Coronavirus disease-2019 (COVID-19), a potentially lethal respiratory illness caused by the coronavirus SARS-CoV-2, emerged in the end of 2019 and has since spread aggressively across the globe. A thorough understanding of the molecular mechanisms of cellular infection by coronaviruses is therefore of utmost importance. A critical stage in infection is the fusion between viral and host membranes. Here, we present a detailed investigation of the role of selected SARS-CoV-2 Spike fusion peptides, and the influence of calcium and cholesterol, in this fusion process. Structural information from specular neutron reflectometry and small angle neutron scattering, complemented by dynamics information from quasi-elastic and spin–echo neutron spectroscopy, revealed strikingly different functions encoded in the Spike fusion domain. Calcium drives the N-terminal of the Spike fusion domain to fully cross the host plasma membrane. Removing calcium, however, reorients the peptide back to the lipid leaflet closest to the virus, leading to significant changes in lipid fluidity and rigidity. In conjunction with other regions of the fusion domain, which are also positioned to bridge and dehydrate viral and host membranes, the molecular events leading to cell entry by SARS-CoV-2 are proposed.
There is growing interest in pharmacological interventions directly targeting the aging process. Pharmacological interventions against aging should be efficacious when started in adults and, ideally, repurpose existing drugs. We show that dramatic lifespan extension can be achieved by targeting multiple, evolutionarily conserved aging pathways and mechanisms using drug combinations. Using this approach in C. elegans, we were able to slow aging and significantly extend healthy lifespan. To identify the mechanism of these drug synergies, we applied transcriptomics and lipidomics analysis. We found that drug interactions involved the TGF-b pathway and recruited genes related with IGF signaling. daf-2, daf-7, and sbp-1 interact upstream of changes in lipid metabolism, resulting in increased monounsaturated fatty acid content and this is required for healthy lifespan extension. These data suggest that combinations of drugs targeting distinct subsets of the aging gene regulatory network can be leveraged to cause synergistic lifespan benefits. Agilent RNA 6000 Nano reagents Agilent Technologies 5067-1511 Deposited Data Raw and Processed RNA-Seq data This paper GEO: GSE108263 Raw and Processed Lipidomics data This Paper MetaboLights: MTBLS648 Experimental Models: Organisms/Strains C. elegans: Strain Bristol wild-type N2 Caenorhabditis Genetics Center N2 C. elegans: Strain daf-2(e1370)III Caenorhabditis Genetics Center CB1370 C. elegans: Strain daf-16(mu86) I Caenorhabditis Genetics Center CF1038 C. elegans: Strain daf-7(e1372) III Caenorhabditis Genetics Center CB1372 C. elegans: Strain eat-2 (ad1116) II Caenorhabditis Genetics Center DA1116 C. elegans: Strain sbp-1 (ep79) III Caenorhabditis Genetics Center CE541 D. melanogaster: Male Bloomington Drosophila Stock Center Oregon-R Oligonucleotides Primer: sbp-1 Forward: gca cgt tct gac atg tgg aa This paper N/A Primer: sbp-1 Reverse: ccg cca aac cca gat tgt ct This paper N/A
OSBP-related protein 8 (ORP8) encoded by Osbpl8 is an endoplasmic reticulum sterol sensor implicated in cellular lipid metabolism. We generated an Osbpl8−/− (KO) C57Bl/6 mouse strain. Wild-type and Osbpl8KO animals at the age of 13-weeks were fed for 5 weeks either chow or high-fat diet, and their plasma lipids/lipoproteins and hepatic lipids were analyzed. The chow-fed Osbpl8KO male mice showed a marked elevation of high-density lipoprotein (HDL) cholesterol (+79%) and phospholipids (+35%), while only minor increase of apolipoprotein A-I (apoA-I) was detected. In chow-fed female KO mice a less prominent increase of HDL cholesterol (+27%) was observed, while on western diet the HDL increment was prominent in both genders. The HDL increase was accompanied by an elevated level of HDL-associated apolipoprotein E in male, but not female KO animals. No differences between genotypes were observed in lecithin:cholesterol acyltransferase (LCAT) or hepatic lipase (HL) activity, or in the fractional catabolic rate of fluorescently labeled mouse HDL injected in chow-diet fed animals. The Osbpl8KO mice of both genders displayed reduced phospholipid transfer protein (PLTP) activity, but only on chow diet. These findings are consistent with a model in which Osbpl8 deficiency results in altered biosynthesis of HDL. Consistent with this hypothesis, ORP8 depleted mouse hepatocytes secreted an increased amount of nascent HDL into the culture medium. In addition to the HDL phenotype, distinct gender-specific alterations in lipid metabolism were detected: Female KO animals on chow diet showed reduced lipoprotein lipase (LPL) activity and increased plasma triglycerides, while the male KO mice displayed elevated plasma cholesterol biosynthetic markers cholestenol, desmosterol, and lathosterol. Moreover, modest gender-specific alterations in the hepatic expression of lipid homeostatic genes were observed. In conclusion, we report the first viable OsbplKO mouse model, demonstrating a HDL elevating effect of Osbpl8 knock-out and additional gender- and/or diet-dependent impacts on lipid metabolism.
