Abstract
Purpose :
The small heat shock protein αB-crystallin (αB) is an important component of the endolysosomal pathway and protein homeostasis in the human retinal pigment epithelial cells (RPE). It is secreted out of the cells via exosomes (ARVO 2009; J Biol Chem, 286:3261, 2011) and the expression of this small heat shock protein is required for exosome secretion (J Biol Chem, 291, 12930, 2016). We have previously reported that αB associates with detergent-resistant membrane microdomains (DRMs), also known as lipid rafts (JBC, 2011). DRMs/lipid rafts are known to be localized proteolipid assemblies that may compartmentalize specific signaling complexes on the plasma membrane and inside the cells, possibly within the nucleus. Here we investigated the role (if any) of αB in the organization and physiology of DRMs/lipid rafts in human RPE19 (hRPE) cells in culture.
Methods :
The DRMs/lipid raft fractions were isolated by centrifugation using Iodixanol (Optiprep density gradient medium, Sigma-Aldrich) gradients and identified by immunoblotting using known protein markers (Caveolin1, Hsp90, Enolase1 and Flotillin1). Specific DRM/lipid raft fractions were isolated from wild type and from hRPE cultures where αB expression was silenced (employing αBshRNA) and subjected to proteomic analysis by Mass spectrophotometry.
Results :
Silencing of αB-crystallin expression leads to loss of lipid raft fractions from the Iodixanol gradients. Interestingly, we also found that exposure of hRPE19 cells to methyl β-cyclodextrin (MBCD), an inhibitor of cholesterol biogenesis also leads to the loss of lipid raft fractions from these gradients; αB-crystallin is known to bind cholesterol. Proteomic analysis reveals highly significant alterations in the composition and concentration of the proteins associated with the lipid raft complexes isolated from WT and αB-crystallin silenced cells .
Conclusions :
The organization and dynamics of the assembly of the lipid rafts have thus far remained focused on the lipid components in the plasma membrane that may offer disparate substrates for proteins with differential lipid affinities. Our data suggest that proteins like αB-crystallin may have an important and a direct role in the functional organization of DRMs and the physiological status of lipid rafts in the RPE.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.