Julio Ashley R, Shikwana Flowreen, Truong Cindy, Burton Nikolas R, Dominguez Emil, Turmon Alexandra C, Cao Jian, Backus Keriann
Department of Biological Chemistry, David Geffen School of Medicine, UCLA, Los Angeles, CA 90095 (USA).
Department of Chemistry and Biochemistry, UCLA, Los Angeles, CA 90095 (USA).
bioRxiv. 2023 Nov 16:2023.10.30.564067. doi: 10.1101/2023.10.30.564067.
Protein homeostasis is tightly regulated, with damaged or misfolded proteins quickly eliminated by the proteasome and autophagosome pathways. By co-opting these processes, targeted protein degradation technologies enable pharmacological manipulation of protein abundance. Recently, cysteine-reactive molecules have been added to the degrader toolbox, which offer the benefit of unlocking the therapeutic potential of 'undruggable' protein targets. The proteome-wide impact of these molecules remains to be fully understood and given the general reactivity of many classes of cysteine-reactive electrophiles, on- and off-target effects are likely. Using chemical proteomics, we identified a cysteine-reactive small molecule degrader of the SARS-CoV-2 nonstructural protein 14 (nsp14), which effects degradation through direct modification of cysteines in both nsp14 and in host chaperones together with activation of global cell stress response pathways. We find that cysteine-reactive electrophiles increase global protein ubiquitylation, trigger proteasome activation, and result in widespread aggregation and depletion of host proteins, including components of the nuclear pore complex. Formation of stress granules was also found to be a remarkably ubiquitous cellular response to nearly all cysteine-reactive compounds and degraders. Collectively, our study sheds light on complexities of covalent target protein degradation and highlights untapped opportunities in manipulating and characterizing proteostasis processes via deciphering the cysteine-centric regulation of stress response pathways.
蛋白质稳态受到严格调控，受损或错误折叠的蛋白质会通过蛋白酶体和自噬体途径迅速清除。通过利用这些过程，靶向蛋白质降解技术能够对蛋白质丰度进行药理学调控。最近，半胱氨酸反应性分子已被添加到降解剂工具库中，这为释放“不可成药”蛋白质靶点的治疗潜力提供了便利。这些分子对蛋白质组的影响仍有待充分了解，鉴于许多类半胱氨酸反应性亲电试剂具有普遍的反应性，脱靶效应很可能存在。我们使用化学蛋白质组学方法，鉴定出一种针对严重急性呼吸综合征冠状病毒2（SARS-CoV-2）非结构蛋白14（nsp14）的半胱氨酸反应性小分子降解剂，它通过直接修饰nsp14和宿主分子伴侣中的半胱氨酸以及激活全局细胞应激反应途径来实现降解。我们发现半胱氨酸反应性亲电试剂会增加全局蛋白质泛素化、触发蛋白酶体激活，并导致宿主蛋白质广泛聚集和消耗，包括核孔复合体的成分。应激颗粒的形成也被发现是几乎所有半胱氨酸反应性化合物和降解剂普遍存在的细胞反应。总体而言，我们的研究揭示了共价靶向蛋白质降解的复杂性，并通过解读以半胱氨酸为中心的应激反应途径调控，突出了在操纵和表征蛋白质稳态过程中尚未开发的机会。
