High density lipoprotein (HDL) cholesterol levels are associated with decreased risk of cardiovascular disease, but not all HDL are functionally equivalent. A primary determinant of HDL functional status is the conformational adaptability of its main protein component, apoA-I, an exchangeable apolipoprotein. Chemical modification of apoA-I, as may occur under conditions of inflammation or diabetes, can severely impair HDL function and is associated with the presence of cardiovascular disease. Chemical modification of apoA-I also impairs its ability to exchange on and off HDL, a critical process in reverse cholesterol transport. In this study, we developed a method using electron paramagnetic resonance spectroscopy (EPR) to quantify HDL-apoA-I exchange. Using this approach, we measured the degree of HDL-apoA-I exchange for HDL isolated from rabbits fed a high fat, high cholesterol diet, as well as human subjects with acute coronary syndrome and metabolic syndrome. We observed that HDL-apoA-I exchange was markedly reduced when atherosclerosis was present, or when the subject carries at least one risk factor of cardiovascular disease. These results show that HDL-apoA-I exchange is a clinically relevant measure of HDL function pertinent to cardiovascular disease.
Cardiovascular disease risk depends on high-density lipoprotein (HDL) function, not HDL-cholesterol. Isolevuglandins (IsoLGs) are lipid dicarbonyls that react with lysine residues of proteins and phosphatidylethanolamine. IsoLG adducts are elevated in atherosclerosis. The consequences of IsoLG modification of HDL have not been studied. We hypothesized that IsoLG modification of apoA-I deleteriously alters HDL function. We determined the effect of IsoLG on HDL structure-function and whether pentylpyridoxamine (PPM), a dicarbonyl scavenger, can preserve HDL function. IsoLG adducts in HDL derived from patients with familial hypercholesterolemia ( = 10, 233.4 ± 158.3 ng/mg) were found to be significantly higher than in healthy controls ( = 7, 90.1 ± 33.4 pg/mg protein). Further, HDL exposed to myeloperoxidase had elevated IsoLG-lysine adducts (5.7 ng/mg protein) compared with unexposed HDL (0.5 ng/mg protein). Preincubation with PPM reduced IsoLG-lysine adducts by 67%, whereas its inactive analogue pentylpyridoxine did not. The addition of IsoLG produced apoA-I and apoA-II cross-links beginning at 0.3 molar eq of IsoLG/mol of apoA-I (0.3 eq), whereas succinylaldehyde and 4-hydroxynonenal required 10 and 30 eq. IsoLG increased HDL size, generating a subpopulation of 16-23 nm. 1 eq of IsoLG decreased HDL-mediated [H]cholesterol efflux from macrophages via ABCA1, which corresponded to a decrease in HDL-apoA-I exchange from 47.4% to only 24.8%. This suggests that IsoLG inhibits apoA-I from disassociating from HDL to interact with ABCA1. The addition of 0.3 eq of IsoLG ablated HDL's ability to inhibit LPS-stimulated cytokine expression by macrophages and increased IL-1β expression by 3.5-fold. The structural-functional effects were partially rescued with PPM scavenging.
Cap hydrolysis is a critical step in several eukaryotic mRNA decay pathways and is carried out by the evolutionarily conserved decapping complex containing Dcp2 at the catalytic core. In yeast, Dcp1 is an essential activator of decapping and coactivators such as Edc1 and Edc2 are thought to enhance activity, though their mechanism remains elusive. Using kinetic analysis we show that a crucial function of Dcp1 is to couple the binding of coactivators of decapping to activation of Dcp2. Edc1 and Edc2 bind Dcp1 via its EVH1 proline recognition site and stimulate decapping by 1000-fold, affecting both the K M for mRNA and rate of the catalytic step. The C-terminus of Edc1 is necessary and sufficient to enhance the catalytic step, while the remainder of the protein likely increases mRNA binding to the decapping complex. Lesions in the Dcp1 EVH1 domain or the Edc1 prolinerich sequence are sufficient to block stimulation. These results identify a new role of Dcp1, which is to link the binding of coactivators to substrate recognition and activation of Dcp2.
