Área Académica de Medicina, Instituto de Ciencias de la Salud, Universidad Autónoma del Estado de Hidalgo, Camino a Tilcuatla s/n Municipio de San Agustín Tlaxiaca. C.P, 42160, Pachuca de Soto, Hidalgo, Mexico.
Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del I.P.N, Avenida Instituto Politécnico Nacional No. 2508, Col. San Pedro Zacatenco, Delegación Gustavo A. Madero, 07360, CDMX, CP, Mexico.
Parasit Vectors. 2017 Oct 18;10(1):500. doi: 10.1186/s13071-017-2461-5.
The abundant number of kinases that Entamoeba histolytica possesses allows us to assume that the regulation of cellular functions by phosphorylation-dephosphorylation processes is very important. However, the kinases responsible for the phosphorylation in Entamoeba spp. vary in the structure of their domains and, therefore, could be responsible for the unusual biological characteristics of this parasite. In higher eukaryotes, Src kinases share conserved structural domains and are very important in the regulation of the actin cytoskeleton. In both Entamoeba histolytica and Entamoeba invadens, the major Src kinase homologue of higher eukaryotes lacks SH3 and SH2 domains, but does have KELCH domains; the latter are part of actin cross-linking proteins in higher eukaryotic cells.
The function of the EhSrc protein kinase of Entamoeba spp. was evaluated using Src inhibitor-1, microscopy assays, Src kinase activity and western blot. In addition, to define the potential inhibitory mechanism of Src-inhibitor-1 for the amoebic EhSrc protein kinase, molecular dynamic simulations using NAnoscale Molecular Dynamics (NAMD2) program and docking studies were performed with MOE software.
We demonstrate that Src inhibitor-1 is able to prevent the activity of EhSrc protein kinase, most likely by binding to the catalytic domain, which affects cell morphology via the disruption of actin cytoskeleton remodeling and the formation of phagocytic structures without an effect on cell adhesion. Furthermore, in E. invadens, Src inhibitor-1 inhibited the encystment process by blocking RhoA GTPase activity, a small GTPase protein of Rho family.
Even though the EhSrc molecule of Entamoeba is not a typical Src, because its divergent amino acid sequence, it is a critical factor in the biology of this parasite via the regulation of actin cytoskeleton remodeling via RhoA GTPase activation. Based on this, we conclude that EhSrc could become a target molecule for the future design of drugs that can prevent the transmission of the disease.
溶组织内阿米巴拥有大量的激酶，这使得我们可以假设磷酸化-去磷酸化过程对细胞功能的调节非常重要。然而，负责磷酸化的激酶在结构上在结构域上有所不同，因此可能是导致这种寄生虫具有异常生物学特性的原因。在高等真核生物中，Src 激酶具有保守的结构域，在调节肌动蛋白细胞骨架方面非常重要。在溶组织内阿米巴和侵袭性内阿米巴中，高等真核生物的主要 Src 激酶同源物缺乏 SH3 和 SH2 结构域，但具有 KELCH 结构域；后者是高等真核细胞中肌动蛋白交联蛋白的一部分。
使用 Src 抑制剂-1、显微镜检测、Src 激酶活性和 Western blot 评估溶组织内阿米巴的 EhSrc 蛋白激酶的功能。此外，为了确定 Src 抑制剂-1 对 EhSrc 蛋白激酶的潜在抑制机制，使用 NAnoscale Molecular Dynamics (NAMD2) 程序进行分子动力学模拟，并使用 MOE 软件进行对接研究。
我们证明 Src 抑制剂-1 能够抑制 EhSrc 蛋白激酶的活性，很可能是通过与催化结构域结合，通过破坏肌动蛋白细胞骨架重塑和形成吞噬结构而不影响细胞黏附来影响细胞形态。此外，在侵袭性内阿米巴中，Src 抑制剂-1 通过抑制 RhoA GTP 酶活性来抑制囊泡形成过程，RhoA GTP 酶是 Rho 家族的一种小 GTP 酶蛋白。
尽管溶组织内阿米巴的 EhSrc 分子不是典型的 Src，但其氨基酸序列的差异，通过调节肌动蛋白细胞骨架重塑并激活 RhoA GTP 酶，成为该寄生虫生物学的关键因素。基于这一点，我们得出结论，EhSrc 可能成为未来设计预防疾病传播的药物的靶标分子。
