Research On The Lipid Transfer Mechanism Of The OSBP-related Protein 10(ORP10) And ORP11

The oxysterol binding protein(OSBP)and OSBP-related proteins(ORPs)have emerged as key mediators and/or regulators of lipid transport at junctions between the ER and other organelles.ORPs are conserved in yeast,plants and mammals,humans have 12 ORP genes,and splicing generates 16 different protein products,named OSBP and ORP1-11.ORP10 and ORP11 contain an OSBP-related domain(ORD)like other members of ORPs,ORD has been implicated in many cellular processes including maintaining the membrane contact sites and nonvesicular phospholipids transfer.Previous studies have shown that ORP10 has an essential role in establishing/maintaining the ER-TGN contact sites.ORP10 depletion induces an increase in TGN PI4P that leads to increased secretion of apoB-100.ORP11 distribute at the Golgi-late endosome interface,however,the lipid transport mechanisms of ORP 11 have remained incompletely understood.In this dissertation,a series of studies were carried out to study the lipids binding and transport ability of the human ORP10 and ORP11 ORD domain.The following work has been completed during the research process:1.ORP10 and ORP 11 ORD proteins were subcloned into pET21b vector by molecular biology methods,the fusion proteins were expressed in E.coli BL21(DE3).2.All proteins were purified over Ni NTA column and then by ion exchange,finally,we obtain the proteins with high purity and concentration.We have been examined the lipids binding ability of ORP10 and ORP11 ORD by various in vitro fluorescence methods.The results showed that ORP10 and ORP 11 can bind different PIPs.Then we examined the lipids transport ability of ORP10 and ORP11 ORD,the results showed that ORP10 and ORP11 ORD can transport PI4P,PI(4,5)P2 and PS.3.According to the in virto biochemical results,we set up crystallization screen of ORP10 and ORP11 ORD with PI4P,PI(4,5)P2 and PS.Finally,the crystals of ORP10 with PS/PI4P,ORP11 with PS/PIP were obtained.