| Objective : The highly conserved eukaryotic ubiquitin-proteasome pathway (UPP) plays a pivotal role in protein homeostasis. The most widespread mechanism of ubiquitin action is probably in the context of protein degradation by poly-ubiquitination and proteasome, which is critical in regulating numerous cellular processes including cell cycle progression and transcriptional activation, immune reaction, oncogenesis and inflammation. It has also become apparent that ubiquitination is a reversible reaction, and the removal of the ubiquitin is catalyzed by processing proteases that have been generically named deubiquitinating enzymes (DUBs). There are 95 deubiquitinating enzymes in human. The family of deubiquitinating enzymes is divided into five subfamilies, including ubiquitin-specific proteases (USPs), ubiquitin C-terminal hydrolases (UCHs), Machado-Joseph disease protein domain proteases (MJDs), autophagins, otubains, ataxin-3/josephin ubiquitin proteases (OTUs), and JAMM isopeptidases.The largest and most diverse of these subfamilies is the ubiquitin-specific protease (USP) group. Ubiquitin specific proteases (USP) can remove ubiquitin from specific protein substrates and allow protein salvage from proteasome degradation to regulate protein half life and function. The current study found that many of the human ubiquitin-specific protease with a variety of physiological and pathological conditions is closely related to the occurrence of the disease. For example, USP2a in prostate cancer has an anti-apoptotic effect, therefore, USP2a as an oncogene present in the human body; UCH-L1 gene mutations cause autosomal dominant hereditary disease genes of Parkinson's disease; HAUSP (USP7) can be specific binding p53 ubiquitination chain and deubiquitinate p53, reversing degradation and thus inhibit tumor cell growth, so HAUSP as a tumor suppressor in the body.In the process of our study, research findings indicate that usp46 is a quantitative trait gene regulating mouse immobile behavior in the tail suspension and forced swimming tests. They found that CS mice show negligible immobility in inescapable situations rather than C57BL/6J mice. The main difference between them is one 3-bp deletion (coding a lysine residue) in usp46. Thus, usp46 affects the immobility in the TST and FST. They make a hypothesis that USP46 may implicate in depression.In this study, we aim to clone a ubiquitin-specific protease, USP46, and investigated the expression level of usp46 of rat tissues, establishment of deubiquitinating enzyme activity detection system, identify wide type USP46 and mutation USP46 (△K92) deubiquitinating enzymatic activity. Try to explore the molecular mechanism of the difference between CS mice and C57BL/6J of immobile behavior in the tail suspension and forced swimming tests for the etiology of depression research clues. At the same time to lay the foundation for molecular epidemiological studies of the deubiquitinating enzymes.Methods:(1) Using the molecular cloning methods to Clone the usp46 gene of rat. Total RNA was obtained from rat brain using TRIzol. The usp46 of rat was amplified and cloned by RT-PCR. The amplified fragment was inserted into pGEM T-Easy vector for DNA sequence analysis.(2) Relative Quantitative real time PCR was performed to study the distribution in rat tissues of usp46. The results derived from qRT-PCR were analyzed based on threshold cycle (Ct) values of a method called 2-ΔΔCt method in accordance with GAPDH gene to standardization, and compare with the expression level of brain.(3) Then usp46 was subcloned in-frame with GST into the expression vector pGEX-6P-1. The GST-USP46 fusion protein was expressed in DH.5αE. coli under the IPTG induction, and detects the solubility of fusion protein.(4) Construction of deubiquitinating enzyme activity assay system and detection of rat USP46 deubiquitinating enzymatic activity. USP46 was assayed basically following the method by using T7-driven isopropyl-β-D- thiogalactoside (IPTG)-inducible USP46 expression plasmid and a plasmid expresses the GST–Ub52 fusion protein substrate. We generate pAC-T7 plasmid and pAC-T7-usp46 plasmid using directional cloning method. For cleavage of the GST-Ub52 substrate, E. coli strain BL21 cells harboring pGEX-Ub52 were further transformed with either pAC-T7-usp46 or pAC-UBP2 (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.(5) Site-directed mutations including usp46 (C44S) and usp46 (△K92), and detect their deubiquitinating enzyme activity following the above system. The Cys44 within the usp46 conserved Cys-box of rat was mutated to serine using deoxyoligonucleotides and the Stratagene Quik-ChangeTM site-directed mutagenesis kit according to the manufacturer's specifications. The deletion of Lys92 of usp46 is same as above. Using BandScan software analysis the deubiquitinating enzyme activity, and use the positive control of the proteolyses products of 36 KDa protein band as a standard (brightness set at 100).(6) Using the molecular cloning methods to clone the usp46 gene of human from TE-1 cell line, generate mutation usp46 (C44S) of human and detect their deubiquitinating enzymatic activity.Results:(1) We have cloned a member of USP family, USP46, from rat brain. USP46 encodes 366 amino acids with a molecular weight of approximately 44 kDa containing highly conserved Cys, Asp, His domains characteristic of the ubiquitin-specific proteases.(2) We also investigated the expression level of usp46 by Real-Time RT-PCR in rat tissues. The result showed that usp46 mRNA moderate expression in various organs including testis and kidney, which is 1.48 and 1.12 times than brain, and weak expression in liver, heart and skeletal muscle, which is 0.59, 0.19 and 2.64 times compared to brain. However, usp46 is expressed strongly in spleen and lung, which is 2.89 and 2.64 times than brain. (3) The GST fusion protein, GST-USP46, expressed in the transformants was approximate 70 kDa as determined in SDS-PAGE analysis and was indissolvable.(4) Deubiquitinating enzyme activity assay system well developed. The assays demonstrated that USP46 of rat has deubiquitinating enzymatic activity. When the wild-type usp46 was co-expressed with GST-Ub52, the amount of the 42 KDa GST-Ub52 proteins was reduced and, concomitantly, a 36 KDa proteolysed band was produced. Thus, usp46 has deubiquitinating enzyme activity.(5) The deubiquitinating enzyme activity of USP46 disappeared when Cys44 was replaced by Ser, interestingly, after deletion of Lys92 of usp46, the deubiquitinating enzyme activity declined from 66.90% to 47.90%. It may be the main cause of the difference between CS mice and C57BL/6J of immobility behavior in the tail suspension and forced swimming tests.(6) We have cloned usp46 gene of human from TE-1 cell line, usp46 of human has 99.7% amino acid identity with rat and 100% amino acid identity with mouse. It has deubiquitinating enzyme activity, while Cys44 was replaced by Ser, the activity disappeared.Conclusions:(1) We have successful cloned a member of USP family, USP46, from rat brain.(2) The expression level of usp46 mRNA varies from brain in defferent tissues of rat.(3) The GST fusion protein, GST-usp46, expressed in the transformants was approximate 70 KDa as determined in SDS-PAGE analysis and was indissolvable.(4) Deubiquitinating enzyme activity assay system well developed. The assays demonstrated that USP46 of rat has deubiquitinating enzymatic activity.(5) The deubiquitinating enzyme activity of USP46 disappeared when Cys44 was replaced by Ser, while deletion of Lys92, the activity of USP46 declined significantly. It may provide the molecular evidence of the main cause of the difference between CS mice and C57BL/6J of immobility behavior in the TST and FST tests for the etiology of depression research clues.(6) We also isolated usp46 of human from TE-1 cell line and proved it also has deubiquitinating enzymatic activity. Lay the foundation for molecular epidemiological studies of the deubiquitinating enzymes.
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