The Functional Study On TBC1D8B Gene And Identification Of Its Interaction Proteins

The TBC domain protein family is a ubiquitous,highly conserved,GTPase activating protein(GAP)in eukaryotes.It has been predicted approximately 40 protein members with TBC domain in the human genome.In the motif analysis,the TBC family proteins usually contain a conserved 200-amino acid-domain,including a GTPase activating structure that specifically recognize the RAB-GTP to catalyze it into the RAB-GDP.Additionally,some other domains closely related to biological functions of cell membranes exist,such as PTT(phosphotyrosime-binding domain),PH(pleckstrin homology domain),CC(coiled-coil)or GRAM(glucosyl transferases)domain.This protein family has also been found involved in the development of cancer.Although a few members in the family have shown their target small GTPase(s)and their roles,the detailed function of TBC1D8B,which has been descripted to be associated with osteoporosis and childhood disintegration disease(CDD)as one of the TBC domain family proteins,are still required to be further elucidated.Based on the above research background,our investigation is going to reveal the target proteins as well as the roles of TBCID8B using bioinformatics analysis combined with a series of functional experiments such as morphological research,co-localization of proteins and yeast two-hybrid.The main results as follow:1.TBC1D8B affects the sub-cellular distribution of mitochondria and transportThe previous reports suggest that the TBC proteins are localized in the cytoplasm and primarily involved in the transport of vesicles orendocytosis.In our current work,it has been shown TBC1D8B fused GFP can co-localize with the mitochondrial structural protein Mito.Furthermore,overexpression of TBC1D8B leads to a significant increase in mitochondria aggregation at the perinuclear nucleus,and a slower mitochondrial axoplasmic transport rate.It can also shorten the outgrowth in nerve cells.2.Screening of RAB substrates interacting with TBC1D8BWhen we analyzed the potential physical interactions with TBC1D8B through the data from websites and literatures,it has been suggested that the RAB proteins,such as ARF1,RAB5C RAB8A,RAB11B,and PDCD10 could bind to TBC1D8B.The experiment of those fused GFP and RFP demonstrates that most of them could co-localize in cytoplasm.But the result of yeast two-hybrid do not support the direct binding between TBC1D8B with the above proteins.3,Overexpression of TBC1D8B promotes cell proliferation,migration,and infiltrationConstructing and liposome-transfecting the recombinant TBC1D8B-pCMV-Tag3C,we screen the Hela cell line that stably over-expressed TBC1D8B.With the assays of hMTT,cell scratch and transwell chamber,it is found that the overexpression of TBC1D8B enhanced cell proliferation,migration,and infiltration,implying that TBC1D8B might be an oncogene.To identify the molecular mechanism involved in oncogenesis of TBC1D8B,the yeast two-hybrid was employed again to verify the interactions between TBC1D8B with HSPA1A,HSPA6,BBS7 and ZBTB7C,but obtained the negative resultsThe innovation of this article:It was first discovered that TBC1D8B can localize to mitochondria and affect mitochondrial distribution and transport.