The lipid distribution of plasma membranes of eukaryotic cells is asymmetric and phospholipid scramblases disrupt this asymmetry by mediating the rapid, nonselective transport of lipids down their concentration gradients. As a result, phosphatidylserine is exposed to the outer leaflet of membrane, an important step in extracellular signaling networks controlling processes such as apoptosis, blood coagulation, membrane fusion and repair. Several TMEM16 family members have been identified as Ca2+-activated scramblases, but the mechanisms underlying their Ca2+-dependent gating and their effects on the surrounding lipid bilayer remain poorly understood. Here, we describe three high-resolution cryo-electron microscopy structures of a fungal scramblase from Aspergillus fumigatus, afTMEM16, reconstituted in lipid nanodiscs. These structures reveal that Ca2+-dependent activation of the scramblase entails global rearrangement of the transmembrane and cytosolic domains. These structures, together with functional experiments, suggest that activation of the protein thins the membrane near the transport pathway to facilitate rapid transbilayer lipid movement.

Ceramides are sphingolipids that modulate a variety of cellular processes via 2 major mechanisms: functioning as second messengers and regulating membrane biophysical properties, particularly lipid rafts, important signaling platforms. Altered sphingolipid levels have been implicated in many cardiovascular diseases, including hypertension, atherosclerosis, and diabetes mellitus–related conditions; however, molecular mechanisms by which ceramides impact endothelial functions remain poorly understood. In this regard, we generated mice defective of endothelial sphingolipid de novo biosynthesis by deleting the Sptlc2 (long chain subunit 2 of serine palmitoyltransferase)—the first enzyme of the pathway. Our study demonstrated that endothelial sphingolipid de novo production is necessary to regulate (1) signal transduction in response to NO agonists and, mainly via ceramides, (2) resting eNOS (endothelial NO synthase) phosphorylation, and (3) blood pressure homeostasis. Specifically, our findings suggest a prevailing role of C16:0-Cer in preserving vasodilation induced by tyrosine kinase and GPCRs (G-protein coupled receptors), except for Gq-coupled receptors, while C24:0- and C24:1-Cer control flow-induced vasodilation. Replenishing C16:0-Cer in vitro and in vivo reinstates endothelial cell signaling and vascular tone regulation. This study reveals an important role of locally produced ceramides, particularly C16:0-, C24:0-, and C24:1-Cer in vascular and blood pressure homeostasis, and establishes the endothelium as a key source of plasma ceramides. Clinically, specific plasma ceramides ratios are independent predictors of major cardiovascular events. Our data also suggest that plasma ceramides might be indicative of the diseased state of the endothelium.

Sphingolipids are both, fundamental structural components of the eukaryotic membranes and signaling molecules regulating a variety of biological functions. Highly bioactive lipids, ceramide and sphingosine-1-phosphate have emerged as important regulators of cardiovascular functions in health and disease. In this review we discuss recent insights into the role of sphingolipids, particularly ceramide and sphingosine-1-phosphate, in the pathophysiology of the cardiovascular system. We also highlight advances into the molecular mechanisms regulating serine palmitoyltransferase, the first and rate-limiting enzyme of de novo biosynthesis, with an emphasis on recently discovered inhibitors of serine palmitoyltransferase, ORMDL and NOGO-B proteins. Understanding the molecular mechanisms regulating this biosynthetic pathway may lead to the development of novel therapeutic approaches for the treatment of cardiovascular diseases.

Background: CD4 and Tetherin are stabilized through intrachain and interchain disulfide bonds, respectively. Results: CD4 and Tetherin retro-translocate from ER to cytosol with oxidized disulfide bridges as folded and multimeric molecules. Conclusion: Cysteines reduction is not a prerequisite for ER to cytosol dislocation. Significance: Our observations challenge the requirements of reduction and unfolding before dislocation.

Background:The AAA-ATPase VCP/p97 and the deubiquitinase YOD1 are required in the endoplasmic reticulum-associated degradation (ERAD) of misfolded proteins. Results: Three ERAD substrates (NHK-␣1〈⌻, NS1-kLC, and Tetherin) become cytosolically exposed independently of p97 and YOD1, whereas MHC-I␣-and CD4-induced retro-translocation requires them. Conclusion: VCP/p97 and YOD1 have distinct substrate-dependent activities in ERAD. Significance: We demonstrate two different levels of p97 and YOD1 requirements in ERAD.

Background Most of the circulating sphingosine‐1‐phosphate (S1P) is bound to ApoM (apolipoprotein M) of high‐density lipoprotein (HDL) and mediates many beneficial effects of HDL on the vasculature via G protein–coupled S1P receptors. HDL‐bound S1P is decreased in atherosclerosis, myocardial infarction, and diabetes mellitus. In addition to being the target, the endothelium is a source of S1P, which is transported outside of the cells by Spinster‐2, contributing to circulating S1P as well as to local signaling. Mice lacking endothelial S1P receptor 1 are hypertensive, suggesting a vasculoprotective role of S1P signaling. This study investigates the role of endothelial‐derived S1P and ApoM‐bound S1P in regulating vascular tone and blood pressure. Methods and Results ApoM knockout (ApoM KO) mice and mice lacking endothelial Spinster‐2 (ECKO‐Spns2) were infused with angiotensin II for 28 days. Blood pressure, measured by telemetry and tail‐cuff, was significantly increased in both ECKO‐Spns2 and ApoM KO versus control mice, at baseline and following angiotensin II. Notably, ECKO‐Spns2 presented an impaired vasodilation to flow and blood pressure dipping, which is clinically associated with increased risk for cardiovascular events. In hypertension, both groups presented reduced flow‐mediated vasodilation and some degree of impairment in endothelial NO production, which was more evident in ECKO‐Spns2. Increased hypertension in ECKO‐Spns2 and ApoM KO mice correlated with worsened cardiac hypertrophy versus controls. Conclusions Our study identifies an important role for Spinster‐2 and ApoM‐HDL in blood pressure homeostasis via S1P‐NO signaling and dissects the pathophysiological impact of endothelial‐derived S1P and ApoM of HDL‐bound S1P in hypertension and cardiac hypertrophy.

Bioactive sphingolipids are emerging as key regulators of vascular function and homeostasis. While most of the clinical studies have been devoted to profile circulating sphingolipids in maternal plasma, little is known about the role of the sphingolipid at the feto-placental vasculature, which is in direct contact with the offspring circulation. Our study aims to compare the sphingolipid profile of normal with preeclamptic (PE) placental chorionic arteries and isolated endothelial cells, with the goal of unveiling potential underlying pathomechanisms in the vasculature. Dihydrosphingosine and sphingomyelin (SM) concentrations (C16:0-, C18:0-, and C24:0- sphingomyelin) were significantly increased in chorionic arteries of preeclamptic placentas, whereas total ceramide, although showing a downward trend, were not statistically different. Moreover, RNA and immunofluorescence analysis showed impaired sphingosine-1-phosphate (S1P) synthesis and signaling in PE vessels. Our data reveal that the exposure to a deranged maternal intrauterine environment during PE alters the sphingolipid signature and gene expression on the fetal side of the placental vasculature. This pathological remodeling consists in increased serine palmitoyltransferase (SPT) activity and SM accrual in PE chorionic arteries, with concomitance impairment endothelial S1P signaling in the endothelium of these vessels. The increase of endothelial S1P phosphatase, lyase and S1PR2, and blunted S1PR1 expression support the onset of the pathological phenotype in chorionic arteries.

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