Ubiquitin Ligase Enzyme Crrf And Tumor Development Of The Relationship Between Functional Studies

Invasion and metastasis of neoplasm cause lethal malignancy and poor therapy.The tumor metastasis is a multiple step and complex process that involved in such as tumor cells motility,angiogenesis,survival from the circulation.Immunohistochemisty has been widely used in the clinical diagnosis.Detection of some of cancer-related molecular markers indicates the malignance of tumor,raising evidence for clinical treatment. However,new molecular cancer-related markers should be explored and applied in order to accurately justify the malignance of patients.CRRF is a novel gene and its expression level is higher in malignant cancers than that of normal tissues according to analysis of genes expression by microarray.At the same time,CRRF protein could be detected in three higher invasive esophageal cancer cells KYSE180,KYSE450 and E.C.9706,but in another three esophageal cancer cells KYSE2,KYSE410 and KYSE510 the expression of CRRF could not be detected.It suggested that CRRF is relative to invasive ability of cancer cells.In order to identify the relationship of CRRF expression and the malignance in cancer patients,CRRF expression was detected in pancreatic cancer and colon cancer tissue arrays.The CRRF expression was significantly positively related with moderate and poor differentiated cancer tissues and not to well differentiated cancer tissues,that indicated that CRRF expression is involved in cancer malignance.The coding region of CRRF gene contains 1146 bases,it encodes 381 amino acids. According to domains analysis,two main domains,N-terminal protease-associated domain(PA domain) and C-terminal RING finger domain were predicted.CRRF protein also contains signal peptide and transmembrane domain.CRRF showed punctuated localization in cytosol and dissolved in Triton X-114,suggested a possible Typeâ… transmembrane protein.In addition,molecular weight of CRRF decreased after cells treated with Tunicamycin and protein samples treated with PNGase F.Using N-glycosylated putative point mutations,Asn88 was identified as the N-glycosylated site of CRRF.In vitro and in vivo ubiquitination assay suggested that CRRF demonstrates E3 ligase activity and UbcH5a and UbcH5c mediates this activity.Mutations in RING finger domain of CRRF abolished the E3 ligase activity,suggested the conservation and importance of RING finger domain for the function of CRRF.CRRF expression is also found to be related with solid tumor and lymphoma cancer cells sensitivity to As2O3.Pancreatic cancer cells and Raji cells,which express higher CRRF,showed sensitive to As2O3.However,pancreatic caner cells and Jurkat cells, which are hard to detect CRRF expression,showed insensitive to As2O3.Snapin was screened and identified as CRRF interacting protein.Proximal C terminal of CRRF mediated the interaction between CRRF and Snapin.Using in vitro ubiquitination assay CRRF ubiquitinates Snapin.Finally,Snapin and CRRF were co-transfected into COS-7 cells to detect the expression relationship between these two proteins,higher Snapin protein level is detected in higher CRRF protein expressed cells. This data suggested CRRF mediating Snapin ubiquitination maybe is involved in Snapin other function and not for proteasome degradation.