Objective The molecular mechanisms underlying sex differences in dyslipidemia are poorly understood. We aimed to distinguish genetic and hormonal regulators of sex differences in plasma lipid levels. Approach and Results We assessed the role of gonadal hormones and sex chromosome complement on lipid levels using the Four Core Genotypes mouse model (XX females, XX males, XY females, and XY males). In gonadally intact mice fed a chow diet, lipid levels were influenced by both male–female gonadal sex and XX–XY chromosome complement. Gonadectomy of adult mice revealed that the male–female differences are dependent on acute effects of gonadal hormones. In both intact and gonadectomized animals, XX mice had higher HDL cholesterol (HDL-C) levels than XY mice, regardless of male–female sex. Feeding a cholesterol-enriched diet produced distinct patterns of sex differences in lipid levels compared to a chow diet, revealing the interaction of gonadal and chromosomal sex with diet. Notably, under all dietary and gonadal conditions, HDL-C levels were higher in mice with two X chromosomes compared to mice with an X and Y chromosome. By generating mice with XX, XY and XXY chromosome complements, we determined that the presence of two X chromosomes, and not the absence of the Y chromosome, influences HDL-C concentration. Conclusions We demonstrate that having two X chromosomes versus an X and Y chromosome complement drives sex differences in HDL-C. It is conceivable that increased expression of genes escaping X-inactivation in XX mice regulates downstream processes to establish sexual dimorphism in plasma lipid levels.
Conformational dynamics in bilobed enzymes can be used to regulate their activity. One such enzyme, the eukaryotic decapping enzyme Dcp2, controls the half-life of mRNA by cleaving the 5′ cap structure, which exposes a monophosphate that is efficiently degraded by exonucleases. Decapping by Dcp2 is thought to be controlled by an open-to-closed transition involving formation of a composite active site with two domains sandwiching substrate, but many details of this process are not understood. Here, using NMR spectroscopy and enzyme kinetics, we show that Trp43 of Schizosaccharomyces pombe Dcp2 is a conserved gatekeeper of this open-to-closed transition. We find that Dcp2 samples multiple conformations in solution on the millisecond-microsecond timescale. Mutation of the gatekeeper tryptophan abolishes the dynamic behavior of Dcp2 and attenuates coactivation by a yeast enhancer of decapping (Edc1). Our results determine the dynamics of the open-to-closed transition in Dcp2, suggest a structural pathway for coactivation, predict that Dcp1 directly contacts the catalytic domain of Dcp2, and show that coactivation of decapping by Dcp2 is linked to formation of the composite active site.enzyme dynamics | methyl groups | mRNA decay | protein NMR C onformational dynamics in enzymes often comprise the ratelimiting step in the catalytic cycle and thus are prime targets for regulatory cofactors (1-5). Bilobed proteins frequently use an open-to-closed transition to coordinate catalysis on their substrates following cellular cues such as posttranslational modifications or macromolecular interactions (6-8). A recent model proposes that the eukaryotic mRNA decapping enzyme Dcp2 is regulated by such a transition, where a composite active site is formed using conserved surfaces on each of the two N-terminal domains (9). According to this model, stimulating or inhibiting this conformational transition could regulate decapping. However, the structural details of this composite active site and the timescale of interconversion between closed and open states of Dcp2 are currently unknown. Moreover, whether coactivators use the composite active site to effect decapping is unclear.Degradation of eukaryotic mRNA is critical to many biological processes including development (10), stress response (11), clearance of the products of pervasive transcription (12), and quality control of gene expression (13). For example, it has been suggested that microRNAs (miRNAs) act primarily by destabilizing messages and it is known that Dcp2 is a vital component of miRNA-induced mRNA decay (14, 15). Further, an entire class of unstable transcripts was recently discovered that is sensitive to the exonuclease Xrn1, whose members are therefore likely products of decapping (12, 16). Each of the variety of pathways that utilize decapping relies on coactivator proteins that are believed to recruit messages to the decapping machinery and activate it.A model of decapping coactivation is emerging following recent work on the Saccharomyces cerevisiae coactivator...
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.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.