The Preliminary Researches Of Shark TBC1D15 Protein Function

Diabetes is a disease of glucose metabolism disorder, which is caused by the interaction between genes and environmental factors, characterized by hyperglycemia because of insulin resistance or insufficient insulin secretion. Disturbance of carbohydrate metabolism, which is extremely easy to induce many complications, has become the third major serious chronic non-communicable disease threating to human health after cardiovascular disease and oncological diseases. Early research has shown that APSL separated from the regenerated liver of Chiloscyllium can inhibit lipid peroxidation, eliminate free radicals, protect and repair the damaged liver or pancreatic β cells. The pharmacological action and pharmacodynamics studies have shown that oral dosage forms of CTB-APSL fusion protein expressed in silkworm pupae was given to type 2 diabetic model mouse by intragastric administration, which can effectively protect the islet cells, recover the damaged cells to achieve hypoglycemic effect by increasing insulin secretion. With the high-throughput sequencing completion of the shark regenerating liver c DNA library, through bioinformatics analysis found that APSL was located in the N-terminus of shark TBC1D15 protein. That is, the APSL whole protein is shark TBC1D15. However, the function research of the APSL whole protein was not carried out.According to the reported function of TBC1D15 protein, the function verification of the APSL whole protein was carried out in this experiment, and the Rab-GAP specificity of the APSL whole protein was also detected. The early stage of the bioinformatics analysis showed that APSL whole protein contains a domain with Rab-GAP activity. There are six typical motifs A–F in domain. TBC domain homology between shark and Homo sapiens is as high as 91%. Previous studies have shown that TBC1D15 protein has Rab-GAP activity for Rab7/Rab11 proteins.Therefore, we expressed APSL whole protein and fusion proteins MBP-Rab7-WT(wild type), MBP-Rab7-Q67L(activated) and MBP-Rab7-T22N(inactivated) in E. coli expression system. Then his pull down experiment was used to detect the interaction between APSL whole protein and the fusion protein Rab7. The results showed that the APSL whole protein can combine with Rab7, and the combined contents in different morphology Rab7 protein is different. This suggested that APSL whole protein has the Rab-GAP activity.In addition, to further study the the specificity of APSL whole protein GAP activity, recombinant plasmid p ETduet-His-sumo-Rab4/Rab5/Rab8/Rab11 were constructed respectively. Meanwhile, recombinant plasmid p ETduet-His-sumo-Rab7-WT was also reconstructed. Then induced expression in E. coli system respectively, and purified fusion proteins His-sumo-Rab4/Rab5/Rab7/Rab8/Rab11. Through detecting the increase rate of Pi content in the GTP-GDP exchange during Rab protein hydrolyzing, it is indirectly reflected that APSL whole protein has peculiarity for Rab proteins. The experimental results confirmed that APSL whole protein has peculiarity for Rab proteins, and it has the strongest Rab-GAP activity for Rab 11, but no for Rab4. According to its sequence structure and function characteristic, we can infer that APSL is the TBC1D15 protein from shark, named “shark TBC1D15” and a new nember of TBC1D15 family.To study the protein expression and regulation of shark TBC1D15 on the basis of our laboratory research platform, the research of mi RNA regulation was carried out to further explore a function which shark TBC1D15 protein may have. The method of dual luciferase reporter test was used to research the regulation of mi RNA on shark TBC1D15 target gene. The present result is that one of mi RNAs which regulates shark TBC1D15 target gene was selected.According to the Rab-GAP functions of TBC1D15 family, This study validated that the APSL whole protein is a novel nember of TBC1D15 family. This is the first to report shark TBC1D15 and add the richness of TBC1D15 family. The research on interaction between mi RNA and TBC family will provide new possibilities to explore the hypoglycemic mechanism of APSL and regulation of glucose metabolism.