SCYL1-BP1 And XPC Interfere In P53-MDM2 Feedback Loop

SCYL1-BP1 and XPC interfere in p53-MDM2 feedback loopThe p53 protein is known as a "guardian of the genome" because of its crucial role in coordinating cellular responses to genotoxic stress. It is the most famous tumor suppression gene and can be mediated by a variety of mechanisms, including cell cycle arrest, apoptosis, and cellular senescence. p53 is tightly regulated, although the precise mechanisms of p53 activation are not fully understood, they are generally thought to involve posttranslational modifications, including ubiquitination, acetylation, phosphorylation, sumoylation, neddylation, methylation, and glycosylation of the p53 polypeptide. And it is well accepted that the ubiquitin-proteasome pathway plays a major part in the scope of p53 regulation.Both Pirh2 and MDM2 are two RING finger E3 Ligases of p53. The SCYL1-BP1 protein was identified as an interacting partner of E3 ligase Pirh2 and MDM2 in the yeast two-hybrid screening. Further investigation suggested there are two interactions involving in different mechanisms. The SCYL1-BP1 can be ubiqutinated and degraded by Pirh2 but not by MDM2, which supported the SCYL1-BP1 protein can be regulated by Pirh2 acting as a substrate. On the contrary, while SCYL1-BP1 binds to ubiquitin E3 ligase MDM2, it promotes MDM2 self-ubiquitination and results in a reduction on MDM2 protein level. Moreover, SCYL1-BP1 has a significant inhibition on MDM2 transcription.SCYL1-BP1 protein levels increasement in cells cause parallel changes in the amount of p53 protein, which may come from the inhibition of MDM2-mediated p53 ubiquitination. SCYL1-BP1 does not alter human papillomavirus E6 mediated p53 ubiquitination, indicating that the effect is specific for MDM2. Increases in SCYL1-BP1 levels in cells led to higher transcriptional activation of p21 and gadd45, a reduced rate of cellular proliferation, an increase in apoptosis and an inhibition in tumorigenicity.CHIP experiments show more p53 stabilized by SCYL1-BP1 are recruited to p21 promoter than mdm2. That means SCYL1-BP1 regulated MDM2-p53 feedback loop in one way, and explains that why SCYL1-BP1 stabilized p53 didn't impair MDM2 degradation by transactivating MDM2. Thus we propose that SCYL1-BP1 is a new regulator of the MDM2-p53 feedback loop and it may be a potential tumor suppressor.Different from SCYL1-BP1 regulates p53 ubiquitinaiton, XPC (Xeroderma Pigmentosum C) functions in post-ubiquitination step of p53 but before proteasome degradation. XPC is one of the most important DNA repair proteins, which can recognize UV-lesion in cells, bind to the DNA damage site and recruit other DNA repair proteins. Here we demonstrated that XPC also functions on ubiquitin-proteasome pathway. First of all, XPC binds to a ubiquitin E4, Ufd2a. Second, a man-made ubiquitination pathway marker- UbG76V-GFP became stabilized when XPC defect. Last not least, we found a natural substrate of XPC in mammalian cells-p53. In XPC defect cells, p53 protein turnover is slower than in XPC wild-type cells, but ubiquitinated p53 is accumulated. It suggests that XPC regulates post-ubiquitination step but before proteasome degradation. p21 and MDM2 turnover are not affected by XPC, indicating XPC is not a general regulator on the ubiquitin-proteasome pathway.We further found that XPC has a selection on ubiquitinated p53. XPC binds to MDM2, the ubiquitin ligase of p53, and helps degradation of MDM2 ubiquitinated p53. However, XPC seems not interested in another ubiquitin ligase of p53, Pirh2 ubiquitinated p53. It has no affection on the degradation of Pirh2 ubiquitinated p53. And we also failed to detect any interaction between XPC and Pirh2. Thus, we suggest XPC is brought to the ubiquitinated substrate through the binding with MDM2, and then XPC functions to transfer the ubiquitinated p53 to the proteasome.Moreover, in the normal condition, overexpression of XPC in XPC wt cells, degradation of p53 is not changed. After UV treatment, p53 stabilizes in cells without XPC overexpression, but becomes degrading when cells have extra XPC. It proves XPC participates in p53 ubiquitinated degradation and indicates that XPC may be a very important protein that connects both DNA repair and ubiquitin-proteasome pathways.