SARS-CoV-2 spike proteins are responsible for the membrane fusion event, which allows the virus to enter the host cell and cause infection. This process starts with the binding of the spike extramembrane domain to the angiotensin-converting enzyme 2 (ACE2), a membrane receptor highly abundant in the lungs. In this study, the extramembrane domain of SARS-CoV-2 Spike (sSpike) was injected on model membranes formed by supported lipid bilayers in presence and absence of the soluble part of receptor ACE2 (sACE2), and the structural features were studied at sub-nanometer level by neutron reflection. In all cases the presence of the protein produced a remarkable degradation of the lipid bilayer. Indeed, both for membranes from synthetic and natural lipids, a significant reduction of the surface coverage was observed. Quartz crystal microbalance measurements showed that lipid extraction starts immediately after sSpike protein injection. All measurements indicate that the presence of proteins induces the removal of membrane lipids, both in the presence and in the absence of ACE2, suggesting that sSpike molecules strongly associate with lipids, and strip them away from the bilayer, via a non-specific interaction. A cooperative effect of sACE2 and sSpike on lipid extraction was also observed.
To better understand the principles underlying the substrate specificity of A-type phospholipases (PLAs), a high throughput mass spectrometric assay was employed to study the effect of acyl chain length and unsaturation of phospholipids on their rate of hydrolysis by three different secretory PLAs in micelles and vesicle bilayers. With micelles, each enzyme responded differently to substrate acyl chain unsaturation and double bond position, probably reflecting differences in the accommodative properties of their substrate binding sites. Experiments with saturated acyl positional isomers indicated that the length of the sn2 chain was more critical than that of the sn1 chain, suggesting tighter association of the former with the enzyme. Only the first 9 -10 carbons of the sn2 acyl chain seem to interact intimately with the active site. Strikingly, no discrimination between positional isomers was observed with vesicles, and the rate of hydrolysis decreased far more with increasing chain length than with micelles, suggesting that translocation of the phospholipid substrate to the active site is rate-limiting with bilayers. Supporting this conclusion, acyl chain structure affected hydrolysis and spontaneous intervesicle transfer, which correlates with lipid efflux propensity, analogously. We conclude that substrate efflux propensity plays a more important role in the specificity of secretory PLA 2 s than commonly thought and could also be a key attribute in phospholipid homeostasis in which (unknown) PLA 2 s are key players.Type A phospholipases (PLAs) 3 are ubiquitous enzymes involved in many biological phenomena, such as signal transduction (1) and phospholipid homeostasis (2), including acyl chain remodeling (3) and degradation (4). However, the identities of the specific proteins involved in these phenomena have often not been established. PLAs are also clinically relevant because they have been implicated in many common diseases or disorders, including atherosclerosis, allergy, Alzheimer disease, and cancer (5).Several PLAs display significant specificity toward the phospholipid acyl chain(s) in vitro (6, 7). However, despite numerous studies, the factors underlying such specificity have remained largely elusive. In principle, two factors contribute to selective hydrolysis of phospholipids by PLA: 1) accommodation of the acyl chains in the protein lipid binding site and 2) the ease of efflux of the phospholipid substrate from the bilayer (8, 9). The understanding of acyl chain accommodation has been greatly hampered by the lack of crystal structures of PLAs complexed with a physiological substrate. Crystal structures have been obtained for some PLAs with a bound noncleavable shortchain substrate analogue (10 -13), but because these analogues have truncated acyl chains, they only provide very limited information on the factors underlying acyl chain specificity. Furthermore, the information could also be biased, since studies with other lipid-binding proteins have shown that the mode of accommodation of an alkyl...
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