Abstract 434: A Novel 5-Step Model for the Mechanism of ABCA1-Mediated Nascent HDL Biogenesis
Objective— Lipid-poor apoA-I 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.
Methods and Results— We propose a novel five-step model of nascent HDL biogenesis: ABCA1 remodeling of the plasma membrane lipids exposing phosphatidylserine and apoA-I binding to ABCA1 are the first two independent steps; third, ABCA1 facilitates apoA-I partial unfolding; forth, partially unfolded apoA-I inserts into the modified plasma membrane resulting in apoA-I lipidation; and fifth, nascent HDL is released from the cell. We created fluorescent apoA-I indicators that can monitor apoA-I unfolding and lipidation states. In cell free assays of reconstituted HDL (rHDL) generation from apoAI and DMPC liposomes, the fluorescent indicators demonstrated apoA-I unfolding and lipidation concurrent with rHDL formation. Next, HEK293 cells were stably transfected with different ABCA1 vectors encoding wild type (WT) and W590S and C1477R Tangier disease mutation isoforms. WT ABCA1 mediated cholesterol efflux to apoA-I (requires all steps) and sodium taurocholate (NaTC, requires only the membrane remodeling step,). Although neither mutant could efflux cholesterol efficiently to apoA-I, they were blocked at different steps. The W590S mutant bound apoAI but could not efflux cholesterol to NaTC, thus it was blocked at the membrane remodeling step. However, the C1477R mutant could not bind apoAI but could efflux cholesterol to NaTC, thus its activity was blocked at the apoAI binding step. When the lipidation indicator apoA-I was incubated with stably transfected HEK cells, we observed cell associated lipidated apoA-I in cells expressing WT ABCA1, but mostly unlipidated apoA-I was associated with the cells expressing W590S ABCA1.
Conclusion— Our results support a novel five-step model for nascent HDL biogenesis: 1, 2) ABCA1 remodeling of the plasma membrane and apoA-I binding to ABCA1, which facilitate 3) apoA-I partial unfolding and 4) and lipidation by the remodeled membrane, followed by 5) the release of nascent HDL.
- © 2012 by American Heart Association, Inc.