Defensins comprise a potent class of membrane disruptive antimicrobial peptides (AMPs) with well-characterized broad spectrum and selective microbicidal effects. By using high-resolution synchrotron small angle x-ray scattering to investigate interactions between heterogeneous membranes and members of the defensin subfamilies, α-defensins (Crp-4), β-defensins (HBD-2, HBD-3), and θ-defensins (RTD-1, BTD-7), we show how these peptides all permeabilize model bacterial membranes but not model eukaryotic membranes: defensins selectively generate saddle-splay (‘negative Gaussian’) membrane curvature in model membranes rich in negative curvature lipids such as those with phosphoethanolamine (PE) headgroups. These results are shown to be consistent with vesicle leakage assays. A mechanism of action based on saddle-splay membrane curvature generation is broadly enabling, since it is a necessary condition for processes such as pore formation, blebbing, budding, vesicularization, all of which destabilize the barrier function of cell membranes. Importantly, saddle-splay membrane curvature generation places constraints on the amino acid composition of membrane disruptive peptides. For example, we show that the requirement for generating saddle-splay curvature implies that a decrease in arginine content in an AMP can be offset by an increase in both lysine and hydrophobic content. This ‘design rule’ is consistent with the amino acid compositions of 1,080 known cationic AMPs.
Although NPC1L1 is required for intestinal cholesterol absorption, data demonstrating mechanisms by which this protein facilitates the process are few. In this study, a hepatoma cell line stably expressing human NPC1L1 was established, and cholesterol uptake was studied. A relationship between NPC1L1 intracellular trafficking and cholesterol uptake was apparent. At steady state, NPC1L1 proteins localized predominantly to the transferrin-positive endocytic recycling compartment, where free cholesterol also accumulated as revealed by filipin staining. Interestingly, acute cholesterol depletion induced with methyl--cyclodextrin stimulated relocation of NPC1L1 to the plasma membrane, preferentially to a newly formed "apical-like" subdomain. This translocation was associated with a remarkable increase in cellular cholesterol uptake, which in turn was dose-dependently inhibited by ezetimibe, a novel cholesterol absorption inhibitor that specifically binds to NPC1L1. These findings define a cholesterol-regulated endocytic recycling of NPC1L1 as a novel mechanism regulating cellular cholesterol uptake.Whole body cholesterol homeostasis is maintained through three major pathways: de novo synthesis, intestinal absorption, and biliary excretion. Mice lacking npc1l1 (Niemann-Pick C1-like 1) have a substantial reduction in intestinal cholesterol absorption and are resistant to high cholesterol diet-induced cholesterol accumulation (1-3). The phenotypes of npc1l1-null mice recapitulate the effect of ezetimibe (1, 2), a novel cholesterol absorption inhibitor (4 -6), indicating that NPC1L1 is in the ezetimibe inhibitory pathway. Although both the annexin-2/caveolin-1 complex and aminopeptidase N have been reported previously to be the direct target of ezetimibe (7, 8), caveoilin-1 knockout mice have a normal percentage of cholesterol absorption (9), and the physiological evidence for aminopeptidase N as the ezetimibe target has yet to be shown. On the other hand, ezetimibe was recently shown to specifically bind to NPC1L1 (10). All these data strongly support that NPC1L1 is the target of ezetimibe and resides within the cholesterol uptake pathway. However, the reconstitution of NPC1L1-dependent cholesterol transport in cultured cell systems has been unsuccessful, and tissue-specific cofactors were speculated to be needed (1), limiting further exploration of the molecular basis for cholesterol absorption.The NPC1L1 gene was initially identified to be a homolog of NPC1 (Niemann-Pick C1) and was predicted to be involved in intracellular cholesterol trafficking (11) based on the fact that mutations in the NPC1 gene result in a lipid storage disease, Niemann-Pick disease type C1 (12, 13). NPC1L1 is widely expressed in many human tissues, with the highest expression in the liver and small intestine (1, 3, 11). The expression pattern varies among species. Mouse and rat npc1l1 mRNAs are much more abundant in the small intestine than in the liver (1, 3). The reason for the different tissue expression patterns among species is unknown.T...
