Proteomics And Biochemical Analyses Reveal The Differential Regulation Of Two ADAM12 Isoforms

Background and Aims:A Disintegrin And Metalloproteinase 12(ADAM 12)is a member of the disintegrin protease family.It has two major splicing variants,one transmembrane long(L)isoform ADAM12L and another secreted short(S)isoform ADAM12S.Although both AD AM 12 isoforms have prodomain,metalloproteinase,disintegrin,cysteine-rich,and EGF-like domains,they have distinct C-termini.ADAM12S has additional 34 amino acids at its C-terminus,which replace the 205-amino acid transmembrane and cytoplasmic tail in ADAM12L.The amino acid sequence difference at their C-termini leads to distinct subcellular localization and different biological functions.Recent studies have reported that ADAM 12 is highly expressed in a variety of cancers,such as small cell lung cancer,breast cancer,liver cancer,and brain cancer.It has also been reported that the expression of ADAM 12 in the urine is closely correlated with the stages of breast and prostate cancer patients.ADAM 12 can promote the proliferation,migration,and metastasis of cancer cells.It has gradually become a biomarker for clinical diagnosis of cancers.As a metalloproteinase,current studies mainly focus on the identification of its proteolytic substrates and elucidation of its proteolytic functions on the cancer development.However,how these proteins are regulated is not explored.In our previous work,we identified ADAM 12L-interacting proteins through high-throughput mass spectrometry screening and discovered several proteins upstream of ADAM12L.These proteins,such as Myoferlin,can modulate the ADAM12L enzymatic activity.However,the ADAM12S-interacting proteins are unknown and whether they regulate the function of their interacting partners is not clear.In this work,ADAM12S-interacting proteins were identified using quantitative proteomic approaches and their regulation on the function is explored.Methods:We expressed ADAM12S-FLAG in HeLa cells and harvest the cells to obtain cell lysate for FLAG M2 immunoprecipitation.The purified samples were subjected to immunoblotting for validation of the purification.The gel bands of the control and experimental samples after immunoprecipitation and silver staining were cut in equal fractions.The proteins were digested with trypsin in-gel,purified and analyzed by LC-MS/MS.The raw MS/MS data were searched using Proteome Discoverer and MaxQuant to obtain potential AD AM12S-interacting proteins.Bioinformatics analysis of these proteins was used to explore the potential biological functions for ADAM12S.Then we verified some interactions through biochemical methods and explored their regulation on the ADAM12S protein level and its biological functions.Results:We identified 16 ADAM12S-interacting proteins through two quantitative proteomic approaches for the analyses of the MS/MS data from two biological replicates of immunoprecipitation and mass spectrometry analyses.Among them,only one protein interacts with ADAM12L.The interaction between ADAM12S and GRP94,P4HB,PDIA6,Vimentin,and CANX were further verified by biochemical approaches.We found that both GRP94 and P4HB specifically interact with ADAM12S,while Vimentin specifically interacts with ADAM12L and CANX interacts with both ADAM 12 isoforms,which are consistent with the data obtained from mass spectrometry analyses.In addition,we identified several chaperones which regulate proteins folding,secretion and promote cancer development.The interaction between GRP94 and ADAM 12 was also evaluated with protein docking.The result showed that GRP94 interacts more strongly with ADAM12S than ADAM12L,which was further confirmed by reverse pulldown assay.Moreover,we discovered that GRP94 expression upregulates ADAM12S while GRP94 knockdown reduces ADAM12S protein level in cells and the secretion of its mature form to culture medium.Although ADAM12S interacts with both P4HB and PDIA6,P4HB does not affect the protein level of ADAM12S.However,PDIA6 upregulates ADAM12S protein level.In addition,using wound-healing experiments,we showed that ADAM12S promotes the migration of cancer cells.Conclusions:We identified the specific interacting partners for ADAM12S by quantitative proteomics and validated their interaction through biochemical methods.We further discovered that GRP94 enhances the ADAM12S protein level and promotes the secretion of mature ADAM12S.Although both PDIs,PDIA6 and P4HB,interact with ADAM12S,only PDIA6 affects its protein level.