As the major pathway mediating specific protein degradation in eukaryotes, ubiquitin-proteasome system (UPS) is involved in various physiological and pathological processes such as cell cycle regulation, immune response, signal transduction and DNA-repair. Deubiquitinases (DUB) maintain the balance of UPS and related physiological processes via reversibly removing ubiquitin from the covalently modified protein substrates, which have been implicated in various disease processes in case of their imbalance expression. Because DUB plays critical regulating roles in the UPS pathway, they may be also the ideal drug targets for severe and intractable human diseases, such as cancer and neurodegenerative disease. With the rapid development of proteomic technology, systematical investigation of specific substrates and interacting proteins of varied DUB via mass spectrometry approach may shed light on these DUB's biological function and regulating roles in the physiological and pathogenic states. In this review, we briefly introduce the characteristics of DUB and summarize the recent application and progresses of proteomics in DUB research.
Humans ; Mass Spectrometry ; Proteasome Endopeptidase Complex ; metabolism ; Proteomics ; Signal Transduction ; Ubiquitin ; metabolism ; Ubiquitin-Specific Proteases ; metabolism

Ubiquitin, a critical small molecular protein, plays an important role in regulating multiple signaling pathways. Ubiquitination is a post-translational modification induced by ubiquitin through an ATP-dependent enzyme catalyzed reaction. A large number of proteins in the complicated signaling network participate in wound healing. This paper reviews the research progress in regulation of ubiquitin and ubiquitination for wound healing processes regarding the recent years.
Signal Transduction ; physiology ; Ubiquitin ; metabolism ; Ubiquitin-Protein Ligases ; physiology ; Ubiquitination ; Wound Healing ; physiology

Humans ; Protein Binding ; genetics ; Protein Structure, Secondary ; Ubiquitin Thiolesterase ; genetics ; metabolism ; Ubiquitin-Specific Peptidase 7

Ubiquitin-Protein Ligases/metabolism* ; Proteolysis ; Biological Products ; Ligands

Cytokines ; metabolism ; Humans ; Leukocytes, Mononuclear ; immunology ; secretion ; Ubiquitin ; blood

The ubiquitin-proteasome system plays an important role in protein degradation. The process of ubiquitination requires ubiquitin activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3 to complete the coordination. Our previous studies have shown that HUWE1 (HECT, UBA and WWE domain containing 1), as an E3 ubiquitin ligase, can degrade epidermal growth factor receptor (EGFR) to inhibit renal tubulointerstitial fibrosis. However, E2 ubiquitin-conjugating enzymes binding to HUWE1 are still unclear. The aim of the present study was to identify E2 ubiquitin-conjugating enzymes of HUWE1. Real-time PCR was used to identify E2 ubiquitin-conjugating enzyme that may interact with HUWE1. The expression of E2 ubiquitin-conjugating enzyme was detected in kidney of unilateral ureteral obstruction (UUO) mice and HK-2 cells treated with transforming growth factor-β (TGF-β). The results showed that the expressions of E2 ubiquitin-conjugating enzyme UBE2Q2 were significantly down-regulated at both RNA and protein levels in UUO kidneys. The expression of UBE2Q2 was also down-regulated in HK-2 cells stimulated with TGF-β, which was consistent with the change in the expression of HUWE1. These findings indicated that UBE2Q2 expression was synergistic with HUWE1 in the injured kidney. Co-immunoprecipitation (Co-IP) experiments showed that HUWE1 interacted with UBE2Q2 in HK-2 cells. The co-localization of UBE2Q2 and HUWE1 was confirmed by cell immunofluorescence staining. After knocking down UBE2Q2 by siRNA, ubiquitin binding to HUWE1 and EGFR was decreased. In sum, our results demonstrated that UBE2Q2, ubiquitin-conjugating enzyme, works with HUWE1 to mediate ubiquitination and degradation of target protein in kidney.
Animals ; Cell Line ; Fibrosis ; Humans ; Kidney Diseases ; Mice ; Ubiquitin-Conjugating Enzymes/metabolism* ; Ubiquitin-Protein Ligases/metabolism* ; Ubiquitination

Ubiquitination is one of the reversible protein post-translational modifications, in which ubiquitin molecules bind to the target protein in a cascade reaction of ubiquitin activating enzymes, ubiquitin conjugating enzymes, and ubiquitin ligases. The deubiquitinating enzymes (DUBs) remove ubiquitin residues from the substrates, which play key roles in the formation of mature ubiquitin, the removal and trimming of ubiquitin chains, as well as the recycling of free ubiquitin chains. Ubp14, a member of the ubiquitin specific proteases family in Saccharomyces cerevisiae, is mainly responsible for the recycling of intracellular free ubiquitin chains. To investigate its global biological function, a ubp14∆ mutant was constructed by homologous recombination technique. The growth rate of ubp14∆ mutant was lower than that of the wild-type (WT) strain. Using stable isotope labeling by amino acids in cell culture (SILAC) combined with deep coverage proteomics analysis, the differentially expressed proteins of ubp14∆ mutant relative to the wild-type strain were systematically analyzed. A total of 3 685 proteins were identified in this study, and 109 differentially expressed proteins were filtered out by statistical analysis. Gene ontology analysis found that differentially expressed proteins caused by Ubp14 loss were mainly involved in amino acid metabolism, REDOX, heat shock stress and etc, which shed light on the broad biological function of this DUB. This study provides highly reliable proteomic data for further exploring the biological functions of the deubiquitination enzyme Ubp14, and further understanding the relationship between the free ubiquitin homeostasis and biological process regulation.
Saccharomyces cerevisiae/metabolism* ; Proteomics ; Endopeptidases/metabolism* ; Ubiquitin/metabolism* ; Ubiquitination ; Proteins/metabolism* ; Deubiquitinating Enzymes/metabolism* ; Biological Phenomena

