A Conserved Arginine-Rich Motif within the Hypervariable N-Domain of Drosophila Centromeric Histone H3 (CenH3CID) Mediates BubR1 Recruitment

Centromere identity is determined epigenetically by deposition of CenH3, a centromere-specific histone H3 variant that dictates kinetochore assembly. The molecular basis of the contribution of CenH3 to centromere/kinetochore functions is, however, incompletely understood, as its interactions with the rest of centromere/kinetochore components remain largely uncharacterised at the molecular/structural level.Here, we report on the contribution of Drosophila CenH3(CID) to recruitment of BubR1, a conserved kinetochore protein that is a core component of the spindle attachment checkpoint (SAC). This interaction is mediated by the N-terminal domain of CenH3(CID) (NCenH3(CID)), as tethering NCenH3(CID) to an ectopic reporter construct results in BubR1 recruitment and BubR1-dependent silencing of the reporter gene. Here, we also show that this interaction depends on a short arginine (R)-rich motif and that, most remarkably, it appears to be evolutionarily conserved, as tethering constructs carrying the highly divergent NCenH3 of budding yeast and human also induce silencing of the reporter. Interestingly, though NCenH3 shows an exceedingly low degree of conservation, the presence of R-rich motives is a common feature of NCenH3 from distant species. Finally, our results also indicate that two other conserved sequence motives within NCenH3(CID) might also be involved in interactions with kinetochore components.These results unveil an unexpected contribution of the hypervariable N-domain of CenH3 to recruitment of kinetochore components, identifying simple R-rich motives within it as evolutionary conserved structural determinants involved in BubR1 recruitment

cAMP effects on polarization of BMDM and RAW264.7 macrophages to proinflammatory or antiinflammatory phenotypes

Trabajo presentado en el Neuroscience 2017, celebrado en Washington del 11 al 15 de noviembre de 2017Infiltrating macrophages are implicated in the neuroinflammatory response in neurodegenerative diseases. There appears to be equilibrium in the lesion site between cells that either exacerbate tissue injury or promote CNS repair. In vitro studies show that this equilibrium can be shifted by chemical stimuli. “Classically activated” M1 macrophages or “M1 spectrum” are considered pro-inflammatory and can be induced by stimuli such as LPS and pro-inflammatory cytokines IFN-gamma or TNF-alpha. The “alternative activation” to M2 macrophages or “M2 spectrum” has anti-inflammatory and tissue reparative phenotype. We studied the influence of cAMP levels on primary Bone Marrow Derived Macrophages (BMDM) and the murine macrophage cell line Raw264.7 to shift to M1 or M2 phenotypes when cells are grown in the presence of pro- or anti-inflammatory stimuli. We tested the implication of different components of the cAMP cascade: adenylyl cyclase, cAMP-PDE4, and PKA. Cells were first pre-incubated 30 min with the different drugs, then stimulated by M1 (10 ng/mL LPS, 20 ng/mL IFN-gamma) or M2 stimuli (20 ng/mL each IL-4/IL-13 for BMDM or 20 ng/mL each IL-4/IL-10 for Raw264.7 for 24 h. Nitric oxide (NO) production or arginase activity were then determined. When BMDM and Raw264.7 cells were pre-treated with the adenylyl cyclase activator forskolin both M1 and M2 cells reverted to M0 phenotype in a concentration-dependent manner. IBMX and some PDE4 inhibitors, rolipram, roflumilast, apremilast and C33 induced a M1 to M0 shift and all potentiated a M2 phenotype in a concentration-dependent manner. cAMP analogs acting on PKA induced a M1 to M2 shift and potentiated the M2 phenotype. In contrast, cAMP analogs acting on Epac did not modify the effects of M1 or M2 stimuli, suggesting that polarization occurs through PKA and not Epac under high cAMP levels. These results were confirmed by immunocytochemistry with phenotype-specific antibodies (iNOS for M1 cells and mannose receptor or arginase 1 for M2 cells). Phenotypic regulation of macrophages with cAMP elevating drugs may be a therapeutic tool for neuroinflammatory diseases.Peer reviewe

A subset of type-II collagen-binding antibodies prevents experimental arthritis by inhibiting FCGR3 signaling in neutrophils

Abstract Rheumatoid arthritis (RA) involves several classes of pathogenic autoantibodies, some of which react with type-II collagen (COL2) in articular cartilage. We previously described a subset of COL2 antibodies targeting the F4 epitope (ERGLKGHRGFT) that could be regulatory. Here, using phage display, we developed recombinant antibodies against this epitope and examined the underlying mechanism of action. One of these antibodies, R69-4, protected against cartilage antibody- and collagen-induced arthritis in mice, but not autoimmune disease models independent of arthritogenic autoantibodies. R69-4 was further shown to cross-react with a large range of proteins within the inflamed synovial fluid, such as the complement protein C1q. Complexed R69-4 inhibited neutrophil FCGR3 signaling, thereby impairing downstream IL-1β secretion and neutrophil self-orchestrated recruitment. Likewise, human isotypes of R69-4 protected against arthritis with comparable efficiency. We conclude that R69-4 abrogates autoantibody-mediated arthritis mainly by hindering FCGR3 signaling, highlighting its potential clinical utility in acute RA

A subset of antibodies targeting citrullinated proteins confers protection from rheumatoid arthritis

Although anti-citrullinated protein antibodies (ACPAs) are a hallmark of rheumatoid arthritis and generally considered pathogenic, their functional relevance is incompletely understood. In this study, the authors describe an ACPA with a protective effect against antibody-induced arthritis in mice