Chaaban, H. | bioRxiv Science

bioRxiv2024

Endothelial RIPK3 minimizes organotypic inflammation and vascular permeability in ischemia-reperfusion injury

Recent studies have revealed a link between endothelial receptor-interacting protein kinase 3 (RIPK3) and vascular integrity. During mouse embryonic development, hypoxia can trigger elevated endothelial RIPK3 that contributes to lethal vascular rupture. However, it is unknown whether RIPK3 regulate endothelial barrier function in adult vasculature under hypoxic injury conditions such as ischemia-reperfusion (I/R) injury. Here we performed inducible genetic deletion of endothelial Ripk3 (RipkiECKO) in mice, which led to elevated vascular permeability in the small intestine and multiple distal organs after intestinal I/R injury. Mechanistically, this vascular permeability correlated with increased endothelial secretion of IL-6 and organ-specific expression of VCAM-1 and ICAM-1 adhesion molecules. Circulating monocyte depletion with clodronate liposomes reduced permeability in organs with elevated adhesion molecules, highlighting the contribution of monocyte adhesion and extravasation to RipkiECKO barrier dysfunction. These results elucidate mechanisms by which RIPK3 regulates endothelial inflammation to minimize vascular permeability in I/R injury. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=185 SRC="FIGDIR/small/625188v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@1b3ef3forg.highwire.dtl.DTLVardef@201e47org.highwire.dtl.DTLVardef@a402e0org.highwire.dtl.DTLVardef@1b8f5cb_HPS_FORMAT_FIGEXP M_FIG C_FIG

pathology↗

bioRxiv2024

Amyloid Morphology is Encoded in H-bonds and Electrostatics Interactions Ruling Protein Phase Separation

Ion-protein interactions regulate biological processes and are the basis of key strategies of modulating protein phase diagrams and stability in drug development. Here, we report the mechanisms by which H-bonds and electrostatic interactions in ion-protein systems determine phase separation and amyloid formation. Using microscopy, small-angle X-ray scattering, circular dichroism and atomistic molecular dynamics (MD) simulations, we found that anions specifically interacting with insulin induced phase separation by neutralising the protein charge and forming H-bond bridges between insulin molecules. The same interaction was responsible for an enhanced insulin conformational stability and resistance to oligomerisation. Under aggregation conditions, the anion-protein interaction translated into the activation of a coalescence process, leading to amyloid-like microparticles. This reaction is alternative to conformationally-driven pathways, giving rise to elongated amyloid-like fibrils and occurs in the absence of preferential ion-protein binding. Our findings depict a unifying scenario in which common interactions dictated both phase separation at low temperatures and the occurrence of pronounced heterogeneity in the amyloid morphology at high temperatures, similar to what has previously been reported for protein crystal growth.

biophysics↗