Autoantibodies to Endothelial Cell Surface ATP Synthase, the Endogenous Receptor for Hsp60, Might Play a Pathogenic Role in Vasculatides

International audienceBACKGROUND: Heat shock protein (hsp) 60 that provides "danger signal" binds to the surface of resting endothelial cells (EC) but its receptor has not yet been characterized. In mitochondria, hsp60 specifically associates with adenosine triphosphate (ATP) synthase. We therefore examined the possible interaction between hsp60 and ATP synthase on EC surface. METHODOLOGY/PRINCIPAL FINDINGS: Using Far Western blot approach, co-immunoprecipitation studies and surface plasmon resonance analyses, we demonstrated that hsp60 binds to the β-subunit of ATP synthase. As a cell surface-expressed molecule, ATP synthase is potentially targeted by anti-EC-antibodies (AECAs) found in the sera of patients suffering vasculitides. Based on enzyme-linked immunosorbent assay and Western blotting techniques with F1-ATP synthase as substrate, we established the presence of anti-ATP synthase antibodies at higher frequency in patients with primary vasculitides (group I) compared with secondary vasculitides (group II). Anti-ATP synthase reactivity from group I patients was restricted to the β-subunit of ATP synthase, whereas those from group II was directed to the α-, β- and γ-subunits. Cell surface ATP synthase regulates intracellular pH (pHi). In low extracellular pH medium, we detected abnormal decreased of EC pHi in the presence of anti-ATP synthase antibodies, irrespective of their fine reactivities. Interestingly, soluble hsp60 abrogated the anti-ATP synthase-induced pHi down-regulation. CONCLUSIONS/SIGNIFICANCE: Our results indicate that ATP synthase is targeted by AECAs on the surface of EC that induce intracellular acidification. Such pathogenic effect in vasculitides can be modulated by hsp60 binding on ATP synthase which preserves ATP synthase activity

Vibrational-rotational structure of the silane molecule in the band of v2+v4 (F2)

In recent years, extensive theoretical studies have been carried out on the silane molecule, namely their vibrational-rotational structure. In this work, we continue our research series and focus on the 28SiD4 isotopologue. The IR-spectrum of the silane molecule was recorded in the range 1250-1450 cm-1 (pentad region) on Bruker IFS 120HR Fourier interferometer. The P, Q, and R branches with Jmax up to 17 were assigned, and spectroscopic constants of the v2+v4 (F2) band were derived for 28SiD4. As a result, a set of spectroscopic parameters was obtained which describe the vibrational-rotational structure of the silane molecule close to the experimental uncertainties

TLR2 is one of the endothelial receptors for beta 2-glycoprotein I.

International audienceDuring the antiphospholipid syndrome, beta2-gpI interacts with phospholipids on endothelial cell (EC) surface to allow the binding of autoantibodies. However, induced-pathogenic intracellular signals suggest that beta2-gpI associates also with a receptor that is still not clearly identified. TLR2 and TLR4 have long been suspected, yet interactions between TLRs and beta2-gpI have never been unequivocally proven. The aim of the study was to identify the TLR directly involved in the binding of beta2-gpI on EC surface. beta2-gpI was not synthesized and secreted by ECs in vitro, but rather taken up from FCS. This uptake occurred through association with TLR2 and TLR4 which partitioned together in the lipid rafts of ECs. After coimmunoprecipitation, mass-spectrometry identification of peptides demonstrated that TLR2, but not TLR4, was implicated in the beta2-gpI retention. These results were further confirmed by plasmon resonance-based studies. Finally, siRNA were used to obtain TLR2-deficient ECs that lost their ability to bind biotinylated beta2-gpI and to trigger downstream phosphorylation of kinases and activation of NFkappaB. TLR4 may upregulate TLR2 expression, thereby contributing to beta2-gpI uptake. However, our data demonstrate that direct binding of beta2-gpI on EC surface occurs through direct interaction with TLR2. Furthermore, signaling for anti-beta2-gpI may be envisioned as a multiprotein complex concentrated in lipid rafts on the EC membrane

Structure-Based Design of Peptidic Inhibitors of the Interaction between CC Chemokine Ligand 5 (CCL5) and Human Neutrophil Peptides 1 (HNP1)

Protein-protein interactions (PPIs) are receiving increasing interest, much sparked by the realization that they represent druggable targets. Recently, we successfully developed a peptidic inhibitor, RRYGTSKYQ ("SKY" peptide), that shows high potential in vitro and in vivo to interrupt a PPI between the platelet-borne chemokine CCL5 and the neutrophil-derived granule protein HNP1. This PPI plays a vital role in monocyte adhesion, representing a key mechanism in acute and chronic inflammatory diseases. Here, we present extensive and detailed computational methods applied to develop the SKY peptide. We combined experimentally determined binding affinities (KD) of several orthologs of CCL5 with HNP1 with in silico studies to identify the most likely heterodimeric CCL5-HNP1 complex which was subsequently used as a starting structure to rationally design peptidic inhibitors. Our method represents a fast and simple approach that can be widely applied to determine other protein-protein complexes and moreover to design inhibitors or stabilizers of protein-protein interactio

