Suppressor Of Fused Is Downregulated By Speckle-type POZ E3 Ubiquitin Ligase Adaptor Protein

The identification of the roles for HH proteins supported the model of developmental biology,in which organizer tissues secrete a morphogen,HH in this case,that diffuses to trigger differential cellular responses according to its concentration.The major components in Hedgehog(Hh)family regulate a variety of developmental processes and tumorigenesis.Dysregulation of the HH pathway was found to be responsible for congenital syndromes and various cancers.Based on its roles in diverse areas of cell and developmental biology,expecially in the development of cancer,members of research of developing small molecular inhibitors targeting Hedgehog signaling pathway have been carried out in the recent years.As a negative regulator of the pathway,Sufu can interact with all Gli proteins in a complex.The Gli proteins are repressed by Sufu through tethering Gli in the cytoplasm.Besides,Sufu facilitate the phosphorylation and proteolysis processes of the full length form of Gli protein into the repressor form;and Sufu protects Gli2 and Gli3 from degradation.So,Sufu is very important to Gli protein functions even regulate the activity of the whole pathway.Speckle-type POZ(pox virus and zinc finger protein)protein(SPOP)is an E3 ubiquitin ligase adaptor protein that is frequently mutated in prostate and endometrial cancers.It usually binds to the E3 ligase cullin 3 and form a complex to promote substrates protein into degradation.In 2009,one group demonstrated that Sufu antagonizes Spop,preventing degradation of full-length Gli2 and Gli3;and the process of Sufu-Spop antagonism is evolutionarily conserved since Drosophila Sufu protects Ci from Hib-mediated degradation through competitive binding to Ci.In 2014,Zhang Qing group found that in Drosophila Hh signaling promotes downregulation of Sufu through its target protein HIB(Hh-induced BTB protein)Epistasis analysis indicates that HIB downregulates Sufu through Crn.Finally,they show that Spop,the mammalian homologue of HIB,can substitute HIB to downregulate Sufu level in DrosophilaTo confirm Spop can regulate Sufu in mammal like Hib in Drosophila,we conduct experiments in cell lines to change the expression of Spop protein and test Sufu expression.Then we found that Spop can reduce Sufu protein expression,but not mRNA expression level.In addition,we realize that Spop does not bind to Sufu derictly by CO-IP assay;and Spop has no relationship with the stability of Sufu.So,Sufu is not one substrate of Spop.Interestingly,Spop can facilitate the ubiquitination degradetion of Sufu.Besides,after treatment of ActD,we found that Spop can destabilize mRNA of Sufu and affect the translation progress of Sufu.Mechanismly,Spop could reduce Sufu expression through Pten.Simultaneously,we detect the level of Spop and Sufu in human renal cell carcinoma tissues and found that Spop is overexpressed while Sufu is downregulated.And Sufu plays its role as a tumor suppressor gene in ccRCC cell lines;and Sufu promote cell apoptosis,inhibit cell proliferation?migration and invasion.In this study,we confirmed for the first time that SPOP can downregulate Sufu protein expression in mammals,and further demonstrated that downregulation of Sufu by SPOP may be accomplished through PTEN.And Sufu protein plays a role in suppressing cancer in renal cell carcinoma.In the subsequent studies,we will further study the detailed mechanism of SPOP downregulation of Sufu and the mechanism of Sufu's role in suppressing cancer in renal cell carcinoma.It is believed that these research results can provide further clues and supplements for Sufu's regulatory models and targeted therapy for renal cell carcinoma.