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Updated: Feb 23, 2026
Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry
Published on: March 23, 2020
Identification of deubiquitinase targets of isothiocyanates using SILAC-assisted quantitative mass spectrometry
Ann P Lawson1, Daniel W Bak2, D Alexander Shannon2
1Department of Biology, Brandeis University, Waltham, MA 02453-9110, USA.
Abstract:
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.
Insights
Phenethyl isothiocyanate (PEITC), found in cruciferous vegetables, inhibits deubiquitinases (DUBs) like USP1. This action impairs DNA repair and enhances cancer cell sensitivity to chemotherapy, revealing new therapeutic mechanisms.
Area of Science:
- Biochemistry and Molecular Biology
- Cancer Research
- Nutritional Science
Background:
- Cruciferous vegetables contain isothiocyanates (ITCs) with known chemopreventative and anticancer properties.
- The precise molecular mechanisms underlying ITC action, particularly their effects on oncogenic proteins, remain largely unelucidated.
- Deubiquitinases (DUBs) play critical roles in cellular processes, and their dysregulation is implicated in diseases like cancer.
Purpose of the Study:
- To identify novel deubiquitinase (DUB) targets of phenethyl isothiocyanate (PEITC).
- To elucidate the molecular mechanisms by which PEITC affects DUB activity and downstream cellular processes.
- To explore the potential of PEITC as a therapeutic agent in cancer treatment.
Main Methods:
- Utilized SILAC (Stable Isotope Labeling by Amino acids in Cell culture) assisted quantitative mass spectrometry to identify PEITC-DUB interactions.
- Investigated the effects of PEITC on USP1 activity, ubiquitination, and degradation.
- Assessed the impact of altered USP1 activity on PCNA ubiquitination and DNA repair capacity.
Main Results:
- Identified nine new PEITC-sensitive DUBs, including USP1, USP3, USP10, USP11, USP16, USP22, USP40, USP48, and VCPIP1.
- Demonstrated that PEITC inhibits USP1 through both direct inhibition and increased ubiquitination/degradation, leading to reduced USP1 activity.
- Observed that decreased USP1 activity results in increased mono-ubiquitinated PCNA, impairing DNA repair, and reduced mono-ubiquitylated histones H2A and H2B.
Conclusions:
- PEITC's dual mechanism of inhibiting and degrading USP1 offers a novel explanation for the enhanced sensitivity of cancer cells to cisplatin.
- PEITC's modulation of USP1 activity and subsequent impact on DNA repair pathways present a promising avenue for cancer therapy development.
- Further understanding of PEITC's mechanism of action can facilitate its application as a therapeutic agent in oncology.
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