PO.BCS01.09 · 生物信息与计算
切割的ATP5A1在氧化应激下介导一个poly(ADP-核糖)依赖的R-loop稳态调控轴
Cleaved ATP5A1 mediates a poly(ADP-ribose)-dependent regulatory axis for R-Loop homeostasis under oxidative stress
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
氧化应激诱导的DNA损伤会促进R-loop的积累，R-loop是一种带有游离DNA链的DNA:RNA杂合结构，通过PARP家族酶以poly(ADP-核糖)（PAR）依赖的方式被解析。PAR化在氧化应激应答期间协调DNA修复因子的募集，此外多种线粒体蛋白也在保护核基因组方面发挥作用。我们此前利用免疫沉淀/质谱（IP/MS）方法，将切割的ATP5A1（一种线粒体应激响应的蛋白质型）鉴定为R-loop解析蛋白（包括DHX9和hnRNPU）的相互作用物。我们进一步在Calu-1（一种非小细胞肺癌细胞模型）中，采用体外实验、细胞培养、分子动力学（MD）模拟和RNA-seq方法，研究了它在R-loop调控中的PAR依赖作用。为鉴定ATP5A1具有生物活性的R-loop解析异构体，分析了来自Peptide Atlas的质谱肽段片段，以识别潜在的钙蛋白酶切割位点（跨越多个氨基酸的切割窗口）。生物信息学分析预测第135位残基为主要切割位点，并经western blot和MD稳定性谱验证。AlphaFold3结构预测和MD模拟证实了PAR链与切割的ATP5A1（残基135-553）的稳定相互作用，RMSD为0.2-0.3 nm，稳定残基的RMSF范围为0.1-0.2 nm，包括ADPR单体和聚合物。IP/MS证实了在电离辐射诱导的氧化应激后，瞬时表达的ATP5A1(135-553)发生PAR化。采用斑点印迹（Slot blot）对野生型、ATP5A1敲除和切割的ATP5A1回补条件下的R-loop进行定量。对照和ATP5A1敲除（KO）细胞的RNA-seq识别出差异表达基因，在切割的ATP5A1关联的R-loop应答特征中富集于细胞代谢、DNA修复和增殖/细胞周期通路。这些发现将切割的ATP5A1鉴定为一种PAR相互作用蛋白，在氧化应激下调节R-loop稳态，将线粒体逆行信号与核应激应答联系起来，并揭示了在氧化应激信号失调的癌症中一个潜在的R-loop治疗靶点。
查看英文原文 English abstract
Oxidative stress-induced DNA damage potentiates accumulation of R-loops, DNA:RNA hybrid structures with a displaced DNA strand that are resolved in a poly (ADP-ribose) (PAR)-dependent manner via PARP family enzymes. PARylation orchestrates recruitment of DNA repair factors during the oxidative stress response, in addition to multiple mitochondrial proteins also playing a role to protect the nuclear genome. We previously identified cleaved ATP5A1, a mitochondrial stress-responsive proteoform, as an interactor of R-loop resolving proteins including DHX9 and hnRNPU using immunoprecipitation/mass spectrometry (IP/MS) approach. We further investigated its PAR-dependent role in R-loop regulation using in vitro assays, cell culture, molecular dynamics (MD) simulation, and RNA-seq approaches in Calu-1, a non-small cell lung cancer cell model. To identify the bioactive R-loop resolving isoform of ATP5A1, mass spectrometry peptide fragments from Peptide Atlas were analyzed to identify potential calpain cleavage sites (cleavage windows that span multiple amino acids). Bioinformatic analyses predicted residue 135 as the primary cleavage site, validated by western blot and MD stability profiles. AlphaFold3 structure prediction and MD simulations demonstrated stable interactions of PAR chains with cleaved ATP5A1 (residues 135-553), with RMSD 0.2-0.3 nm and RMSF of stable residues ranging 0.1-0.2 nm, including ADPR monomers and polymers. IP/MS confirmed PARylation of transiently expressed ATP5A1(135-553) following ionizing radiation-induced oxidative stress. Slot blot was used to quantify R-loops in wild-type, ATP5A1 knockout, and cleaved ATP5A1 rescue conditions. RNA-seq of control and ATP5A1 knock-out (KO) cells identified differentially expressed genes enriching for cellular metabolism, DNA repair, and proliferation/cell cycle pathways in a cleaved ATP5A1-linked R-loop response signature. These findings identify cleaved ATP5A1 as a PAR-interacting protein that modulates R-loop homeostasis under oxidative stress, linking mitochondrial retrograde signaling to nuclear stress responses and revealing a potential R-loop therapeutic target in cancers with dysregulated oxidative stress signaling.
利益披露 Disclosure
A. Mukherjee, None..
K. Goel, None..
W. Lei, None..
N. Hill, None..
R. Greer, None..
N. Pfister, None.