Dong Xiaoshu, Yu Xinqian, Lu Minghao, Xu Yaxin, Zhou Liyan, Peng Tao
State Key Laboratory of Chemical Oncogenomics, School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, Shenzhen, 518055, China.
Cell Death Discov. 2024 Oct 29;10(1):456. doi: 10.1038/s41420-024-02225-7.
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.
十字花科蔬菜中天然存在的异硫氰酸酯（ITCs），如苄基异硫氰酸酯（BITC）、苯乙基异硫氰酸酯（PEITC）和萝卜硫素（SFN），因其在体外和体内具有可观的抗癌活性而引起了广泛的研究关注。虽然细胞凋亡的诱导被认为在ITCs的抗癌作用中起关键作用，但ITCs诱导细胞凋亡的具体蛋白质靶点及相关上游事件仍不清楚。在本研究中，我们提出了一组化学探针，它们衍生自BITC、PEITC和SFN，并配备了生物正交炔基手柄，以系统地分析活癌细胞中ITCs的靶蛋白。使用基于竞争的定量化学蛋白质组学方法，我们鉴定了一系列富含凋亡等生物学过程的ITCs候选靶蛋白。我们发现，Bcl-2家族的凋亡调节因子BID在其N端半胱氨酸上被ITCs共价修饰。功能表征表明，PEITC与BID的N端半胱氨酸共价结合会导致蛋白质构象变化，并破坏BID N端和C端区域之间的自抑制相互作用，从而释放出高活性的C端片段以发挥下游促凋亡作用。一致地，PEITC促进BID的切割和线粒体易位，从而强烈诱导细胞凋亡。我们进一步表明，N端半胱氨酸的突变会损害BID的N端和C端相互作用，解除自抑制并增强其凋亡活性。总体而言，我们的化学蛋白质组学分析和功能研究不仅揭示了BID是PEITC介导凋亡诱导上游事件的主要靶点，还揭示了一种涉及BID第一螺旋内N端半胱氨酸调节其促凋亡潜力的新分子机制。
