Dissecting The SR-B1 And LIMP-2 Roles In Cholesterol Transport

Cholesterol homeostasis is of central importance for life.Mammalian cells acquire most cholesterol through receptor-mediated lipoprotein derived-cholesterol uptake to meet their cholesterol needs.However,the mechanisms and regulation of this cholesterol transport remain poorly understood.In this study,we focus on the roles of the Scavenger Receptor class B?SR-B?family members-LIMP-2?Lysosomal Integral Membrane Protein-2,also known as SCARB2?and SR-B1?also known as SCARB1?-in cholesterol uptake and transport.LIMP-2 is the only one of the SR-B family members that is localized in the intracellular lysosomal membrane.SR-B1 and SR-B3?also known as CD36 or SCARB3?are plasma membrane?PM?proteins.These three proteins have similar secondary structures.Both SR-B1 and SR-B3 are known to directly mediate lipid transport.However,it is unclear whether LIMP-2 can participate in lipid transport.The LIMP-2 structural information obtained in our previous study reveals a hydrophobic tunnel traverses the molecule.In this paper,we clarify a new function of LIMP-2 in lipid transport,revealing that it mediates a new mechanism of lysosomal LDL-derived cholesterol export.Using molecular dynamic modeling,we found that this hydrophobic tunnel can accommodate a cholesterol molecule.Moreover,electron microscope experiments showed that there were cholesterol-like inclusion bodies in LIMP-2 knockout mice.Therefore,we speculated that LIMP-2 may be involved in cholesterol transport.Besides,click chemistry,microscale thermophoresis,liposomes and other experiments show that the LIMP-2 lysosomal lumen domain can bind and transport cholesterol in vitro.Furthermore,the results of cell-based experiments show that the hydrophobic channel in the luminal domain of LIMP-2 can bind and transport LDL-derived cholesterol to the lysosomal membrane,and then to the endoplasmic reticulum?ER?and stored in lipid droplets?LD?.Moreover,depletion of LIMP-2 alters sterol regulatory element binding protein?SREBP2?-mediated cholesterol regulation and LDL receptor?LDLR?levels,thereby affecting cholesterol homeostasis.In addition,our data indicate that the lysosomal cholesterol export pathway mediated by LIMP-2 works in parallel with the known NPC1-mediated cholesterol transport pathway.In addition,we investigate the molecular and regulation mechanism of SR-B1,a homologous protein of LIMP-2 localized in the plasma membrane to mediate cholesterol transport from high-density lipoprotein?HDL?.SR-B1 is the main receptor for HDL and has important physiological functions.It is the key to prevent atherosclerosis.SR-B1transports excess cholesterol in the blood circulation from HDL-cholesterol?HDL-C?to steroidogenic tissues and the liver.At present,the role of SR-B1 in HDL-C metabolism is unknown.Organelle interactions are essential for life activities,especially in lipid metabolism.Here,we verify that SR-B1 transports HDL-derived cholesterol to the plasma membrane through its intramolecular hydrophobic tunnel similar to LIMP-2.Furthermore,we find that the PM-localized SR-B1 can interact with the ER membrane protein VAPB to form membrane contact sites?MCSs?.Moreover,depletion of SR-B1 or VAPB can significantly impair PM cholesterol to ER and LD transport.Finally,we find that blocking Ca²⁺signaling can significantly inhibit HDL-derived cholesterol transport.In addition,the Ca²⁺-binding protein E-Syt1 can interact with SR-B1 and VAPB and quickly respond to HDL addition,facilitate the formation of MCSs.In summary,our study reveals a new mechanism for lysosomal cholesterol export,which is expected to provide new targets for the treatment of lysosomal storage disorders such as Niemann-Pick disease caused by inactivation of NPC1.Moreover,our study shows that SR-B1 mediates HDL-derived cholesterol transport through the formation of ER-PM MCSs and reveals that Ca²⁺signaling is an important regulator of this process.