Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas (ICB), Universidade Federal de Minas Gerais, Belo Horizonte, MG 30161-970, Brazil.
Laboratório Especial de Ciclo Celular, Instituto Butantan, São Paulo, MG, São Paulo, SP 05503-900, Brazil.
DNA Repair (Amst). 2024 Sep;141:103726. doi: 10.1016/j.dnarep.2024.103726. Epub 2024 Jul 27.
Trypanosoma cruzi is the etiological agent of Chagas disease and a peculiar eukaryote with unique biological characteristics. DNA damage can block RNA polymerase, activating transcription-coupled nucleotide excision repair (TC-NER), a DNA repair pathway specialized in lesions that compromise transcription. If transcriptional stress is unresolved, arrested RNA polymerase can activate programmed cell death. Nonetheless, how this parasite modulates these processes is unknown. Here, we demonstrate that T. cruzi cell death after UV irradiation, a genotoxic agent that generates lesions resolved by TC-NER, depends on active transcription and is signaled mainly by an apoptotic-like pathway. Pre-treated parasites with α-amanitin, a selective RNA polymerase II inhibitor, become resistant to such cell death. Similarly, the gamma pre-irradiated cells are more resistant to UV when the transcription processes are absent. The Cockayne Syndrome B protein (CSB) recognizes blocked RNA polymerase and can initiate TC-NER. Curiously, CSB overexpression increases parasites' cell death shortly after UV exposure. On the other hand, at the same time after irradiation, the single-knockout CSB cells show resistance to the same treatment. UV-induced fast death is signalized by the exposition of phosphatidylserine to the outer layer of the membrane, indicating a cell death mainly by an apoptotic-like pathway. Furthermore, such death is suppressed in WT parasites pre-treated with inhibitors of ataxia telangiectasia and Rad3-related (ATR), a key DDR kinase. Signaling for UV radiation death may be related to R-loops since the overexpression of genes associated with the resolution of these structures suppress it. Together, results suggest that transcription blockage triggered by UV radiation activates an ATR-dependent apoptosis-like mechanism in T. cruzi, with the participation of CSB protein in this process.
克氏锥虫是恰加斯病的病原体，是一种具有独特生物学特性的特殊真核生物。DNA 损伤可阻断 RNA 聚合酶，激活转录偶联核苷酸切除修复（TC-NER），这是一种专门修复转录过程中受损的 DNA 修复途径。如果转录应激未得到解决，受阻的 RNA 聚合酶可激活程序性细胞死亡。然而，这种寄生虫如何调节这些过程尚不清楚。在这里，我们证明了 T. cruzi 在紫外线照射后（一种产生可通过 TC-NER 修复的损伤的遗传毒性剂）的细胞死亡依赖于活跃的转录，并且主要通过类似凋亡的途径进行信号传递。用α-鹅膏蕈碱（一种选择性 RNA 聚合酶 II 抑制剂）预处理寄生虫可使其对这种细胞死亡产生抗性。同样，当转录过程不存在时，γ预辐照细胞对 UV 更具抗性。Cockayne 综合征 B 蛋白（CSB）识别受阻的 RNA 聚合酶，并能启动 TC-NER。奇怪的是，CSB 的过表达会在暴露于 UV 后不久增加寄生虫的细胞死亡。另一方面，在照射后相同的时间，CSB 单敲除细胞对相同的处理表现出抗性。UV 诱导的快速死亡通过膜外磷脂酰丝氨酸的暴露来信号传递，表明主要通过类似凋亡的途径发生细胞死亡。此外，WT 寄生虫在预先用共济失调毛细血管扩张症和 Rad3 相关（ATR）抑制剂处理后，这种死亡被抑制，ATR 是一种关键的 DDR 激酶。UV 辐射死亡信号可能与 R 环有关，因为与这些结构的解决相关的基因的过表达会抑制它。综上所述，结果表明，UV 辐射引发的转录阻断在 T. cruzi 中激活了依赖 ATR 的类似凋亡的机制，CSB 蛋白参与了这一过程。
