Identification of deubiquitinase targets of isothiocyanates using SILAC-assisted quantitative mass spectrometry

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.

Computational and biochemical studies of isothiocyanates as inhibitors of proteasomal cysteine deubiquitinases in human cancer cells

“…The docking data was further confirmed by Ub‐VS assay in which BITC only at 50 μM could inhibit the USP14′s activity (Figure ). Our data may have provided an explanation for why USP14 was not found to be a target of ITCs in previous studies under their experimental conditions …”

Phenethyl Isothiocyanate Suppresses Stemness in the Chemo- and Radio-Resistant Triple-Negative Breast Cancer Cell Line MDA-MB-231/IR Via Downregulation of Metadherin

“…Once a protein is conjugated with a poly-ubiquitin chain, it is recognized and degraded by the proteasome system [56]. PEITC has been reported to induce ubiquitination through the inhibition of deubiquitinase activity [57]. MicroRNAs may also play a role in the regulation of MTDH expression by PEITC.…”

Phenethyl Isothiocyanate, a Dual Activator of Transcription Factors NRF2 and HSF1

“…PEITC also decreases the levels of DUBs by initiating their degradation 68. Another mechanism by which PEITC could regulate the epigenome is by altering the expression of a number of microRNAs (miRNAs) 69.…”

“…Recently, Lawson and colleagues found that, possibly through the modification of their cysteines, PEITC targets various deubiquitinases (DUBs) many of which (e.g., VCPIP1, USP1, USP3, USP16, and USP48) are involved in chromatin remodeling and DNA repair. PEITC also decreases the levels of DUBs by initiating their degradation . Another mechanism by which PEITC could regulate the epigenome is by altering the expression of a number of microRNAs (miRNAs) .…”