Évaluation régionale des connaissances sur les services rendus par la biodiversité au fonctionnement des socio-écosystèmes des paysages herbagers (prairies permanentes et bocages)

Après une brève présentation des caractéristiques générales des socio-écosystèmes des paysages herbagers en Région Nouvelle-Aquitaine (section 1), et un résumé des résultats de synthèse bibliographique des connaissances régionales sur les relations entre biodiversité et socio-écosystèmes herbagers (section 2), ce chapitre propose un état des lieux des connaissances sur le rôle de la biodiversité dans le fonctionne-ment des écosystèmes herbagers et des paysages associés, incluant la fourniture de services écologiques variés (section 3). La section suivante (section 4) introduit la perception des valeurs, marchandes et non marchandes de la biodiversité pour les usagers et acteurs du territoire. Un état des principales pressions environnementales (section 5) permet de dresser les principales menaces qui pèsent sur la biodiversité des paysages herbagers et sur les services qui y sont associés. Les principales ac-tions et instruments des politiques publiques, permettant le maintien ou la restaura-tion de cette biodiversité, sont enfin évoqués (section 6

Évaluation régionale des connaissances sur les services rendus par la biodiversité au fonctionnement des socio-écosystèmes des paysages herbagers (prairies permanentes et bocages)

Après une brève présentation des caractéristiques générales des socio-écosystèmes des paysages herbagers en Région Nouvelle-Aquitaine (section 1), et un résumé des résultats de synthèse bibliographique des connaissances régionales sur les relations entre biodiversité et socio-écosystèmes herbagers (section 2), ce chapitre propose un état des lieux des connaissances sur le rôle de la biodiversité dans le fonctionne-ment des écosystèmes herbagers et des paysages associés, incluant la fourniture de services écologiques variés (section 3). La section suivante (section 4) introduit la perception des valeurs, marchandes et non marchandes de la biodiversité pour les usagers et acteurs du territoire. Un état des principales pressions environnementales (section 5) permet de dresser les principales menaces qui pèsent sur la biodiversité des paysages herbagers et sur les services qui y sont associés. Les principales ac-tions et instruments des politiques publiques, permettant le maintien ou la restaura-tion de cette biodiversité, sont enfin évoqués (section 6

ATP synthase is a receptor for soluble hsp60.

<p><b>A-</b> HUVEC membrane-enriched protein were electrophoresed on bidimensional gels. Left panel: staining with Coomassie blue. Middle panel: WB with anti-hsp60 mAb. Right panel: WB with recombinant hsp60 revealed with anti-hsp60 mAb. <b>B-</b> Mass spectrometry data of proteins bound to hsp60. <b>C</b>- Hsp60 and ATP synthase were co-immunoprecipitated from membrane-enriched protein extracts of EAhy926 cells with anti-ATP synthase and anti-hsp60mAbs, respectively. Proteins were analysed by WB using polyclonal anti-hsp60 or anti-ATP synthase Abs. MW markers are on the left. <b>D-</b> Increasing amounts of ATP synthase were passed over hsp60 immobilized onto a sensor chip. Sensogram of each amount of analyte with substraction of non-specific binding represent resonance units. Specific binding is shown (Δ). <b>E-</b> EAhy926 cells, permeabilized or not, were incubated with a primary anti-ATP synthase Ab revealed by FITC-conjugated anti-Ig Ab, and intracellular as well as cell-surface expression of ATP synthase analyzed by fluorescence microscopy. Propidium iodide was used to visualize nuclei, and isotype control staining performed as negative control.</p

Multireactivity of anti-ATP synthase Ab positive sera.

<p>Sera from group I (WG, PAN, MPA, CSS), group II (SLE, pSS, RA) patients and controls positive for the α-, β-, or γ-subunit of ATP synthase were evaluated for their reactivity against all subunits. Densitometry measurements of reactivity by Western blots are shown. Broken lines show the cut off level for positivities against α-subunit (3.9.10⁵ AU), β-subunit (4.3.10⁵ AU) and γ-subunit (3.9.10⁵ AU), corresponding to the mean+2 SD of control values. WG = Wegener's granulomatosis; PAN = polyarteritis nodosa; MPA = microscopic polyangiitis; CSS = Churg-Strauss syndrome; SLE = systemic lupus erythematosus; pSS = primary Sjögren's syndrome; RA = rheumatoid arthritis.</p