BRCA2和RAD51是在同源重组（HR）和DNA双链断裂（DSB）修复中起关键作用的两种蛋白质。BRCA2通过与其八个BRC重复结合来协助RAD51的纤维化和解聚，其中BRC4是最高效和最好的角色。小分子大大减少了RAD51的活性，被认为是一种损害BRCA2/RAD51结合并最终损害HR通路的策略，旨在使癌细胞对PARP抑制剂（PARPi）更敏感。这种策略模仿了合成致死（SL）方法，在体外使用特制的肌酰化衍生的BRC4（myr-BRC4）成功实施，其设计旨在更有效地进入细胞。本研究采用质谱技术（MS）蛋白质组学方法，对经过myr-BRC4肽处理的细胞进行蛋白质指纹图谱的获取。 （数据可通过ProteomeXchange，标识符为PXD042696，进行访问。）我们对myr-BRC4处理的与未处理的BxPC-3胰腺癌细胞进行了比较蛋白质谱分析，并评估了蛋白质的差异表达。在鉴定的蛋白质中，我们将注意力集中在RAD51和BRCA2相互作用组共有的蛋白质上，并对那些减少显示高统计学意义的蛋白质关注。我们鉴定了三种被下调的蛋白质（FANCI，FANCD2和RPA3），并通过免疫印迹分析确认了蛋白质的下调，从而验证了MS方法。我们的结果表明，由于myr-BRC4处理的直接结果，检测到FANCD2，FANCI和RPA3的下调可用作监测HR损伤的指标。意义：由于其在SL方法中的可能应用，RAD51的抑制越来越受到关注。破坏RAD51和BRCA2之间的蛋白质-蛋白质相互作用（PPIs），或者破坏其某些配体蛋白质的蛋白质-蛋白质相互作用，可以增强PARPi引起的DNA损伤诱导的细胞死亡。这对于难治性癌症，如具有BRCA兼容性的和奥拉帕尼（PARPi）耐药的胰腺腺癌具有应用潜力。尽管RAD51是一个广泛研究的靶点，但研究人员仍然缺乏详细的机制信息。这限制了该领域的进展，只有少数RAD51抑制剂被识别出来，并且没有获得监管批准。尽管如此，BRC4肽是到目前为止最专一和最好的RAD51结合剂和抑制剂之一。我们的研究是第一个报告cellular 中使用myr-BRC4处理后的蛋白质指纹的研究，为在DNA损伤修复中发现特定的蛋白质/通路改变提供了参考。我们的结果表明，作为myr-BRC4处理的直接结果，最终作用于BRCA2/RAD51的破坏，检测到FANCD2，FANCI和RPA3的下调可用作监测DNA损伤修复受损的指标，并因此用于促进新的有效治疗策略的发展。版权所有 © 2023. Elsevier B.V. 发表。

BRCA2 and RAD51 are two proteins that play a central role in homologous recombination (HR) and DNA double strand break (DSB) repair. BRCA2 assists RAD51 fibrillation and defibrillation through binding with its eight BRC repeats, with BRC4 being one of the most efficient and best characterized. RAD51 inactivation by small molecules has been proposed as a strategy to impair BRCA2/RAD51 binding and, ultimately, the HR pathway, with the aim of making cancer cells more sensitive to PARP inhibitors (PARPi). This strategy, which mimics a synthetic lethality (SL) approach, has been successfully performed in vitro by using the myristoylated derivative of BRC4 (myr-BRC4), designed for a more efficient cell entry. The present study applies a method to obtain a proteomic fingerprint after cellular treatment with the myr-BRC4 peptide using a mass spectroscopy (MS) proteomic approach. (Data are available via ProteomeXchange with identifier PXD042696.) We performed a comparative proteomic profiling of the myr-BRC4 treated vs. untreated BxPC-3 pancreatic cancer cells and evaluated the differential expression of proteins. Among the identified proteins, we focused our attention on proteins shared by both the RAD51 and the BRCA2 interactomes, and on those whose reduction showed high statistical significance. Three downregulated proteins were identified (FANCI, FANCD2, and RPA3), and protein downregulation was confirmed through immunoblotting analysis, validating the MS approach. Our results suggest that, being a direct consequence of myr-BRC4 treatment, the detection of FANCD2, FANCI, and RPA3 downregulation could be used as an indicator for monitoring HR impairment. SIGNIFICANCE: RAD51's inhibition has gained increasing attention because of its possible implications in personalized medicine through the SL approach. Chemical disruption of protein-protein interactions (PPIs) between RAD51 and BRCA2, or some of its partner proteins, could potentiate PARPi DNA damage-induced cell death. This could have application for difficult to treat cancers, such as BRCA-competent and olaparib (PARPi) resistant pancreatic adenocarcinoma. Despite RAD51 being a widely studied target, researchers still lack detailed mechanistic information. This has stifled progress in the field with only a few RAD51 inhibitors having been identified, none of which have gained regulatory approval. Nevertheless, the peptide BRC4 is one of the most specific and best characterized RAD51 binder and inhibitor reported to date. Our study is the first to report the proteomic fingerprint consequent to cellular treatment of myr-BRC4, to offer a reference for the discovery of specific protein/pathway alterations within DNA damage repair. Our results suggest that, being a direct consequence of myr-BRC4 treatment, and ultimately ofBRCA2/RAD51 disruption, the detection of FANCD2, FANCI, and RPA3 downregulation could be used as an indicator for monitoring DNA damage repair impairment and therefore be used to potentiate the development of new effective therapeutic strategies.Copyright © 2023. Published by Elsevier B.V.