The Shraga Segal Department of Microbiology, Immunology and Genetics, Faculty of Health Sciences and the Cancer Research Center, Ben Gurion University of the Negev, P.O.B. 653, Beer Sheva 84105, Israel.
The Shraga Segal Department of Microbiology, Immunology and Genetics, Faculty of Health Sciences and the Cancer Research Center, Ben Gurion University of the Negev, P.O.B. 653, Beer Sheva 84105, Israel..
Cell Signal. 2019 Oct;62:109340. doi: 10.1016/j.cellsig.2019.06.005. Epub 2019 Jun 5.
Protein kinase C (PKC)-interacting cousin of thioredoxin (PICOT; also termed glutaredoxin 3 (Glrx3)) is a ubiquitously expressed protein that possesses an N-terminal monothiol thioredoxin (Trx) domain and two C-terminal tandem copies of a monothiol Glrx domain. It has an overall highly conserved amino acid sequence and is encoded by a unique gene, both in humans and mice, without having other functional gene homologs in the entire genome. Despite being discovered almost two decades ago, the biological function of PICOT remains largely ill-defined and its ramifications are underestimated considering the fact that PICOT-deficiency in mice results in embryonic lethality. Since classical Glrxs are important regulators of the cellular redox homeostasis, we tested whether PICOT participate in the stress-induced DNA-damage response, focusing on nuclear proteins that function as integral components of the DNA repair machinery. Using wild type versus PICOT-deficient (PICOT-KD) Jurkat T cells we found that the anti-oxidant mechanism in PICOT-deficient cells is impaired, and that these cells respond to genotoxic drugs, such as etoposide and camptothecin, by increased caspase-3 activity, a reduced survival and a slower and diminished phosphorylation of the histone protein, H2AX. Nevertheless, the effect of PICOT on the drug-induced phosphorylation of H2AX was independent of the cellular levels of reactive oxygen species. PICOT-deficient cells also demonstrated reduced and slower γH2AX foci formation in response to radiation. Furthermore, immunofluorescence staining using PICOT- and γH2AX-specific Abs followed by confocal microscopy demonstrated partial localization of PICOT at the γH2AX-containing foci at the site of the DNA double strand breaks. In addition, PICOT knockdown resulted in inhibition of phosphorylation of ATR, Chk1 and Chk2 kinases, which play an essential role in the DNA-damage response and serve as upstream regulators of γH2AX. The present data suggest that PICOT protects cells from DNA damage-inducing agents by operating as an upstream positive regulator of ATR-dependent signaling pathways. By promoting the activity of ATR, PICOT indirectly regulates the phosphorylation and activation of Chk1, Chk2, and γH2AX, which are critical components of the DNA damage repair mechanism and thereby attenuate the stress- and replication-induced genome instability.
蛋白激酶 C（PKC）相互作用的硫氧还蛋白表亲（PICOT；也称为谷氧还蛋白 3（Glrx3））是一种广泛表达的蛋白质，具有 N 端单硫醇硫氧还蛋白（Trx）结构域和两个 C 端串联的单硫醇 Glrx 结构域。它具有高度保守的氨基酸序列，并且在人和小鼠中由一个独特的基因编码，整个基因组中没有其他功能基因同源物。尽管 PICOT 是在近二十年前发现的，但它的生物学功能仍然很大程度上不明确，而且考虑到 PICOT 缺失会导致小鼠胚胎致死，它的影响被低估了。由于经典的 Glrx 是细胞氧化还原稳态的重要调节剂，我们测试了 PICOT 是否参与应激诱导的 DNA 损伤反应，重点是作为 DNA 修复机制整体组成部分的核蛋白。使用野生型与 PICOT 缺失（PICOT-KD）Jurkat T 细胞，我们发现 PICOT 缺失细胞的抗氧化机制受损，这些细胞对依托泊苷和喜树碱等细胞毒性药物的反应是 caspase-3 活性增加、存活率降低以及组蛋白 H2AX 的磷酸化减少和缓慢。然而，PICOT 对药物诱导的 H2AX 磷酸化的影响与细胞内活性氧水平无关。PICOT 缺失细胞对辐射的γH2AX 焦点形成也减少和缓慢。此外，使用 PICOT 和 γH2AX 特异性 Abs 进行免疫荧光染色，然后通过共聚焦显微镜观察，证明 PICOT 部分定位于 DNA 双链断裂部位含有 γH2AX 的焦点处。此外，PICOT 敲低导致 ATR、Chk1 和 Chk2 激酶的磷酸化抑制，ATR、Chk1 和 Chk2 激酶在 DNA 损伤反应中发挥重要作用，是 γH2AX 的上游调节剂。本数据表明，PICOT 通过作为 ATR 依赖性信号通路的上游正调节剂，保护细胞免受 DNA 损伤诱导剂的侵害。通过促进 ATR 的活性，PICOT 间接调节 Chk1、Chk2 和 γH2AX 的磷酸化和激活，Chk1、Chk2 和 γH2AX 是 DNA 损伤修复机制的关键组成部分，从而减弱应激和复制诱导的基因组不稳定性。
