Departamento de Microbiologia, Imunologia e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, Brazil.
Instituto Carlos Chagas, Fundação Oswaldo Cruz-Fiocruz, Curitiba, Brazil.
Front Cell Infect Microbiol. 2020 Apr 16;10:149. doi: 10.3389/fcimb.2020.00149. eCollection 2020.
The integrated stress response in eukaryotic cells is an orchestrated pathway that leads to eukaryotic Initiation Factor 2 alpha subunit (eIF2α) phosphorylation at ser51 and ultimately activates pathways to mitigate cellular damages. Three putative kinases (k1, k2, and k3) are found in the genome, the flagellated parasite that causes Chagas disease. These kinases present similarities to other eukaryotic eIF2α kinases, exhibiting a typical insertion loop in the kinase domain of the protein. We found that this insertion loop is conserved among kinase 1 of several strains but differs among various Kinetoplastidae species, suggesting unique roles. Kinase 1 is orthologous of GCN2 of several eukaryotes, which have been implicated in the eIF2α ser51 phosphorylation in situations that mainly affects the nutrients levels. Therefore, we further investigated the responses to nutritional stress of devoid of K1 generated by CRISPR/Cas9 gene replacement. In nutrient-rich conditions, replicative epimastigotes depleted of K1 proliferate as wild type cells but showed increased levels of polysomes relative to monosomes. Upon nutritional deprivation, the polysomes decreased more than in K1 depleted line. However, eIF2α is still phosphorylated in K1 depleted line, as in wild type parasites. eIF2α phosphorylation increased at longer incubations times, but KO parasites showed less accumulation of ribonucleoprotein granules containing ATP-dependent RNA helicase involved in mRNA turnover (DHH1) and Poly-A binding protein (PABP1). Additionally, the formation of metacyclic-trypomastigotes is increased in the absence of k1 compared to controls. These metacyclics, as well as tissue culture trypomastigotes derived from the K1 knockout line, were less infective to mammalian host cells, although replicated faster inside mammalian cells. These results indicate that GCN2-like kinase in affects stress granule formation, independently of eIF2α phosphorylation upon nutrient deprivation. It also modulates the fate of the parasites during differentiation, invasion, and intracellular proliferation.
真核细胞的综合应激反应是一条协调的途径，导致真核起始因子 2 阿尔法亚基（eIF2α）丝氨酸 51 位磷酸化，并最终激活减轻细胞损伤的途径。在引起恰加斯病的鞭毛寄生虫基因组中发现了三个假定的激酶（k1、k2 和 k3）。这些激酶与其他真核 eIF2α 激酶相似，在蛋白质的激酶结构域中具有典型的插入环。我们发现，该插入环在几种 株的激酶 1 中是保守的，但在不同的动基体科物种中不同，提示其具有独特的作用。激酶 1 是几种真核生物 GCN2 的同源物，GCN2 在主要影响营养水平的情况下被认为与 eIF2α 丝氨酸 51 位磷酸化有关。因此，我们进一步研究了用 CRISPR/Cas9 基因替换生成的无 K1 的 对营养应激的反应。在营养丰富的条件下，缺乏 K1 的复制性 epimastigotes 像野生型细胞一样增殖，但相对于单核糖体，多核糖体的水平增加。在营养剥夺时，多核糖体的减少量超过了 K1 耗尽的系。然而，在 K1 耗尽的系中，eIF2α 仍然被磷酸化，就像在野生型寄生虫中一样。eIF2α 磷酸化在更长的孵育时间增加，但 KO 寄生虫中含有 ATP 依赖性 RNA 解旋酶（DHH1）和多聚 A 结合蛋白（PABP1）的核糖核蛋白颗粒的积累较少。此外，与对照相比，无 k1 时的循环期-转化体的形成增加。这些循环期-转化体以及源自 K1 敲除系的组织培养 转化体，对哺乳动物宿主细胞的感染性降低，尽管在哺乳动物细胞内复制更快。这些结果表明， 中的 GCN2 样激酶在营养缺乏时独立于 eIF2α 磷酸化影响应激颗粒的形成。它还调节寄生虫在分化、入侵和细胞内增殖过程中的命运。
