Purification Of HOPS Protens And The Interaction With Autophagic Snare Proteins

Autophagy is a self-clearance and an evolutionarily conserved process in eukaryotes,depending on lysosomal degradation pathway.When cells are stimulated,autophagosome engulfs long-lived proteins and damaged organelles,allowing cells to recycle energy under stressful conditions.This is critical for cells to survive in starvation and maintain intracellular homeostasis.Studies have shown that autophagy plays an important role in immune defense,inhibition of neurodegenerative diseases,cancers and so on.Therefore,analysis of the structure of proteins related to autophagy will provide potential drug targets for the treatment of diseases.Autophagy is a highly dynamic,multi-step process that primarily takes place through the following stages:induction of autophagy;formation of phagophore;maturation of autophagosome;fusion of autophagosome with lysosome and degradation of substrates.The fusion stage of autophagosome and lysosome is a key process of autophagy and requires the participation of multiple proteins.It is known that SNAREs belong to membrane-anchored proteins,the main role is to mediate the membrane fusion of vesicles with targets.STX17 belongs to t-SNAREs and localizes to autophagosome,VAMP8 belongs to v-SNAREs and mainly located on lysosome.Studies have shown that VAMP8 interacts with SNAP29 and STX17,which is recruited on the outer membrane of completed autophagosomes,to form a ternary complex of STX17-SNAP29-VAMP8 that mediates the autophagosome-lysosome fusion.HOPS is a protein complex consisting of VPS11,VPS16,VPS18,VPS33,VPS39,and VPS41,which regulates membrane transport in vivo through membrane fusion mechanisms,one of the functions is to regulate the "assembly" of the SNARE complex.The evidence suggests that the HOPS complex as a fusion factor interacts with STX17 to facilitate the membrane fusion process.Therefore,this experiment intends to use the purified proteins to determine that the HOPS complex directly interacts with the STX17 protein in vitro,and then explore whether the HOPS complex also interacts with the SNAP29 and VAMP8 proteins.Firstly,the coding sequence of the six genes were amplified from the existing plasmids by PCR,and ligated to the prokaryotic expression vector pGEX 4T-1-GST or pET-His-NusA.After the identification though colony PCR and DNA sequencing,this study had successfully constructed pET-His-NusA-TEV-HA-VPS11,pGEX 4T-1-GST-TEV-Flag-VPS16,pET-His-NusA-TEV-HA-VPS18,pGEX 4T-1-GST-TEV-Flag-VPS33,pET-His-NusA-TEV-HA-VPS39 and pGEX 4T-1-GST-TEV-Flag-VPS41 recombinant plasmids,and then transferred into E.coli BL21(DE3),the recombinant proteins were induced by IPTG.After determining the solubility of the six recombinant proteins,they were purified by glutathione sepharose 4B and nickel column.TEV enzyme was used to cut the GST-tag and NusA-tag off.The results of coomassie brilliant blue rapid staining and western blot showed that:the recombinant plasmids pET-His-NusA-TEV-HA-VPS11,VPS18 and VPS39 were highly expressed under the induction condition(0.2 mmol/L IPTG for 16 hours at 22?),the right-sized recombinant proteins with HA-tag were obtained(105 kDa,108 kDa and 97 kDa);the recombinant plasmids pGEX 4T-1-GST-TEV-Flag-VPS16 VPS33 and VPS41 were highly expressed under another induction condition(0.2 mmol/L IPTG for 5 hours at 28?),the right-sized recombinant proteins with Flag-tag were obtained(97 kDa,70 kDa and 98 kDa).In vitro GST pull-down method confirmed that STX17,SNAP29 and VAMP8 interact directly with the HOPS proteins.In summary,six recombinant proteins consisting of human HOPS complex were successfully purified,and their interaction with the SNARE proteins STX17,SNAP29 and VAMP8 were verified in vitro,which provides a practical experimental pathway for revealing the mechanism of HOPS complex in the process of autophagosome and lysosome membrane fusion,and lays the foundation for further study on the biological function of HOPS complex.