Identification of deubiquitinase targets of isothiocyanates using SILAC-assisted quantitative mass spectrometry
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Ann P. Lawson1, Daniel W. Bak2, D. Alexander Shannon2, Marcus J.C. Long3,7, Tushara Vijaykumar4,8, Runhan Yu5, Farid El Oualid6, Eranthie Weerapana2 and Lizbeth Hedstrom1,5
1Department of Biology, Brandeis University, Waltham, MA 02453-9110, USA
2Department of Chemistry, Merkert Center, Boston College, Chestnut Hill, MA 02467-3860, USA
3Graduate Program in Biochemistry and Biophysics, Brandeis University, Waltham, MA 02453-9110, USA
4Graduate Program in Molecular and Cellular Biology, Brandeis University, Waltham, MA 02453-9110, USA
5Department of Chemistry, Brandeis University, Waltham, MA 02453-9110, USA
6UbiQ, 1098 XH Amsterdam, The Netherlands
7Current address: Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA
8Current address: Sanofi Genzyme, Framingham, MA 01701, USA
Lizbeth Hedstrom, email: firstname.lastname@example.org
Keywords: PEITC, USP1, cisplatin, deubiquitinase, cruciferous vegetable
Received: June 20, 2016 Accepted: March 22, 2017 Published: April 20, 2017
Cruciferous vegetables such as broccoli and kale have well documented chemopreventative and anticancer effects that are attributed to the presence of isothiocyanates (ITCs). ITCs modulate the levels of many oncogenic proteins, but the molecular mechanisms of ITC action are not understood. We previously reported that phenethyl isothiocyanate (PEITC) inhibits two deubiquitinases (DUBs), USP9x and UCH37. DUBs regulate many cellular processes and DUB dysregulation is linked to the pathogenesis of human diseases including cancer, neurodegeneration, and inflammation. Using SILAC assisted quantitative mass spectrometry, here we identify 9 new PEITC-DUB targets: USP1, USP3, USP10, USP11, USP16, USP22, USP40, USP48 and VCPIP1. Seven of these PEITC-sensitive DUBs have well-recognized roles in DNA repair or chromatin remodeling. PEITC both inhibits USP1 and increases its ubiquitination and degradation, thus decreasing USP1 activity by two mechanisms. The loss of USP1 activity increases the level of mono-ubiquitinated DNA clamp PCNA, impairing DNA repair. Both the inhibition/degradation of USP1 and the increase in mono-ubiquitinated PCNA are new activities for PEITC that can explain the previously recognized ability of ITCs to enhance cancer cell sensitivity to cisplatin treatment. Our work also demonstrates that PEITC reduces the mono-ubiquityl histones H2A and H2B. Understanding the mechanism of action of ITCs should facilitate their use as therapeutic agents.
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