Proteomic Identification Of A Disintegrin And Metalloproteinase 12(ADAM12) Interacting Proteins And Its Functional Study

Background and Aims: It is reported that a group of enzymes, A Disintegrin And Metalloproteinases(ADAMs), are zinc ion-dependent transmembrane glycoproteins that contain multiple functionally similar domains, such as prodomain, metalloproteinase domain, disintegrin domain, cysteine-rich domain, EGF-like domain, and cytoplasmic tail. They participate in a variety of biological processes, including proteolysis, signal transduction, cell adhesion, migration, invasion, and tumorigenesis. Human ADAM12 is located on chromosome 10q26.3 and has a typical sequence of the ADAM family structure. In previous research, it has been found that ADAM12 is highly expressed in multiple cancer cells. As a hydrolase, ADAM12 could cleave specific substrates, such as Heparin-binding EGF-like growth factor(HB-EGF), Delta-like 1, and Ephrin-A1. Small molecular weight inhibitor was developed to inhibit the catalytic activity of ADAM12, which indicates the importance of its catalytic activity in the treatment of diseases. But the mechanism how ADAM12 regulates its downstream proteins and promotes the proliferation and metastasis of tumor cells is still scarce. In this thesis, we use a mass spectrometry-based quantitative proteomic approach to explore the ADAM12 interacting proteins and study the regulation between ADAM12 and its interacting proteins.Methods: Firstly, we constructed the plasmids of ADAM12’s two isoforms, ADAM12-L and ADAM12-S, in a pCDF vector, with a FLAG tag at the C-terminus. Then, we expressed ADAM12 in a cervical cancer cell line, HeLa, and purified ADAM12-L, ADAM12-S, and their interacting proteins through immunoprecipitation by an anti-FLAG antibody. After evaluation of protein expression and the efficiency of FLAG immunoprecipitation by Western blotting, we ran SDS-PAGE and carried out a silver stain to identify the different bands between the experimental and control samples. The proteins were digested by trypsin in-gel. The resulting peptides were analyzed by LC-MS/MS. The data generated were searched against the UniProt protein database to obtain their identity and relative abundance. Through bioinformatics analysis, we explored the known functions of these interacting proteins and analyzed their biological functions and biological processes. At last, we verified these interactions and explored their regulation through biochemical approaches.Results: In total we identified 297 ADAM12-L interacting proteins from two biological replicates. Among them, 33 proteins were identified in both replicates. We also found 149 ADAM12-S interacting proteins and 13 of them were identified in both replicates. Among them, 11 proteins are only interacted with ADAM12-S. Many ADAM12 interacting proteins play important roles in the proliferation and metastasis of cancer cells. Some proteins are differentially interacted with the two isoforms, suggesting these two isoforms may have different functions. We also biochemically verified three ADAM12-L or ADAM12-S interacting proteins, including myoferlin, vimentin, endoplasmin(also named as GRP94). Furthermore, we found vimentin expression was upregulated by both wild type ADAM12-L and its catalytic inactive mutant ADAM12-L-E351 Q in HeLa cells. In contrast, myoferlin could upregulate the protein level of premature form of ADAM12-L through enhancing its stability. However, the expression and stability of the mature form were not influenced. Knockdown of myoferlin by siRNA reduces the protein levels of both the premature and mature form of ADAM12-L. We also found endoplasmin could regulate the secretion of ADAM12-S. Knockdown of endoplasmin by siRNA, significantly reduces the secretion of ADAM12-S.Conclusions: We identified 297 ADAM12-L interacting proteins and 149 ADAM12-S interacting proteins by LC-MS/MS in total, verified three of these interaction biochemically, and revealed their relative regulation through protein expression, siRNA knockdown and Western blotting analyses. ADAM12-L is probably a key protein that links myoferlin and vimentin, which are both highly expressed in tumor cells. The information obtained here might be useful for the development of new strategies to modulate the signaling pathway targeting ADAM12-L and its interacting proteins. At the same time, GRP94 could regulate the secretion of ADAM12-S, which might help to reveal the molecular mechanism by which ADAM12-S increases tumor cell invasion via degradating extracelluar matrix.