| Objective:The modification of cellular proteins by ubiquitin is an essential regulatory mechanism in many biological processes such as cell cycle progression, DNA-repair, signal transduction and transcriptional activation. This post-translational modification is a dynamic and reversible process controlled by multiple ubiquitin-conjugating and deubiquitinating enzymes. Deubiquitinating enzymes can specifically cleave ubiquitin from ubiquitin-conjugated protein substrates.Deubiquitinating enzymes are a large family of proteases consisting of approximately 95 members, and they are divided into five subfamilies, including the Ubiquitin C-terminal Hydrolases (UCHs), the Ubiquitin Specific Proteases (USPs), the Machado-Joseph Disease Protein Domain Proteases (MJDs), the Ovarian tumour Proteases (OTUs) and the JAMM Motif Proteases. USPs are the largest family that vary greatly in size and structural complexity, they consist in human of approximately 54 members and contain several conserved regions in their amino acid sequence, including the Cys, His, and Asp/Asn residues.In this study, we have identified a novel gene encoding a deubiquitinating enzyme, usp13, from rat brain and human TE-1 cell, and investigated the expression level of usp13 in different rat tissues. We also established two deubiquitinating enzyme activity detection systems, to explore the best method for identifying deubiquitinating enzymatic activity of USP13, at the same time to lay a foundation for the further study on usp13.Methods:(1) The molecular cloning method was used to clone the usp13 gene of rat. Total RNA was isolated from rat brain using TRIzol. In order to clone easily, the usp13 gene was divided into two parts: usp13-5′and usp13-3′first strand cDNA was synthesized from total RNA using Quant Reverse Transcriptase and PCR was performed with the usp13-specific primers. The amplified fragment was subcloned into pGEM-T easy vector for DNA sequence analysis.(2) Relative Quantitative real time PCR was performed to study the tissues distribution of rat usp13. The results were analyzed based on threshold cycle (Ct) values, and expression differences were determined by raising 2 to the -ΔΔCt power, in accordance with GAPDH gene to standardization, and compare with the expression level of brain.(3) Expression of the rat USP13 fusion protein. First, usp13-5′gene was subcloned in-frame with GST into the expression vector pGEX-6P-1 to obtain the pGEX-usp13-5′plasmid, then, usp13-3′fragment was inserted into the pGEX-usp13-5′plasmid, to obtain the pGEX-usp13 plasmid. GST-USP13 fusion protein was expressed in DH5αE. coli under the IPTG induction.(4) Deubiquitinating enzyme activity assay systems.①T7-USP13/ GST-Ub52 system: We generated pAC-T7-usp13 plasmid, which is T7-USP13 expression plasmid. For cleavage of the GST-Ub52 substrate, E. coli strain BL21 cells harboring pGEX-Ub52 which can express the GST–Ub52 fusion protein substrate were further transformed with either pAC-T7-usp13 or pAC-T7-usp46 (positive control) induced by IPTG. GST fusion proteins and their proteolysed products were purified by GSH-Sepharose-TM Resin system and detected by 10% SDS-PAGE.②GST-USP/Ub-Met-β-gal system: Plasmid pAC-M-β-gal expresses the Ub-Met-β-gal fusion protein substrate in a pACYC184 Cmr replicon. Escherichia coli BL21 bacteria harboring pGEX-usp13 were transformed with pAC-M-β-gal, Ampr Cmr colonies were grown and induced with IPTG, and total protein extracts were analyzed by Western blotting with anti-β-gal antibody, pGEX-usp46 was used as a positive control(5) Molecular cloning and engymatic avtivity analysis of usp13 gene from human TE-1 cell line.Results:(1) We cloned usp13 gene open reading frame from rat brain, it consists of 2,577 bp and encodes 858 amino acids with a predicted molecular weight of 96 kDa and contains the conserved Cys, His and Asp domains defined as one of characteristics for deubiquitinating enzymes.(2) We also investigated the expression level of usp13 by Real-Time RT-PCR in rat tissues. The result showed that usp13 mRNA moderate expression in various organs including heart, liver and spleen, which is 1.04, 1.02 and 0.91 times than brain, and weak expression in testis and skeletal muscle,. However, usp13 is expressed a little higher in lung and kidney, which is 1.17 and 1.13 times than brain.(3) The GST-USP13 fusion protein, expressed in the E. coli DH5αwith a molecular weight of approximately 120 kDa as determined in 10% SDS-PAGE analysis. The fusion protein was indissoluable when induced with IPTG at 37℃for 4 hours. However, when the temperature lowered to 15℃and induced with IPTG for 40 hours, the fusion protein was partly dissoluable.(4) Two deubiquitinating enzyme activity assay systems were well established. Each of them can investigate the deubiquitinating enzyme activity of positive control, expression of USP13 and model substrate can also be observed. But both systems are not suitable for investigating the deubiquitinating enzyme activity of USP13.(5) We cloned usp13 gene open reading frame from human TE-1 cell line, usp13 of human consists of 2,592 bp and encodes 863 amino acids with a predicted molecular weight of 97 kDa. The deubiquitinating enzyme activity of human USP13 can not be investigated by the two systems.Conclusions:(1) We have successful cloned a novel ubiquitin specific protease, USP13, from rat brain. (GenBank accession number: GU459226)(2) The expression levels of usp13 mRNA vary in defferent tissues of rat.(3) The GST-USP13 fusion protein, expressed in the E. coli DH5αand it was indissolvable induced with IPTG at 37℃for 4 h, but partly dissoluable at 15℃for 40 h.(4) Two deubiquitinating enzyme activity assay systems were well established. But both systems were not suitable for investigating the deubiquitinating enzyme activity of USP13.(5) We also isolated usp13 from human TE-1 cell line (GenBank accession number: HM138083), and the deubiquitinating enzyme activity of human USP13 was not detected by the two deubiquitinating enzyme activity assay systems.
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