The present study demonstrates that two different forms of the intracellular cholesterol esterification enzyme acyl-CoA:cholesterol acyltransferase (ACAT) are present in the nonhuman primate hepatocyte; one is similar to that originally cloned from human genomic DNA, here termed ACAT1, while a second gene product, termed ACAT2, is reported here. The primate ACAT2 gene product was cloned from an African green monkey liver cDNA library. Sequence analysis of an isolated, full-length clone of ACAT2 cDNA identified an open reading frame encoding a 526-amino acid protein with essentially no sequence similarity to the ACAT1 cDNA over the N-terminal 101 amino acids but with 57% identity predicted over the remaining 425 amino acids. Transfection of the cloned ACAT2 cDNA into two different mammalian cell types resulted in the production of abundant ACAT activity which was sensitive to ACAT inhibitors. Northern blot analysis showed that the ACAT2 mRNA was expressed primarily in liver and intestine in monkeys. In contrast, ACAT1 mRNA was expressed in almost all tissues examined. Topologic predictions from the amino acid sequence of ACAT2 indicates that it has seven trans-membrane domains in a configuration that places the putative active site of the enzyme in the lumen of the endoplasmic reticulum. This orientation of ACAT2 in the endoplasmic reticulum membrane, in addition to its expression only in liver and intestine, suggests that this enzyme may have as a primary function, the secretion of cholesteryl esters into apoB-containing lipoproteins.The intracellular formation of cholesteryl esters catalyzed by the action of the enzyme acyl-CoA:cholesterol acyltransferase (ACAT; EC 2.3.1.26) 1 appears to be nearly ubiqitous in mammalian cells (1). Elucidation of the details of the structure and catalytic mechanism of ACAT and of the regulation of its activity have been stymied by the difficulty in isolating and purifying an active form of this membrane-associated enzyme. It has taken the isolation of a cDNA for ACAT from human genomic DNA, accomplished through functional complementation of mutant Chinese hamster ovary cells lacking ACAT activity, to initiate progress in understanding the biochemistry of ACAT function (2). The mRNA for this ACAT is expressed in most human tissues and cDNAs with nearly identical ACAT sequences have likewise been found in a variety of tissues from mouse, hamster, and rabbit (3-5).Several functions can be attributed to cholesterol esterification by ACAT. The enzyme appears to modulate the potentially toxic effects of cholesterol in cell membranes. By attaching a fatty acid to the free hydroxyl group of cholesterol, physical properties of the cholesterol molecule are changed and the solubility of esterified cholesterol in the lipids of the cell membrane is limited. Cholesteryl esters accumulate in lipid droplets in the cytoplasm, and maintenance of a balance between the free and esterified forms of cholesterol in a cell is believed to be a component of regulation of cholesterol signaling pathways (6...
Glucose metabolism in pancreatic β cells stimulates insulin granule exocytosis, and this process requires generation of a lipid signal. However, the signals involved in lipid amplification of glucose-stimulated insulin secretion (GSIS) are unknown. Here we show that in β cells, glucose stimulates production of lipolysis-derived long-chain saturated monoacylglycerols, which further increase upon inhibition of the membrane-bound monoacylglycerol lipase α/β-Hydrolase Domain-6 (ABHD6). ABHD6 expression in β cells is inversely proportional to GSIS. Exogenous monoacylglycerols stimulate β cell insulin secretion and restore GSIS suppressed by the pan-lipase inhibitor orlistat. Whole-body and β-cell-specific ABHD6-KO mice exhibit enhanced GSIS, and their islets show elevated monoacylglycerol production and insulin secretion in response to glucose. Inhibition of ABHD6 in diabetic mice restores GSIS and improves glucose tolerance. Monoacylglycerol binds and activates the vesicle priming protein Munc13-1, thereby inducing insulin exocytosis. We propose saturated monoacylglycerol as a signal for GSIS and ABHD6 as a negative modulator of insulin secretion.
