Functional Analysis Of FAT10 Functional Domain

A family of small proteins related to ubiquitin called ubiquitin like proteins (UBLs) has been identified in recent years. FAT10is one member of the UBLs family, possessing two ubiquitin-like domains, and constitutively expressed in lymphoid organs like spleen, gut, lymph nodes and thymus. FAT10was also over-expressed in several epithelial cancers like hepatocellular, colon, ovary and uterus carcinomas. It is reported that the overexpression of FAT10causes apoptosis in a murine fibroblast cell line, and FAT10exhibits a close relationship with p53. These findings strongly suggest that FAT10might play important roles in cell-cycle control and tumorigenesis. It was further reported that FAT10can target proteins for degradation via covalent conjugation. However, the functional role of FAT10protein is poorly understood. Further studies are needed to clarify the mechanism of FAT10, which is probably a new target for anticancer therapy.In our present work, the coding sequences of wild-type FAT10, carboxyl-terminal diglycine deficient mutant FAT10â–³GG, and two separate ubiquitin domains FAT10-N, FAT10-C were cloned into pCMV-HA expression vector respectively. After transfected with these FAT10constructs, HEK293T cells were treated with26S proteasome inhibitor MG132before harvest. The whole cell lysates were then subjected to Western blotting analysis. We find both carboxyl-terminal diglycine motif and two ubiquitin like domains are crucial to the substrate-binding ability of FAT10. FAT10-conjugated proteins undergo degradation in a26S proteasome-dependent manner. For further study, we constructed EGFP-fused FAT10and its two ubiquitin like domains, and found that FAT10, FAT10-N and FAT10-C all could enhance the degradation rate of EGFP. We also found FAT10and its two ubiquitin domains caused apoptosis as indicated by annexin V cell surface staining detection. In conclusion, both carboxyl-terminal diglycine motif and two ubiquitin-like domains are crucial to FAT10-substrate binding, degrading and cell apoptosis.