Quantitative chemical proteomics reveals that phenethyl isothiocyanate covalently targets BID to promote apoptosis

TL;DR: Phenethyl isothiocyanate covalently targets BID, an apoptosis regulator, by modifying its N-terminal cysteines, disrupting self-inhibition and promoting apoptosis through conformational changes and mitochondrial translocation, revealing a novel molecular mechanism.

Abstract: Abstract Naturally occurring isothiocyanates (ITCs) found in cruciferous vegetables, such as benzyl isothiocyanate (BITC), phenethyl isothiocyanate (PEITC), and sulforaphane (SFN), have attracted significant research interest for their promising anti-cancer activity in vitro and in vivo. While the induction of apoptosis is recognized to play a key role in the anti-cancer effects of ITCs, the specific protein targets and associated upstream events underlying ITC-induced apoptosis remain unknown. In this study, we present a set of chemical probes that are derived from BITC, PEITC, and SFN and equipped with bioorthogonal alkynyl handles to systematically profile the target proteins of ITCs in live cancer cells. Using a competition-based quantitative chemical proteomics approach, we identify a range of candidate target proteins of ITCs enriched in biological processes such as apoptosis. We show that BID, an apoptosis regulator of the Bcl-2 family, is covalently modified by ITCs on its N -terminal cysteines. Functional characterization demonstrates that covalent binding to N -terminal cysteines of BID by PEITC results in conformational changes of the protein and disruption of the self-inhibitory interaction between N - and C -terminal regions of BID, thus unleashing the highly active C -terminal segment to exert downstream pro-apoptotic effects. Consistently, PEITC promotes the cleavage and mitochondrial translocation of BID, leading to a strong induction of apoptosis. We further show that mutation of N -terminal cysteines impairs the N - and C -terminal interaction of BID, relieving the self-inhibition and enhancing its apoptotic activity. Overall, our chemical proteomics profiling and functional studies not only reveal BID as the principal target of PEITC in mediating upstream events for the induction of apoptosis, but also uncover a novel molecular mechanism involving N -terminal cysteines within the first helix of BID in regulating its pro-apoptotic potential.