Antimicrobial peptides (AMPs) are cationic amphiphiles that comprise a key component of innate immunity. Synthetic analogues of AMPs, such as the family of phenylene ethynylene antimicrobial oligomers (AMOs), recently demonstrated broad-spectrum antimicrobial activity, but the underlying molecular mechanism is unknown. Homologues in this family can be inactive, specifically active against bacteria, or nonspecifically active against bacteria and eukaryotic cells. Using synchrotron small-angle X-ray scattering (SAXS), we show that observed antibacterial activity correlates with an AMO-induced topological transition of small unilamellar vesicles into an inverted hexagonal phase, in which hexagonal arrays of 3.4-nm water channels defined by lipid tubes are formed. Polarized and fluorescence microscopy show that AMO-treated giant unilamellar vesicles remain intact, instead of reconstructing into a bulk 3D phase, but are selectively permeable to encapsulated macromolecules that are smaller than 3.4 nm. Moreover, AMOs with different activity profiles require different minimum threshold concentrations of phosphoethanolamine (PE) lipids to reconstruct the membrane. Using ternary membrane vesicles composed of DOPG:DOPE:DOPC with a charge density fixed at typical bacterial values, we find that the inactive AMO cannot generate the inverted hexagonal phase even when DOPE completely replaces DOPC. The specifically active AMO requires a threshold ratio of DOPE:DOPC = 4:1, and the nonspecifically active AMO requires a drastically lower threshold ratio of DOPE:DOPC = 1.5:1. Since most gram-negative bacterial membranes have more PE lipids than do eukaryotic membranes, our results imply that there is a relationship between negative-curvature lipids such as PE and antimicrobial hydrophobicity that contributes to selective antimicrobial activity.
Background-Stearoyl-coenzyme A desaturase 1 (SCD1) is a well-known enhancer of the metabolic syndrome. The purpose of the present study was to investigate the role of SCD1 in lipoprotein metabolism and atherosclerosis progression. Methods and Results-Antisense oligonucleotides were used to inhibit SCD1 in a mouse model of hyperlipidemia and atherosclerosis (LDLr Ϫ/Ϫ Apob 100/100 ). In agreement with previous reports, inhibition of SCD1 protected against diet-induced obesity, insulin resistance, and hepatic steatosis. Unexpectedly, however, SCD1 inhibition strongly promoted aortic atherosclerosis, which could not be reversed by dietary oleate. Further analyses revealed that SCD1 inhibition promoted accumulation of saturated fatty acids in plasma and tissues and reduced plasma triglyceride, yet had little impact on low-density lipoprotein cholesterol. Because dietary saturated fatty acids have been shown to promote inflammation through toll-like receptor 4, we examined macrophage toll-like receptor 4 function. Interestingly, SCD1 inhibition resulted in alterations in macrophage membrane lipid composition and marked hypersensitivity to toll-like receptor 4 agonists. Conclusions-This study demonstrates that atherosclerosis can occur independently of obesity and insulin resistance and argues against SCD1 inhibition as a safe therapeutic target for the metabolic syndrome.
Background-Two acyl-coenzyme A:cholesterol acyltransferase (ACAT) genes, ACAT1 and ACAT2, have been identified that encode 2 proteins responsible for intracellular cholesterol esterification. Methods and Results-In this study, immunohistology was used to establish their cellular localization in human liver biopsies. ACAT2 protein expression was confined to hepatocytes, whereas ACAT1 protein was found in Kupffer cells only. Studies with a highly specific ACAT2 inhibitor, pyripyropene A, in microsomal activity assays demonstrated that ACAT2 activity was highly variable among individual human liver samples, whereas ACAT1 activity was more similar in all specimens. ACAT2 provided the major cholesterol-esterifying activity in 3 of 4 human liver samples examined. Conclusions-The data suggest that in diseases in which dysregulation of cholesterol metabolism occurs, such as hypercholesterolemia and atherosclerosis, ACAT2 should be considered a target for prevention and treatment.
SUMMARY The serine hydrolase α/β hydrolase domain 6 (ABHD6) has recently been implicated as a key lipase for the endocannabinoid 2-arachidonylglycerol (2-AG) in the brain. However, the biochemical and physiological function for ABHD6 outside of the central nervous system has not been established. To address this we utilized targeted antisense oligonucleotides (ASOs) to selectively knock down ABHD6 in peripheral tissues to identify in vivo substrates and to understand ABHD6's role in energy metabolism. Here we show that selective knockdown of ABHD6 in metabolic tissues protects mice from high fat diet-induced obesity, hepatic steatosis, and systemic insulin resistance. Using combined in vivo lipidomic identification and in vitro enzymology approaches we show that ABHD6 can hydrolyze several lipid substrates, positioning ABHD6 at the interface of glycerophospholipid metabolism and lipid signal transduction. Collectively, these data suggest that ABHD6 inhibitors may serve as novel therapeutics for obesity, nonalcoholic fatty liver disease, and type II diabetes.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
334 Leonard St
Brooklyn, NY 11211
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.