The serine protease domain of MASP-3: enzymatic properties and crystal structure in complex with ecotin.

International audienceMannan-binding lectin (MBL), ficolins and collectin-11 are known to associate with three homologous modular proteases, the MBL-Associated Serine Proteases (MASPs). The crystal structures of the catalytic domains of MASP-1 and MASP-2 have been solved, but the structure of the corresponding domain of MASP-3 remains unknown. A link between mutations in the MASP1/3 gene and the rare autosomal recessive 3MC (Mingarelli, Malpuech, Michels and Carnevale,) syndrome, characterized by various developmental disorders, was discovered recently, revealing an unexpected important role of MASP-3 in early developmental processes. To gain a first insight into the enzymatic and structural properties of MASP-3, a recombinant form of its serine protease (SP) domain was produced and characterized. The amidolytic activity of this domain on fluorescent peptidyl-aminomethylcoumarin substrates was shown to be considerably lower than that of other members of the C1r/C1s/MASP family. The E. coli protease inhibitor ecotin bound to the SP domains of MASP-3 and MASP-2, whereas no significant interaction was detected with MASP-1, C1r and C1s. A tetrameric complex comprising an ecotin dimer and two MASP-3 SP domains was isolated and its crystal structure was solved and refined to 3.2 Å. Analysis of the ecotin/MASP-3 interfaces allows a better understanding of the differential reactivity of the C1r/C1s/MASP protease family members towards ecotin, and comparison of the MASP-3 SP domain structure with those of other trypsin-like proteases yields novel hypotheses accounting for its zymogen-like properties in vitro

Caractérisation fonctionnelle structurale de la protease MASP-3 associée à la MBL et aux ficolines

La voie lectine du complément, élément majeur de la défense innée, est déclenchée par la fixation sur des pathogènes de complexes associant une protéine de reconnaissance oligomérique, MBL ou ficolines, et la protéase MASP-2. MASP-1 et -3, ainsi qu'un fragment tronqué de MASP-2 (MAp19) sont également associés aux protéines de reconnaissance mais leurs fonctions sont inconnues. Le travail porté principalement sur la caractérisation de l'assemblage MBUMASP-3. Il a été montré que les 2 formes oligomériques majeures de lai MBL circulent dans le sérum sous forme trimérique et tétramérique et qu'elles fixent les MASPs de manière comparable avec une stoechiométrie MBL:MASP de 1 :2. Par ailleurs, la structure de la région d'interaction CUB1-EGF-CUB2 de MASP-3 a été résolue à 2,35 A et montre un homodimère disposé de manière tête-bêche dans lequel tous les modules sont localisés dans le même plan. Chaque module EGF possède un site de liaison du Ca2+ stabilisant l'interface du dimère et un 2nd ion Ca2+ est fixé à la partie distale de chaque module CUB1. De plus, un 3ème ion Ca2+, fixé dans chaque module CUB2, stabilise la protéine. La caractérisation de mutants ponctuels a mis en évidence des résidus impliqués dans l'interaction MBUMASP-3: K55 de la MBL et y56, F103, H218 et y225 de MASP-3, ces derniers étant également impliqués dans l'interaction avec les ficolines. Ces résidus définissent 2 sites d'interaction dans les modules CUB1 et CUB2 de chaque monomère stabilisés par un ion Ca2+. Ces sites sont homologues et localisés dans le même plan, ce qui suggère qu'un dimère de MASP-3 interagit avec des sites homologues sur 4 triples hélices de collagène de la MBL ou des ficolines.oligomeric recognition protein, MBL or ficolins, and the MASP-2 protease. MASP-1 and -3 and a truncated form of MASP-2 (Map19) are also associated to the recognition protein but their function is unknown. The aim of this work was to characterize the MBUMASP-3 complex assembly. We provide evidence that the two major forms of MBL in human serum are trimers and tetramers and bind the MASP and MAp19 in similar ways with a 1:2 stoichiometry. ln addition, the x-ray structure of the CUB1-EGF-CUB2 interaction domain of MASP was solved and refined to a resolution of 2.35 À. The structure reveals a head-to-tail homodimer in which ail six modules are located within the same plane. A Ca2+ ion bound to each EGF module stabilizes the dimer interface and a second Ca2+ ion is bound to the distal part of each CUB1 module. Moreover, a third Ca2+ ion is bound to each CUB2 module and stabilizes the protein. Using site-directed mutagenesis, we have identified several residues involved in the interaction between MBL and MASP-3: K55 of MBL and y56, F103, H218 an y225 of MASP-3. These residues are also involved in the interaction with the ficolins. These mutations define two binding sites in the CUB and CUB2 modules of each monomer, both stabilized by Ca2+ ions. The fact that t'Jese sites are homo logo us and located within the same plane strongly suggests that a MASP-3 dimer interacts with homologous interaction sites on 4 collagen triple helices of MBL or ficolins.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Potentially inappropriate prescription of antidepressants in old people: characteristics, associated factors, and impact on mortality

