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Article Open Access

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

  • Authors:
    • Jianmin Wang
    • Yingli Liu
    • Lijuan Tang
    • Sufen Qi
    • Yingjun Mi
    • Dianwu Liu
    • Qingbao Tian
  • View Affiliations / Copyright

    Affiliations: Department of Epidemiology and Statistics, School of Public Health, Hebei Medical University, Shijiazhuang, Hebei 050017, P.R. China
    Copyright: © Wang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Pages: 47-56
    |
    Published online on: May 10, 2017
       https://doi.org/10.3892/ijmm.2017.2984
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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 spec­troscopy analysis of gel spots identified 7 proteins, including 4 proteins with an increased expression, namely vinculin, thimet oligopeptidase, cleavage and polyadenylation specific factor 3, and methylosome protein 50, and 3 proteins with a decreased expression, namely adenylosuccinate synthetase, annexin and phosphoglycerate mutase. In addition, in the samples of 293T cell lines after the overexpression of USP13 and USP13 C345S, vinculin exhibited an increased expression, suggesting that it may be a candidate substrate of USP13. However, sufficient follow-up validation studies are required in order to determine whether vinculin protein directly interacts with USP13.
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Copy and paste a formatted citation
Spandidos Publications style
Wang J, Liu Y, Tang L, Qi S, Mi Y, Liu D and Tian Q: Identification of candidate substrates of ubiquitin-specific protease 13 using 2D-DIGE . Int J Mol Med 40: 47-56, 2017.
APA
Wang, J., Liu, Y., Tang, L., Qi, S., Mi, Y., Liu, D., & Tian, Q. (2017). Identification of candidate substrates of ubiquitin-specific protease 13 using 2D-DIGE . International Journal of Molecular Medicine, 40, 47-56. https://doi.org/10.3892/ijmm.2017.2984
MLA
Wang, J., Liu, Y., Tang, L., Qi, S., Mi, Y., Liu, D., Tian, Q."Identification of candidate substrates of ubiquitin-specific protease 13 using 2D-DIGE ". International Journal of Molecular Medicine 40.1 (2017): 47-56.
Chicago
Wang, J., Liu, Y., Tang, L., Qi, S., Mi, Y., Liu, D., Tian, Q."Identification of candidate substrates of ubiquitin-specific protease 13 using 2D-DIGE ". International Journal of Molecular Medicine 40, no. 1 (2017): 47-56. https://doi.org/10.3892/ijmm.2017.2984
Copy and paste a formatted citation
x
Spandidos Publications style
Wang J, Liu Y, Tang L, Qi S, Mi Y, Liu D and Tian Q: Identification of candidate substrates of ubiquitin-specific protease 13 using 2D-DIGE . Int J Mol Med 40: 47-56, 2017.
APA
Wang, J., Liu, Y., Tang, L., Qi, S., Mi, Y., Liu, D., & Tian, Q. (2017). Identification of candidate substrates of ubiquitin-specific protease 13 using 2D-DIGE . International Journal of Molecular Medicine, 40, 47-56. https://doi.org/10.3892/ijmm.2017.2984
MLA
Wang, J., Liu, Y., Tang, L., Qi, S., Mi, Y., Liu, D., Tian, Q."Identification of candidate substrates of ubiquitin-specific protease 13 using 2D-DIGE ". International Journal of Molecular Medicine 40.1 (2017): 47-56.
Chicago
Wang, J., Liu, Y., Tang, L., Qi, S., Mi, Y., Liu, D., Tian, Q."Identification of candidate substrates of ubiquitin-specific protease 13 using 2D-DIGE ". International Journal of Molecular Medicine 40, no. 1 (2017): 47-56. https://doi.org/10.3892/ijmm.2017.2984
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