptides potentiate atherosclerosis through the immune Neu1-PI3Kγ pathway.

AIMS: Elastin is degraded during vascular ageing and its products, elastin-derived peptides (EP), are present in the human blood circulation. EP binds to the elastin receptor complex (ERC) at the cell surface, composed of elastin-binding protein (EBP), a cathepsin A and a neuraminidase 1. Some in vitro functions have clearly been attributed to this binding, but the in vivo implications for arterial diseases have never been clearly investigated. METHODS AND RESULTS: Here, we demonstrate that chronic doses of EP injected into mouse models of atherosclerosis increase atherosclerotic plaque size formation. Similar effects were observed following an injection of a VGVAPG peptide, suggesting that the ERC mediates these effects. The absence of phosphoinositide 3-kinase γ (PI3Kγ) in bone marrow-derived cells prevented EP-induced atherosclerosis development, demonstrating that PI3Kγ drive EP-induced arterial lesions. Accordingly, in vitro studies showed that PI3Kγ was required for EP-induced monocyte migration and ROS production and that this effect was dependent upon neuraminidase activity. Finally, we showed that degradation of elastic lamellae in LDLR(-/-) mice fed an atherogenic diet correlated with atherosclerotic plaque formation. At the same time, the absence of the cathepsin A-neuraminidase 1 complex in cells of the haematopoietic lineage abolished atheroma plaque size progression and decreased leucocytes infiltration, clearly demonstrating the role of this complex in atherogenesis and suggesting the involvement of endogenous EP. CONCLUSION: Altogether, this work identifies EP as an enhancer of atherogenesis and defines the Neuraminidase 1/PI3Kγ signalling pathway as a key mediator of this function in vitro and in vivo.

Interaction between the elastin peptide VGVAPG and human elastin binding protein.

The elastin binding protein (EBP), a spliced variant of lysosomal ß-galactosidase, is the primary receptor of elastin peptides that have been linked to emphysema, aneurysm and cancer progression. The sequences recognized by EBP share the XGXXPG consensus pattern found in numerous matrix proteins, notably in elastin where the VGVAPG motif is repeated. To delineate the elastin binding site of human EBP, we built a homology model of this protein and docked VGVAPG on its surface. Analysis of this model suggested that Gln-97 and Asp-98 were required for interaction with VGVAPG because they contribute to the definition of a pocket thought to represent the elastin binding site of EBP. Additionally, we proposed that Leu-103, Arg-107, and Glu-137 were essential residues because they could interact with VGVAPG itself. Site-directed mutagenesis experiments at these key positions validated our model. This work therefore provides the first structural data concerning the interaction of the VGVAPG with its cognate receptor. The present structural data should now allow the development of EBP-specific antagonists.

Platelet type III collagen binding protein (TIIICBP) presents high biochemical and functional similarities with kindlin-3.

Type III collagen binding protein (TIIICBP) was previously described as a platelet membrane protein that recognizes the KOGEOGPK peptide sequence within type III collagen. In order to better characterize this protein, we performed different approaches including mass spectrometry sequencing and functional experiments. This study leads to identify high biochemical and functional similarities between TIIICBP and kindlin-3, a member of a family of focal adhesion proteins. Indeed, mass spectrometry surveys indicated that TIIICBP contains several peptides identical to kindlin-3, covering 41% of the amino acid sequence. Polyclonal antibodies raised against a kindlin-3 specific N-terminal sequence, recognized and immunoprecipitated TIIICBP from platelet lysates. Electron microscopy and flow cytometry experiments showed that kindlin-3, as well as TIIICBP, were present associated to platelet membrane and a translocation of cytosolic kindlin-3 to the platelet membrane was observed after platelet activation. Similarly to anti-TIIICBP antibodies and the KOGEOGPK peptide, anti-kindlin-3 antibodies inhibited platelet interactions with type III collagen under flow conditions and slowed down platelet aggregation induced by glycoprotein VI agonists; e.g. collagen-related peptides and convulxin. In addition, the anti-kindlin-3 antibody inhibited platelet aggregation induced by low - but not high - doses of ADP or thrombin which depends on α(IIb)ß(3) integrin function. In conclusion, our results show that the peptides identified by mass spectrometry from purified TIIICBP correspond to the kindlin-3 protein and demonstrate biochemical and functional similarities between TIIICBP and kindlin-3, strengthening a key role for TIIICBP/kindlin-3 in platelet interactions with collagen by cooperating with glycoprotein VI activation and integrin clustering in focal adhesion complexes.

Peptide affinity purification for the isolation and identification of GPCR-associated protein complexes.

Protein networks and their dynamic regulation play a fundamental role in biological systems. Seven transmembrane-spanning G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors controlling the flow of information from the extracellular environment into cells by inducing intracellular signaling pathways. Several GPCR-associated protein complexes (GAPCs), particularly those binding to the intracellular carboxyl-terminus (C-terminus), have been identified over the last 20 years. Recent optimizations in purification protocols and advances in mass spectrometry-based protein identification techniques have considerably accelerated the identification of GAPCs. We will concentrate here on a description of the latest version of the peptide affinity purification approach dedicated to the purification of GAPCs interacting with GPCR C-termini or any other soluble receptor subdomain.

Requirements for membrane attack complex formation and anaphylatoxins binding to collagen-activated platelets.

BACKGROUND: The activation of complement during platelet activation is incompletely understood. OBJECTIVES: We sought to explore the formation of C5b-9 and anaphylatoxins binding to collagen-activated platelets. METHODS: C5b-9, anaphylatoxins C3a, C4a and C5a, and anaphylatoxin receptors C3aR1 and C5aR were measured by flow cytometry and/or confocal microscopy. Platelet microparticles were quantified by flow cytometry, and their C5b-9 content was determined by western blot analyses. In all experiments, sodium citrate was used for blood anticoagulation. RESULTS: C5b-9 rapidly formed on the platelet surface following activation with collagen, TRAP, ADP or A23187, but was surprisingly restricted to a subset of platelets (1 to 15%) independently of P-selectin or phosphatidylserine exposure. Following collagen activation, C5b-9-positive platelets in thrombi were found associated with collagen fibres. C5b-9 formation was obliterated by Mg(2+)-EGTA and significantly reduced by the thrombin inhibitor hirudin (-37%, p<0.05), but was unaffected by chondroitinase, compstatin, SCH79797 (PAR-1 inhibitor), or in the PRP of a MBL-deficient donor. Compstatin and Mg(2+)-EGTA, but not hirudin, SCH79797 or chondroitinase, inhibited the formation of collagen-induced microparticles (-71% and -44%, respectively, p<0.04). These microparticles contained greater amounts of C5b-9 compared with the other agonists. Platelet activation by collagen or convulxin resulted in the strong binding of anaphylatoxins and the exposure of receptors C3aR1 and C5aR (CD88) on their surface. CONCLUSIONS: C5b-9 formation on collagen-activated platelets is i) partially controlled by thrombin, ii) restricted to a subset of platelets, and iii) can occur without P-selectin expression or phosphatidylserine exposure. Activated platelets bind anaphylatoxins on their surface and express C3a and C5a receptors, which may contribute to the localization of inflammatory processes during thrombosis.