Dissertation > Excellent graduate degree dissertation topics show

A Study on High Density Lipoprotein and Apolipoprotein AI

Author: WangShuHui
Tutor: ZhaoShuiPing
School: Central South University
Course: Internal Medicine
Keywords: high-density lipoprotein apoliporotein AI lipid raft dendritic cells atherosclerosisABCA1 apoAI unfolding nascent HDL reversecholesterol transport
CLC: R543.5
Type: PhD thesis
Year: 2012
Downloads: 65
Quote: 0
Read: Download Dissertation

Abstract

ObjectiveDepletion of cholesterol by methyl-β-cyclodextrin (MCD) on peptide-loaded antigen presentation cells (APCs) inhibits antigen presentation. However, whether membrane cholesterol efflux induced by high-density lipoprotein (HDL) and apolipoprotein AI (apoAI) also results in inhibition of antigen presentation is still unknown.MethodsDendritic cells (DCs) were incubated with oxidized low density lipoprotein (ox-LDL) to induce the maturation. Matured DCs were loaded with antigen, then incubated with HDL and apoAI. Antigen presentaion ability and cholesterol quantity in lipid rafts was assayed. Futhermore, major histocompatibility (MHC) class Ⅱ protein content in lipid rafts was demonstrated by confocal microscopy.ResultsOx-LDL was able to induce the maturation of bone marrow-derived murine DCs. Incubation of antigen loaded DCs with HDL and apoAI led to decreased antigen presentation and T cells activation abilities, and the decrease was consistent with an increase in cellular cholesterol efflux. Cholesterol repletion reversed the inhibitory effects of HDL and apoAI, demonstrating that the observed reduction in T cell activation was mediated through cholesterol. Furthermore, lipid raft analysis showed that HDL and apoAI reduced cholesterol and major histocompatibility (MHC) class Ⅱ protein content in lipid rafts, suggesting that cholesterol efflux from DCs to HDL and apoAI inhibits antigen presentation and T cell activation by disrupting lipid rafts in DCs.ConclusionHDL and apoAI inhibit MHC-Ⅱ mediated antigen presentation via the cholesterol efflux activity, and ensuing disruption of lipid rafts in DCs. ObjectiveLipid-poor apoAI acts as an acceptor for cell cholesterol and phospholipids via the cell membrane protein ABCA1, generating nascent HDL. However, the mechanism of this process is not understood at the molecular level, therefore, the aim is to gain insight into the mechanism by which ABCA1generates nascent HDL.MethodsHEK293cells were stably transfected with ABCA1vectors encoding wild type (WT) and W590S and C1477R Tangier disease mutation isoforms. We generated fluorescent ratiometric apoAI indicators that monitor apoAI N-terminal unfolding and lipidation.ResultsWT and W590S ABCA1cells had increased apoAI binding vs. control cells, while WT and C1477R ABCA1cells had increased cell surface phosphatidylserine vs. control cells, indicating that these two mutants block separate activities of ABCA1. Cholesterol efflux to apoAI was impaired by～50%by either mutation, implying that neither activity is required for lipid efflux, but that both activities together promote more robust efflux. In cell free assays of reconstituted HDL (rHDL) generation from apoAI and DMPC liposomes, the fluorescent indicators demonstrated apoAI unfolding and lipidation concurrent with rHDL formation. In cell studies, WT and these two mutant ABCA1isoforms led to apoAI unfolding on the cell surface, but not on control cells. However, apoAI lipidation was not detected on ABCA1expressing cells, but in the conditioned medium, consistent with rapid release of nascent HDL from ABCA1expressing cells.ConclusionDistinct apoAI binding and membrane remodeling activities of ABCA1are sufficient to promote partial unfolding of apoAI on the cell surface prior to the formation and release of nascent HDL. Our results support a novel four-step model for nascent HDL biogenesis:1,2) ABCA1remodeling of the plasma membrane and apoAI binding to ABCA1, either of which facilitates3) apoAI partial unfolding,4) apoAI lipidation by the remodeled membrane, followed by the release of nascent HDL.

Related Dissertations

CLC: > Medicine, health > Internal Medicine > Heart, blood vessels ( circulatory ) disease > Vascular disease > Artery disease
© 2012 www.DissertationTopic.Net Mobile