Identification Of Proteins Interacting With Two Variants Of DNAJC12 By Tandem Affinity Purification And Mass Spectrometry

Study of interacting proteins is the most important method to investigate functions of genes. As the rapid development of proteomics, large-scale screening interacting proteins in different biological system has become more and more important.Heat shock protein (heat schok proteins, HSPs) is highly conserved and rich in cells. Under normal circumstances, as molecular chaperones, HSPs are involved in various metabolic activities of cells, including promoting the synthesis and folding of new peptides, preventing the inappropriate folding and aggregation of protein subunits before correct polymers are formed, assisting other protein molecules in transporting into cells and through the cell membranes, and promoting the recovery of damaged or denatured proteins otherwise accelerate their degradation. Under stressful conditions, such as high temperature, oxidation and other stimuli, the produced HSPs can enhance the tolerance of damage in cells to maintain normal metabolism and increase the cellular survival rate. DNAJC12, as a member of the family of Hsp40 proteins, participates in protein folding, transportation, translation initiation and so on. Previous study indicated that DNAJC12 protein was overexpressed in a variety of tumor cells. It was also proved that DNAJC12 protein was overexpressed in cancers, which has great correlations with the occurrence and development of cancers, but it is still uncertain about the roles it plays in the occurrence and development of cancers. Usually, proteins function through interacting with other proteins. The research of dynamic changes of protein complexes including DNAJC12 protein formed in signaling pathways may contribute to comprehensively reveal the underlying molecular mechanism of regulatory functions of DNAJC12 protein in the occurrence and development of tumors.As proteomics develop rapidly, most cellular biological functions are completed by protein complexes, not by single protein. Thus, it is indispensable to identify and analyze the components of protein complexes. Tandem affinity purification (TAP) is a new technology for study of protein complexes. It adds two specific protein tags to the C- or N-terminal of the target protein. After two-step specific affinity purifications, proteins which interact with the target protein under physiological conditions could be obtained and identified by mass spectrometry. Compared with other purification techniques, the biggest advantage is that by integrating classical affinity purification with co-immunoprecipitation, it can acquire interacting protein complexes with high concentration and purity and lower false positive rate.In this study, we selected tandem affinity purification system with the strptavidin binding protein (SBP) and calmodulin binding protein (CBP) as tags. SBP tag, which is composed of 45 amino acids, is a new streptavidin binding peptide. Proteins tagged by SBP tag can be purified by immobilized stretavidin and then eluted by biotin. Conditions of elution are quite mild. There is in no need of tobacco etch virus (TEV) protease for digestion, which can reduce the influence on target proteins during the process of TEV protease digestion and contamination on purified protein complexes caused by macromolecular substance. Meanwhile, SBP tag is smaller than ProtA tag, which can effectively reduce the influence of TAP tag on exact folding of bait protein and formation of the bait protein complex, and avoid impact of expression of TAP tag on formation of target protein complexes.In this study, to avoid the overexpression of fusion proteins caused by transient transfection and the false positive identification results caused by unnatural or unspecific protein-protein interactions, we built MCF-7-pCTAP-DNAJC12-V1 and MCF-7-pCTAP-DNAJC12-V2 cell lines which can stably express fusion proteins through G418. To eliminate false positive resulted from unspecific binding of TAP tags, we also built MCF-7-pCTAP-A cell lines which can stably transfect blank vectors as the negative control system.Classical experimental strategy is that protein complexes purified by TAP is separated by SDS-PAGE gel electrophoresis. In this study, purified proteins were first separated by SDS-PAGE gel electrophoresis and then silver stained, after that, differential protein bands were cut and identified by mass spectrometry.After analyzing differential protein bands between negative control group and target proteins, we found 14 differential protein bands which specifically binded to DNAJC12-V1,3 differential protein bands which specifically binded to DNAJC12-V2, and then clearly stained bands were identified by mass spectrometry. Identification by mass spectrometry and database search are also the key steps to decide the reliability of experimental results. In this study, parameters and search standard library were strictly set, and 4 candidate proteins of DNAJC12-V1 protein complexes were obtained, including nonmuscle myosin heavy chain(MYH9), myosin regulatory light chain 12B(MRCL2), hypothetical protein and myosin regulatory light chain. The discovery of these proteins provides some function clues and screening molecules for uncovering roles which DNAJC12 plays in the occurrence and development of breast cancer.In conclusion, we separated DNAJC12-V1 protein complexes in MCF-7-pCTAP-DNAJC12-V1 cells and DNAJC12-V2 protein complexes in MCF-7-pCTAP-DNAJC12-V2 cells through tandem affinity purification, and then utilized SDS-PAGE gel electrophoresis to separate protein complexes and analyze differential protein bands, which were then cut and identified by MALDI TOF/TOF. Through the study, we draw the following conclusions:1. The eukaryotic expression vectors for two variants of DNAJC12, pCTAP-DNAJC12-V1 and pCTAP-DNAJC12-V2, were successfully constructed.2. We screened stable expression cell lines of MCF-7-pCTAP, MCF-7-pCTAP-DNAJC12-V1 and MCF-7-pCTAP-DNAJC12-V2 by G418.3. SDS-PAGE gel electrophoresis was used to analyze protein complexes and differential protein bands, and then 7 differential proteins specifically banding with DNAJC12-V1 and 1 differential protein with DNAJC12-V2 were identified by mass spectrometry. At last, we got 4 candidate proteins of DNAJC12-V1 protein complexes. 4. The discovery of these differential proteins has great significance in understanding functions of DNAJC12 in tumor development. Meanwhile the discovery of differential proteins bingding with two variants of DNAJC12 provides the basis for research of its functions.In summary, tandem affinity purification technique is used under near physiological conditions to study the proteins which interact with DNAJC12. Many potential interacting proteins of DNAJC12 have been found, which can lay a good foundation for further research in biological function of DNAJC12 protein and its molecular mechanisms.