Research On The Lipid Transfer Function Of LAMs Protein

The correct distribution of intracellular sterols is critical for many biological functions of mammalian cells,including membrane composition,and signal transduction.The most abundant sterol in animal cells is cholesterol,and its regulation mechanisms are multiple,including absorption,synthesis,efflux and transportation,and the transportation is divided into vesicular transport and nonvesicular transport.The nonvesicular transport is mediated by lipid transfer proteins(LTPs),one member of LTPs is the StARKin superfamily,the StARKin family includes StART(steroidogenic acute regulatory protein-related lipid transfer),LAMs(LTPs anchored at membrane contact sites)and other proteins which contains StART domain(SD).Membrane Contact Sites(MCS)is a closely adjacent region between two organelles or cell membranes within 30 nm.It is a hotspot research of eukaryotic cell lipid exchange process.As a newly discovered member of the StARKin superfamily,the LAMs proteins contain a sterol-specific lipid transfer domain,StART domain.The StART domain can transfer sterols at the MCSs between ER and other membranes.The LAMs family proteins are conserved from yeast Saccharomyces cerevisiae(6 members,Lam1-Lam6)to mammals(3 members,LAMa-c/GramD1a-1c).In this paper,a series of studies were carried out to study the lipid binding and transport ability of the human LAMs protein’s(hLAMb,hLAMc)StART domain.The following work has been completed during the research process:(1)Construct hLAMb,hLAMc recombinant clones,test-expression in E.coli system and screen for soluble proteins.(2)After purification by affinity chromatography,ion exchange chromatography,and gel filtration chromatography,proteins with good purity and concentration of hLAMb and hLAMc were obtained.(3)We examined the lipids(including sterol(DHE),phosphoinositide(PIPs),phosphatidylserine(PS))binding ability of hLAMb and hLAMc by various in vitro fluorescence methods.The results showed that hLAMb and hLAMc can bind DHE,but not PIPs and PS.(4)To further illustrate the mechanisms underlying cholesterol binding by hLAMb and hLAMc,I prepared the complex of hLAMb and hLAMc with the sterol according to the in virto biochemical results.Then I screened the crystallization conditions using the crystal kits in our labrory,and the apo form(no sterols)was also screened.(5)Till now,I have only obtained the crystals of the hLAMc protein during the initial cryatallization screening.