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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.
Highlights
Isothiocyanates (ITCs) are the chemoprotective natural products found in cruciferous vegetables such as broccoli, kale and watercress [1,2,3,4,5,6]
USP9x and UCH37 have distinctive molecular weights that facilitated their identification as phenethyl isothiocyanate (PEITC) targets in these gel-based activity profiling experiments [23]
It’s important to note that the reaction with the probe is time-dependent and irreversible, so high concentrations of PEITC enable the reaction to proceed long enough to label the greatest number of DUBs, yet still observe inhibition of PEITCsensitive DUBs
Summary
Isothiocyanates (ITCs) are the chemoprotective natural products found in cruciferous vegetables such as broccoli, kale and watercress [1,2,3,4,5,6]. ITCs have numerous well-documented anticancer activities, including inhibition of proliferation, induction of cell cycle arrest, apoptosis and autophagic cell death, and reduction of the inflammatory response [2, 7,8,9]. Phenethylisothiocyanate (PEITC) is among the best characterized ITCs due to its potent anticancer activity and low inherent toxicity. A 100 g serving of watercress releases at least 12 mg (80 μmole) of PEITC, resulting in low micromolar concentrations in human plasma [10]. Cells can accumulate ITCs to concentrations 100-200 times those found in plasma [2]
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