Mechanisms of nonvesicular cholesterol transport

ABSTRACT Our long-term objective is to define cellular pathways that regulate cellular cholesterol flux and to elucidate their impact on metabolism and pathology. Most of the free cholesterol in mammalian cells resides in the plasma membrane (PM). We previously showed that the Aster family of nonvesicular lipid transporters are

critical for the movement of cholesterol from the PM to the ER in most if not all mammalian cells. Asters are ER-anchored proteins that bind cholesterol and facilitate the formation of ER-PM contacts in response to elevated accessible PM cholesterol levels. Although they are required for efficient PM to ER transport, Asters

almost certainly do not act alone. Other factors are very likely to be involved in the spatial organization of accessible PM cholesterol, the formation and stabilization of PM-ER contacts, the movement of Aster proteins from ER to PM, and the channeling of PM cholesterol to specific regions of the ER for SREBP regulation or

esterification by ACAT. The identity of such factors is currently unknown. A complete understanding of how cellular cholesterol is transported in vascular cells through nonvesicular pathways will fill important knowledge gaps and may uncover new opportunities for therapeutic intervention in cholesterol movement in the setting of

cardiovascular disease. Specific Aim 1 will identify new players in nonvesicular lipid transport. We have devised proximity labeling strategies to identify proteins that localize with Asters to ER-PM contacts in a cholesterol-dependent manner. Specific Aim 2 will define the physiological functions of Aster interactors in

cellular and systemic lipid transport. We will validate the functional importance of Snap23 and other factors for lipid metabolism and inflammation in cell culture and animal models. Specific Aim 3 will elucidate the mechanisms and physiological consequences of Aster phosphorylation. We have discovered that Aster-A is

phosphorylated in response to cholesterol loading or LPS stimulation. Dissecting the molecular mechanisms that control PM cholesterol levels in cells, and thereby impact lipid metabolism and inflammation, is central to understanding cell physiology and is expected to provide insight into the etiology and future therapy of

metabolic disease.