Rodrigues João Paulo Ferreira, Loch Leonardo, Onofre Thiago Souza, Yoshida Nobuko
Departamento de Microbiologia, Imunologia, e Parasitologia, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo 04039-032, Brazil.
Pathogens. 2022 Nov 4;11(11):1294. doi: 10.3390/pathogens11111294.
Na/H exchanger isoform 1 (NHE1), a member of a large family of integral membrane proteins, plays a role in regulating the cortical actin cytoskeleton. , the agent of Chagas disease, depends on F-actin rearrangement and lysosome mobilization to invade host cells. To determine the involvement of NHE1 in metacyclic trypomastigote (MT) internalization, the effect of treatment in cells with NHE1 inhibitor amiloride or of NHE1 depletion was examined in human epithelial cells. MT invasion decreased in amiloride-treated and NHE1-depleted cells. The phosphorylation profile of diverse protein kinases, whose activation is associated with remodeling of actin fibers, was analyzed in amiloride-treated and NHE1-depleted cells. In amiloride-treated cells, the phosphorylation levels of protein kinase C (PKC), focal adhesion kinase (FAK) and Akt were similar to those of untreated cells, whereas those of extracellular signal-regulated protein kinases (ERK1/2) increased. In NHE1-deficient cells, with marked alteration in the actin cytoskeleton architecture and in lysosome distribution, the levels of phospho-PKC and phospho-FAK decreased, whereas those of phospho-Akt and phospho-ERK1/2 increased. These data indicate that NHE1 plays a role in MT invasion, by maintaining the activation status of diverse protein kinases in check and preventing the inappropriate F-actin arrangement that affects lysosome distribution.
钠/氢交换体同工型1（NHE1）是一大类整合膜蛋白家族的成员，在调节皮质肌动蛋白细胞骨架中发挥作用。克氏锥虫病的病原体克氏锥虫，依赖于F-肌动蛋白重排和溶酶体动员来侵入宿主细胞。为了确定NHE1在循环后期锥鞭毛体（MT）内化过程中的作用，在人上皮细胞中检测了用NHE1抑制剂阿米洛利处理细胞或耗尽NHE1的效果。在阿米洛利处理的细胞和NHE1耗尽的细胞中，MT入侵减少。在阿米洛利处理的细胞和NHE1耗尽的细胞中，分析了多种蛋白激酶的磷酸化谱，这些蛋白激酶的激活与肌动蛋白纤维重塑相关。在阿米洛利处理的细胞中，蛋白激酶C（PKC）、粘着斑激酶（FAK）和Akt的磷酸化水平与未处理细胞相似，而细胞外信号调节蛋白激酶（ERK1/2）的磷酸化水平增加。在NHE1缺陷细胞中，肌动蛋白细胞骨架结构和溶酶体分布有明显改变，磷酸化PKC和磷酸化FAK水平降低，而磷酸化Akt和磷酸化ERK1/2水平增加。这些数据表明，NHE1通过控制多种蛋白激酶的激活状态并防止影响溶酶体分布的不适当F-肌动蛋白排列，在MT入侵中发挥作用。
