Molecular Mechanism Of Ubiquitin Ligase CHIP Regulatory Nucleotide Synthase CAD

CAD is a single multi-enzyme protein formed by aspartate transcarbamylase(ATC),dihydrowheyase(DHO)and carbamoyl phosphate synthase 2(CPS-2),which participates in pyrimidine nucleosides Acid synthesis.CAD mutations can lead to neurological diseases and hereditary metabolic diseases,and excessive activation of CAD can induce cancer.Ubiquitination of protein is a common post-translational modification method,which participates in almost all the regulation of life activities.It is indispensable in protein degradation,metabolism and functional regulation.As an E3 ubiquitin ligase,CHIP can participate in the ubiquitination modification of a variety of substrate proteins and promote the degradation of substrate proteins.At present,the ubiquitination modification mechanism of CAD protein is still unclear.This article aims to explore the molecular mechanism of ubiquitin ligase CHIP regulating the degradation of CAD protein,and the tumor suppressor effect of CHIP in colon cancer.In this paper,HEK293 T cells and HCT116 cells were used as research materials,and a stable transgenic cell line was constructed through the method of lentivirus packaging.In this study,an interaction between CHIP protein and CAD protein was detected through the co-immunoprecipitation experiment(CO-IP).Through the construction of CHIP and CAD domain vectors and CO-IP test,the experimental results It shows that CAD interacts with the TPR domain of CHIP through the GLN/CPSⅡ domain,ATC domain and the TPR domain of CHIP;We further adopted the Denature IP experimental method and found that CHIP can significantly promote the ubiquitination of CAD;In addition,Western blot experiments show that CHIP can promote the degradation of CAD protein in colon cancer cell HCT116 through the proteasome pathway;Studies have shown that CHIP has two functional sites,K30(Hsc70 interaction site)and H260(catalytically active site).Therefore,we constructed a mutant of the two functional sites of CHIP and found that the two functional sites of CHIP are necessary for CHIP to regulate CAD;Studies have confirmed that the up-regulation of CAD protein levels is closely related to the occurrence of cancer.Therefore,we used clone formation experiments,cell proliferation experiments,and cell scratch experiments to detect the effects of CHIP on the clonal formation,proliferation and migration of HCT116 cells.No significant effect on cell migration.In summary,CHIP is the E3 ubiquitin ligase of CAD protein,which degrades CAD through the proteasome pathway in colon cancer cells,and CHIP inhibits colon cancer cell clone formation and cell proliferation.This study provides a theoretical basis for further exploration of the molecular mechanisms regulating CAD,and provides new ideas for tumor treatment.