International audienceBACKGROUND:The increasing use of antidepressants (ADs) has raised concerns about their inappropriate use in old people.OBJECTIVE:To examine the prevalence of potentially inappropriate prescribing (PIP) of ADs, their associated factors, and their impact on mortality in a sample of old people in France.METHODS:The analysis used data from the SIPAF study, a cross-sectional study consisting of 2,350 people aged ≥ 70 years. Trained nurses interviewed participants at home between 2008 and 2010. Information was collected concerning socio-demographic and health characteristics, including medication use. The study population consisted of the 318 AD users from the SIPAF study (13.5%). PIP of ADs was defined according to national and international criteria. Factors associated with PIP of ADs were assessed using a multivariate logistic regression model. The influence of PIP of ADs on mortality was assessed using a Cox model (median follow-up 2.8 years).RESULTS:Among the SIPAF study, 71% of AD users were female and the mean age was 84 ± 7 years. Selective serotonin reuptake inhibitors (SSRIs) were the most prescribed ADs (19.8%). We found PIP of ADs in 36.8% of the study population, mainly the co-prescription of diuretics with SSRIs (17.6%) and the prescription of tricyclics (12.9%). PIP of ADs was associated with polypharmacy (aOR5-9 drugs 2. 61, 95% CI 1.11-6.16 and aOR≥10 drugs 2.69, 95% CI 1.06-6.87) and comorbidity (aOR3-4 chronic diseases 2.59, 95%CI 1.04-6.44 and aOR≥5 chronic diseases 2.33, 95%CI 0.94-5.79), and increased the risk of mortality during follow-up (aHR 2.30, 95%CI 1.28-4.12).CONCLUSIONS:This study shows that more than one third of AD prescriptions may be inappropriate in old people. PIP of ADs was related to polypharmacy and comorbidity and increased mortality among AD users

Modular structure of the proteases of the C1r/C1s/MASP family and their role in complement activation.

<p>(<b>A</b>) The generic modular structure of the proteases of this family is shown. MASP-1 and -3, the two proteases coded by the <i>MASP1</i> gene, exhibit a common core (yellow) and unique serine protease (SP) domains. The target of the activation cleavage and the functional domain subdivision are illustrated. The N-terminal interaction domain mediates the binding to the cognate recognition protein and calcium-dependent protease dimerization. In C1r, C1s and MASP-2, the catalytic activity of the C-terminal SP domain is modulated by the preceding CCP1 and CCP2 modules involved in substrate recognition or dimerization <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067962#pone.0067962-BudayovaSpano2" target="_blank">[22]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067962#pone.0067962-Rossi1" target="_blank">[31]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067962#pone.0067962-Kidmose1" target="_blank">[63]</a>. ap: activation peptide. CCP: complement control protein module; CUB: complement C1r/C1s, Uegf, Bmp1 module (<b>B</b>) The activation cascades triggering the lectin and classical complement (C) pathways, and their inhibition by C1-inhibitor, are illustrated. The proteases are associated in large complexes with collagen defence recognition proteins. A possible role for MASP-3 in the activation of the complement alternative pathway needs to be confirmed and its other possible implications outside complement are to be deciphered.</p

Ecotin-induced displacement and proposed conformational equilibrium of MASP-3.

<p>(<b>A</b>) Displacements of the homologous Phe549 and Phe529 in MASP-1 and MASP-2 (in cyan and blue for the free enzyme, respectively) upon SGMI inhibitor binding (left in orange). (<b>B</b>) The position of the homologous Tyr531 in MASP-3 (green) clusters with the ‘displaced’ positions and not with the ‘free’ positions of MASP-1 and MASP-2. The homologous side-chain in free C1r (dark blue) perfectly fits between those of free MASP-1 and MASP-2. It is also the case for C1s (not shown). (<b>C</b>) Scheme illustrating the proposed conformational equilibrium between an inactive E* conformation and the active E conformation observed upon interaction with ecotin. The segment <i>215–217,</i> which collapses in the substrate-binding site in the E* or Z* states is shown in grey. (<b>D</b>) Position of the segment 667–671 (in grey) homologous to <i>215–217</i>, in close proximity to Tyr531 and the substrate binding site (see ecotin in yellow). This segment includes the specific MASP-3 insertion in loop-2 illustrated in Fig. 6.</p

Kinetic and dissociation constants for the interaction of selected proteases with immobilized ecotin.

<p>Kinetic and dissociation constants for the interaction of selected proteases with immobilized ecotin.</p