Department of Molecular Medicine, Jamia Hamdard, New Delhi 110062, India.
Center for Virology, Jamia Hamdard, New Delhi 110062, India.
ACS Infect Dis. 2024 Sep 13;10(9):3273-3288. doi: 10.1021/acsinfecdis.4c00351. Epub 2024 Aug 7.
Previously, we have demonstrated that deletion of a growth-regulating gene () in the parasite () attenuated the parasite's intracellular amastigote growth but not the growth of extracellular promastigotes. parasites were found to be safe and efficacious against homologous and heterologous species as a vaccine candidate in animal models. The reason for the differential growth of between the two stages of the parasite needed investigation. Here, we report that interacts with a novel Ras-associated binding protein in (LdRab2) to compensate for the growth of promastigotes. LdRab2 was isolated by protein pull-down from the parasite lysate, followed by nano-LC-MS/MS identification. The RAB domain sequence and the functional binding partners of the LdRab2 protein were predicted via Search Tool for the Retrieval of Interacting Proteins (STRING) analysis. The closeness of the LdRab2 protein to other reported centrin-binding proteins with different functions in other organisms was analyzed via phylogenetic analysis. Furthermore, and analyses revealed that LdRab2 also interacts with other centrins 3-5. Since centrin is a calcium-binding protein, we further investigated calcium-based interactions and found that the binding of LdRab2 to LdCen1 and LdCen4 is calcium-independent, whereas the interactions with LdCen3 and LdCen5 are calcium-dependent. The colocalization of LdCen1 and LdRab2 at the cellular basal-body region by immunofluorescence supports their possible functional association. The elevated expression of the LdRab2 protein in the mutant promastigotes suggested a probable role in compensating for the promastigote growth of this mutant strain, probably in association with other parasite centrins.
先前，我们已经证明，在 寄生虫（）中删除一个生长调节基因（）减弱了寄生虫的细胞内无鞭毛体生长，但不影响细胞外前鞭毛体的生长。研究发现，在动物模型中，作为候选疫苗，对同源和异源 物种的寄生虫都是安全有效的。需要调查寄生虫的两个阶段之间的生长差异的原因。在这里，我们报告 与一种新型 Ras 相关结合蛋白在 （LdRab2）相互作用，以补偿 前鞭毛体的生长。LdRab2 通过从寄生虫裂解物中进行蛋白质下拉，然后通过纳升 LC-MS/MS 鉴定来分离。LdRab2 蛋白的 RAB 结构域序列和功能结合伙伴通过 Search Tool for the Retrieval of Interacting Proteins (STRING) 分析进行预测。通过系统发育分析分析了 LdRab2 蛋白与其他报道的 centrin 结合蛋白的接近程度，这些蛋白在其他生物体中具有不同的功能。此外， 和 分析表明，LdRab2 还与其他 中心体 3-5 相互作用。由于 centrin 是一种钙结合蛋白，我们进一步研究了基于钙的相互作用，发现 LdRab2 与 LdCen1 和 LdCen4 的结合不依赖于钙，而与 LdCen3 和 LdCen5 的相互作用依赖于钙。免疫荧光共定位显示 LdCen1 和 LdRab2 在细胞基底体区域的共定位支持它们可能的功能关联。突变体前鞭毛体中 LdRab2 蛋白的高表达表明，它可能在补偿该突变株前鞭毛体的生长中发挥作用，可能与其他寄生虫中心体有关。
