Deubiquitinase USP10 Stabilizes Smurf1 And Inhibits TGF-β/BMP Signaling

Backgrounds:Ubiquitin modification is a process in which ubiquitin that a small protein containing 76amino acids,covalently bands to the substrate protein by ubiquitin activating enzyme,ubiquitin conjugating enzyme and ubiquitin ligase.It can participate in a variety of cellular processes such as gene transcription,DNA damage repair,cell apoptosis and immune response by regulating the stability,activity,location and protein-protein interaction of modified proteins.Ubiquitination is an inducible and reversible process that dynamically affects functional proteins in response to intracellular and external stimuli.Deubiquitinase（DUB）is a class of ubiquitin-removing superfamily isopeptidases,which depends on its deubiquitination enzyme activity to remove ubiquitins from substrate proteins,so as to reverse the regulation effect of ubiquitinization modification on substrate proteins.Currently,the human genome encodes more than 100 DUB molecules,which can be divided into seven subfamilies（USPs,UCHs,MJDs,OTUs,JAMMs,MINDYs,ZUFSP）based on protein sequences and domain similarity characteristics.Ubiquitination modification enzymes and deubiquitination enzymes collectively regulate the homeostasis of ubiquitin in organisms.Hence,deubiquitination modification and ubiquitination modification are equally important for the regulation of substrate proteins.However,the identification of substrates of deubiquitination enzymes and their regulatory mechanisms still need to be further explored.Ubiquitin specific protease 10（USP10）belongs to the USP subfamily with the largest number of members.It is composed of the USP domain of the catalytic center in the C-terminal core and the disordered amino acid sequence in the N-terminal.Its full length contains 798 amino acids.USP10 is a cysteine hydrolase that performs a variety of functions at the cellular level by hydrolyzing the isopeptide bond formed between glycine at the C-terminal of ubiquitin and lysine on the substrate protein to remove ubiquitination modifications on the substrate protein.Current literatures have revealed that deubiquitinating enzymes USP10 interacted with G3BP1/2,TP53,AMPKα,MDM2,BECN1,NICD1,SIRT6,TRAF6 to involve in stress particle formation,metabolic regulation,autophagy activation,vascular morphology,acute kidney injury and inflammation regulation and other processes.However,the physiological function of the deubiquitination enzyme USP10 is not absolutely understood.In 2022,Li,Hongchang et al.constructed Usp10 knockout mice and explored its physiological function and molecular mechanism.USP10 interacted with the ubiquitin ligase MDM2to remove its K27 chain polyubiquitination modification through the deubiquitination activity.Activated MDM2 interacted and ubiquitination degraded p53.p53 interfered ATG12-ATG5 conjugated complex in cytoplasm and activated autophagy.When Usp10gene was deficiency in mice,cytoplasmic p53 could not be degraded,and neonatal mice died within 36 h after birth which results of autophagy defects and metabolic imbalance.In this study,Usp10 knockout neonatal mice had significantly smaller body weight and body size than wild-type neonatal mice,especially in kidney tissue.This suggests that the deubiquitination enzyme USP10 is also involved in the regulation of mouse histological development,but its specific regulatory mechanism remains unclear.Here we analyzed the gene microarray data GSE198574 from the kidney tissues of Usp10^+/+and Usp10^-/-neonatal mice.We found the TGF-β/BMP signaling pathway was activated in the kidney tissues of Usp10^-/-neonatal mice after pathway enrichment of differentially expressed genes.GEO2R and Metascape analyzed transcription factors enrichment of differentially expressed genes（the top 200 genes with the lowest P value）.Most of the top 200 differentially expressed genes were regulated by Smad1 and Smad5.Then we analyzed the changes in protein levels and m RNA levels of Smad1 and Smad5by comparing with wild-type Usp10 neonatal mice.The protein expression levels of Smad1 and Smad5 were significantly upregulated in the kidney tissues of Usp10knockout neonatal mice.We supposed that USP10 may indirectly regulate Smad1 and Smad5 protein levels by regulating negative regulators Smurf1 and/or Smurf2 of the TGF-β/BMP signaling pathway.Western blotting analyzed the expression changes of Smurf1 and Smurf2 in the kidney tissues of neonatal mice.The results showed that the expression of Smurf1 was downregulated in the kidney tissues of Usp10 gene knockout neonatal mice,while the expression of Smurf2 was unchanged.In addition,we constructed USP10^+/+and USP10^-/-HEK293T cell lines and overexpressed Smurf1 in the USP10 knockout HEK293T cells.Western blotting detected the changes of Smurf1,Smad1 and Smad5 protein levels in HEK293T cells,the results were consistent with mouse level.Therefore,Smurf1 may be the substrate protein of the deubiquitination enzyme USP10.USP10 interacted with Smurf1 by co-immunoprecipitation and GST-pull down experiments.USP10 could partially attenuate the polyubiquitination modification of Smurf1 by ubiquitination experiments.Previous studies have shown that USP10 regulates cell proliferation.The cell cycle regulator p21 is one of the downstream target genes of Smad1/5 transcriptional regulation.It was found that the protein level of p21 was increased in kidney tissues of Usp10 gene knockout neonatal mice.Finally,it was found that compared with normal HEK293T cells,the proliferation rate and clonal formation ability of USP10 knockout HEK293T cells were inhibited by CCK-8 and cell clonal formation experiments,while the proliferation ability of Smurf1overexpression cells was part restored by overexpressed Smurf1 in USP10 knockout HEK293T cells.These data indicated the deubiquitination enzyme USP10 inhibits TGF-β/BMP signaling pathway by stabilizing Smurf1,thus maintaining cell proliferation.Research contents:1.GEO2R and Metascape analyze the differentially expressed genes and pathway enrichment in microarray（GSE198574）of kidney tissues from Usp10^+/+and Usp10^-/-neonates.2.Western blotting and immunohistochemical techniques analyze the expression of core transcription factors in this pathway.3.The expression of the signaling pathway and candidate molecules are analyzed by immunoblotting and immunohistochemistry.4.Co-immunoprecipitation and GST-pull down experiments verify the interaction between USP10 and candidate molecules.5.The deubiquitination effect of USP10 on substrate molecules was confirmed by ubiquitination experiments.6.The expressions of p21 and Cleaved-caspase 3 were detected by western blotting.7.The effect of USP10 on cell capacity proliferation was analyzed by CCK-8 and clonal formation assay.Research results:1.TGF-β/BMP signaling pathway was activated in kidney tissues from Usp10^-/-neonates.2.The protein levels of Smad1 and Smad5 were upregulated in kidney tissues from Usp10^-/-neonates.3.The protein level of Smurf1 was downregulated in kidney tissues from Usp10^-/-neonates.4.USP10 interacted with Smurf1.5.USP10 removed part polyubiquitination modification through its deubiquitination enzyme activity.6.The expression of p21 was upregulated in kidney tissues from Usp10^-/-neonates,and the Cleaved-caspase 3 expression was unchanged.7.The proliferation rate and cloned numbers of USP10 knockout HEK293T cells were inhibited,which were partially recovered by overexpressing Smurf1.Conclusion:1.The protein levels of Smad1 and Smad5 are upregulated,which activated TGF-β/BMP signaling pathway in kidney tissues from Usp10^-/-neonates.2.USP10 stabilizes Smurf1 protein levels.3.USP10 interacts and deubiquitinates Smurf1.4.USP10 maintains cell proliferation by inhibiting TGF-β/BMP signaling pathway.