Identification of candidate substrates of ubiquitin-specific protease 13 using 2D-DIGE.

International Journal of Molecular Medicine
Jianmin WangQingbao Tian

Abstract

The present study aimed to identify candidate substrates of ubiquitin-specific protease (USP)13 using two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). USP13 is a well-characterized member of the USP family, which regulates diverse cellular functions by cleaving ubiquitin from ubiquitinated protein substrates. However, existing studies indicate that USP13 has no detectable hydrolytic activity in vitro. This finding implies that USP13 likely has different substrate specificity. In this study, a USP cleavage assay was performed using two different types of model substrates (glutathione S-transferase-Ub52 and ubiquitin-β-galactosidase) to detect the deubiquitinating enzyme (DUB) activity of USP13. In addition, a proteomic approach was taken by using 2D-DIGE to detect cellular proteins whose expressoin is significantly altered in 293T cell lines following the overexpression of USP13 or its C345S mutant (the catalytically inactive form). The data indicated that USP13 still has no detectable DUB activity in vitro nor does C345S. The results of 2D-DIGE demonstrated that the expression of several proteins increased or decreased significantly in 293T cells following the overexpression of USP13. Mass spec-troscopy an...Continue Reading

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Citations

Mar 22, 2019·Toxins·Konstantinos KalogeropoulosChristopher T Workman
Jun 29, 2021·Frontiers in Molecular Biosciences·Shuaishuai HuXinsheng Wu
Apr 16, 2019·Biomedicine & Pharmacotherapy = Biomédecine & Pharmacothérapie·Yue WuShuyan Liu

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Methods Mentioned

BETA
ubiquitination
cleavage assay
electrophoresis
deubiquitination
co-immunoprecipitation

Software Mentioned

DeCyder
Mascot
SPSS
Odyssey

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