Molecular Mechanism Of Stability Of P21 By Ubiquitin-Specific Protease 11

Ubiquitin-specific pro tease11(USP11),a member of the deubiquitinating enzyme family,belongs to cysteine protease.Recent studies have revealed that USP11 can mediate the regulation of stability and functions of cellular proteins,including viral RNA replication proteins,DNA repair proteins and proteins involved in the TGF? and NF-?B signaling pathways.Meanwhile,USP11 plays an important role in the development and progression of diseases.p21,encoded by CDKNIA,belongs to CIP/KIP family.The first identified protein belongs to the cell cycle inhibitory(CKI),which are important for cell cycle and differentiation,cell migration,cytoskeletal dynamics,DNA repair and transcription,apoptosis,onset of senescence and aging.The expression and protein level of p21 are tightly regulated at transcription and post transcriptional level.The transcription of p21 can be regulated by p53-dependent or p53-independent pathways.With a half-life of only 20-60 min,p21 can be degraded in the ubiquitin-proteasome pathway.Though many intracellular proteins can stabilize p21 by different mechanisms,these proteins do not have the activity and function of cysteine protease.It is not clear whether exists a deubiquitinating enzyme,which can directly reverse the ubiquitination of p21 and stabilize p21.Using the LTQ Orbitrap Velos Rro,we find that USP11 may interacts with p21.We use the immunofluorescence,IP and GST pull-down experiments to show that USP11 directly interacts with p21.Employing the uspll knockdown assay and the overexpression of usp11 assay?the real-time PCR?the proteasome inhibitor MG 132 and the half-life of protein assay indicate that the USP11 can greatly increase the steady state level of p21 through extends the half-life of p21 and inhibit the degradation of p21 through the ubiquitin-proteasome pathway.p21,an important regulation factor of cell cycle,mainly regulates the cell cycle progression of G1/S phase.we find that interference of USP11 increases the proportion of S-phase cells,whereas overexpression of p21 counteracts this change in USP11 knockdown cell,which suggests that USP11 regulates the G1/S transition in p21-dependent manner.In order to clarify the biological function of USP11,we use MTT assays and colony formation assays to detect the effect of USP11 on the nonsmall cell lung cancer proliferation;Our data suggests that the interference of USP11 accelerate the proliferation of cells,whereas overexpression of USP11 restrains the proliferation of cells,which suggests that USP11 is very likely to inhibit the proliferation of the non small cell lung cancer.The study will reveal the potential molecular mechanism on the stabilization of p21 protein in the cell and may further broaden our understanding on the theory of cell cycle regulation.The clarification of the molecular function of uspll in non small cell lung cancer may provide a new target and the starting point for the prevention and treatment of the non small cell lung cancer.