The ubiquitin-related modifier Urm1 can be covalently conjugated to lysine residues of other proteins, such as yeast Ahp1 and human MOCS3, through a mechanism involving the E1-like protein Uba4 (MOCS3 in humans). Similar to ubiquitination, urmylation requires a thioester intermediate and forms isopeptide bonds between Urm1 and its substrates. In addition, the urmylation process can be significantly enhanced by oxidative stress. Recent findings have demonstrated that Urm1 also acts as a sulfur carrier in the thiolation of eukaryotic tRNA via a mechanism that requires the formation of a thiocarboxylated Urm1. This role is very similar to that of prokaryotic sulfur carriers such as MoaD and ThiS. Evidence strongly supports the hypothesis that Urm1 is the molecular fossil in the evolutionary link between prokaryotic sulfur carriers and eukaryotic ubiquitin-like proteins. In the present review, we discuss the dual role of Urm1 in protein and tRNA modification.
Animals ; Humans ; Models, Biological ; RNA, Transfer ; metabolism ; Sulfur ; metabolism ; Ubiquitin ; metabolism ; Ubiquitins ; metabolism

OBJECTIVETo explore action mechanism of electroacupuncture (EA) on treatment and prevention of Parkinson's disease (PD).

METHODSFifty clean-grade SD rats were randomly divided into a normal group, a control group, a model group, a pretreatment group and a treatment group, ten rats in each one. The PD model was established by subcutaneous injection of rotenone in neck-back skin (2 mg/kg, dissolved in sun-flower oil, 2 mg/mL in density). The equal-volume sun-flower oil that didn't include rotenone was applied in the control group at the same area as the model group. EA was applied in the treatment group at "Fengfu" (GV 16) and "Taichong" (LR 3) with interrupted wave, 2 Hz in frequency, 1 mA in density, for 20 min. The treatment was given once day for conti-nuous 28 days. Rats in the pretreatment group received the same EA as the treatment group for 7 days, and then put into model establishment. After the model establishment, the rats received no treatment and were sacrificed after 28 days. No EA was given in the normal group, model group and control group. The ethology changes were observed and scored. The expression of Parkin, ubiquitin C terminal hydrolase-L1 (UCH-L1) and ubiquitin activating enzyme-1 (UBE1) in substantia nigra was tested by Western-blot method. The positive cell numbers of alpha-synuclein, ubiquitin (UB) and tyrosine hydroxylase (TH) in substantia nigra was tested by immunohistochemical method.

RESULTSThere were abnormal ethology manifestation such as yellow and coarse hair, arched back, weaken behavior of resisting arrest and slow movement, which was more relieved in the treatment group and pretreatment group. Compared with normal group and control group, the expression of Parkin, UCH-L1, UBE1, UB, TH in the model group was obviously decreased while alpha-synuclein was obviously increased (all P<0.01). After EA or pretreatment, the expression of Parkin, UCH-L1, UBE1, UB, TH in the treatment group and pretreatment group was higher than that in the model group while expression of alpha-synuclein in the treatment group and pretreatment group was lower than that in the model group (all P<0.01).

CONCLUSIONEA or pretreatment could not only have protective effect for rats with PD, but also increase function of ubiquitin-proteasome system, indicating action mechanism of EA on treatment and prevention of PD may be related with ubiquitin-proteasome system.

Animals ; Electroacupuncture ; Humans ; Male ; Parkinson Disease ; metabolism ; therapy ; Proteasome Endopeptidase Complex ; metabolism ; Rats ; Rats, Sprague-Dawley ; Ubiquitin ; metabolism ; Ubiquitin-Protein Ligases ; metabolism ; alpha-Synuclein ; metabolism

OBJECTIVE: To explore the interaction between BCL2-associated athanogene 5 (BAG5) and Parkin protein,and the regulatory mechanism of BAG5 protein on the level of Parkin protein. METHODS: We performed GST pull-down assay to identify which domain of PINK1 interacted with Parkin, and generated different deletions of BAG5 to identify the domains. Chase time experiment was done to determine the effect of co-regulation of BAG5 protein on the ubiquitination. We further examined the possible interaction between Parkin and PINK1 in 293A cells by co-immunoprecipitation method. RESULTS: BAG5 directly interacted with the Parkin, and all the 4 BAG domains interacted with the Parkin. BAG5 stabilized the Parkin by interfering its degradation via the ubiquitin-mediated proteasomal pathway. CONCLUSION BAG5 directly interacts with the Parkin, and BAG5 stabilizes the Parkin via the ubiquitin-mediated proteasomal pathway.
Adaptor Proteins, Signal Transducing ; metabolism ; Humans ; Parkinson Disease ; metabolism ; Protein Binding ; Ubiquitin-Protein Ligases ; metabolism ; Ubiquitination