Lipid Transfer Mechanism Of The OSBP-related Protein 8(ORP8) And OSBP-related Protein 5(ORP5)

Eukaryotic intracellular membrane is composed of specific lipids to ensure its normal function.However,it is still poorly understood how these particular lipids are sensed and transported.There is a group of proteins,called lipid transfer protein(LTPs)plays a very important role in these processes.ORPs(Oxysterol-binding protein(OSBP)-related proteins)is a large family of LTPs,highly conserved from yeast to humans.ORPs participate in many intracellular activities,including signaling,vesicular transport,lipid metabolism and non-vesicular lipid transport.hORP8 and hORP5 belong to subfamily ?of human ORPs and contains OSBP-related domain(ORD)which commonly exist and are highly conserved in ORPs family.ORD has a hydrophobic pocket that binds single lipid,also contains other parts that bind phosphoinositides and regulates lipid binding.Our group constructed ORP8 recombinant clones via molecular cloning,and then expressed and purified target protein in vitro in order to select high expression and purity clones.After we obtained suitable clones,through biochemical experiments and structure analysis,it showed that highly conserved ORD of ORP8 can specifically bind phosphatidylserine(PS)and phosphatidylinositol phosphate(PIP),but not cholesterol.At the same time,we also studied another protein of the same subfamily--ORP5,which shares high sequence identity with ORP8.ORP5 has similar biochemical properties to ORP8.In the lipid transport assays,hORP5 and hORP8-ORD showed high transport efficiency towards PI(4,5)P2,and PI(4,5)P2 can drive PS transport efficiently.However their ORD can't transfer PI4P.And the PI(4,5)P2 gradient between two membranes is critical for PI(4,5)P2 stimulated PS transfer.Besides,we found that different constructs of ORP8-ORD had different ability of PS transfer.According to biochemical results of ORP5 and ORP8-ORD with different binding abilities to specific lipids,we set up the crystallization screen of ORP8 and ORP5 binding various PIPs and PS ligands.Finally,we obtained the crystals of ORP8-ORD apo and PI(4,5)P2-complexed forms.These results pave the way to clarify the mechanism of how ORP8 and ORP5-ORD interact with lipids.