Braden Dennis C, Adbel-Salam Mostafa A L, Asan Alparslan, Skoko John, Lu Huiming, Conrads Thomas P, Freeman Bruce A, Schopfer Francisco J, Saini Ishu, Kuper Jochen, Kisker Caroline, Uboveja Apoorva, Tangudu Naveen K, Aird Katherine M, Davis Anthony J, Neumann Carola A
Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA, 15261, USA; Women's Cancer Research Center, University of Pittsburgh Cancer Institute, Pittsburgh, PA, 15213, USA; UPMC Hillman Cancer Center, Pittsburgh, PA, 15213, USA.
Department of Radiation Oncology, UT Southwestern Medical Center, Dallas, TX75390, USA.
Res Sq. 2025 Mar 26:rs.3.rs-6141403. doi: 10.21203/rs.3.rs-6141403/v1.
Nitroalkenes are endogenous products generated by the metabolism of unsaturated fatty acids. They are generated under oxidative stress conditions, mediating important anti-inflammatory signaling activities through covalent modification of protein cysteine thiols. Despite being cytoprotective in benign tissue, nitroalkenes display single-agent anti-proliferative activity in breast cancer cells and sensitize them to multiple DNA-damaging agents. Initial mechanistic evidence suggested that nitroalkene anti-cancer activities are partially mediated by inhibition of homologous recombination (HR) through the recombinase RAD51 at Cys319. However, nitroalkenes are multi-target agents, and thus, it is likely that other important DNA repair targets beyond RAD51 are modified by nitroalkenes, contributing to their anti-cancer effects. We, therefore, conducted a global proteomics analysis to address this question. This analysis led to the identification of the recQ helicase RECQL4 with a nitro-alkylation at Cys1052. This modification was further confirmed by click chemistry-based chemoproteomics and determined to be DNA damage-dependent. Functional analyses demonstrated that nitroalkene modification inhibits RECQL4 ATP-dependent helicase activity and disrupts DSB end resection and downstream homology-dependent repair. Furthermore, experiments with C1052S mutant RECQL4 revealed that RECQL4 is a major mediator of nitroalkene effects on end resection, DSB formation, and repair. The evidence presented here denotes RECQL4 as an important nitroalkene target conferring DSB repair inhibition and supports further evaluation of nitroalkenes as therapeutic agents in RECQL4-amplified cancers.
硝基烯烃是不饱和脂肪酸代谢产生的内源性产物。它们在氧化应激条件下产生，通过共价修饰蛋白质半胱氨酸硫醇介导重要的抗炎信号活动。尽管在良性组织中具有细胞保护作用，但硝基烯烃在乳腺癌细胞中显示出单药抗增殖活性，并使它们对多种DNA损伤剂敏感。初步的机制证据表明，硝基烯烃的抗癌活性部分是通过重组酶RAD51在Cys319处抑制同源重组（HR）介导的。然而，硝基烯烃是多靶点药物，因此，RAD51以外的其他重要DNA修复靶点很可能被硝基烯烃修饰，从而产生抗癌作用。因此，我们进行了一项全局蛋白质组学分析来解决这个问题。该分析导致鉴定出在Cys1052处发生硝基烷基化的recQ解旋酶RECQL4。这种修饰通过基于点击化学的化学蛋白质组学得到进一步证实，并确定为DNA损伤依赖性。功能分析表明，硝基烯烃修饰抑制RECQL4依赖ATP的解旋酶活性，破坏双链断裂（DSB）末端切除和下游同源依赖性修复。此外，对C1052S突变体RECQL4的实验表明，RECQL4是硝基烯烃对末端切除、DSB形成和修复作用的主要介质。此处提供的证据表明RECQL4是赋予DSB修复抑制作用的重要硝基烯烃靶点，并支持进一步评估硝基烯烃作为RECQL4扩增癌症治疗药物的可能性。
