分选nexin(SNX)家族的蛋白质呈现出具有phox同源性(PX)磷酸肌醇(PI)结合域和其他PX结构域的模块化结构,赋予他们多种重要的真核细胞功能,从信号转导到膜变形和货物结合。尽管SNX在人类和酵母中得到了很好的研究,他们在原生生物中的调查很少。在这里,介绍了在由LdBPK_352470基因编码的利什曼原虫寄生虫中鉴定的第一个SNX的表征。在计算机二级和三级结构预测中,该蛋白的N末端一半上有一个PX结构域,C末端一半上有一个Bin/shiphiphysin/Rvs(BAR)结构域,利用这些特征将其分类为SNX的SNX-BAR亚家族。我们将LdBPK_352470.1基因产物命名为LdSNXi,因为这是在利什曼原虫中发现的第一个SNX(L.)多诺瓦尼。其表达在不同细胞周期阶段的多诺瓦尼乳杆菌中得到证实,它被证明在细胞外培养基中分泌。使用体外脂质结合测定法,证明了用谷胱甘肽-S-转移酶(GST)标记的重组(r)LdSNXi(rGST-LdSNXi)与PtdIns3P和PtdIns4PI结合。使用特异性a-LdSNXi抗体和免疫荧光共聚焦显微镜,内源性LdSNXi的细胞内定位在多诺瓦尼乳杆菌和轴性阿马斯泰格中进行了分析。此外,用增强的绿色荧光蛋白(rLdSNXi-EGFP)标记的rLdSNXi在转染的HeLa细胞中异源表达,并检查其定位。所有观察到的定位都表明功能与LdSNXi的假定SNX身份兼容。Sequence,结构,和进化分析显示LdSNXi与人类SNX2之间具有高度同源性,而基于STRING(v.11.5)的蛋白质-蛋白质相互作用的研究预测了利什曼原虫LdSNXi的推定分子伴侣。
Proteins of the sorting nexin (SNX) family present a modular structural architecture with a phox homology (PX) phosphoinositide (PI)-binding domain and additional PX structural domains, conferring to them a wide variety of vital eukaryotic cell\'s functions, from signal transduction to membrane deformation and cargo binding. Although SNXs are well studied in human and yeasts, they are poorly investigated in protists. Herein, is presented the characterization of the first SNX identified in Leishmania protozoan parasites encoded by the LdBPK_352470 gene. In silico secondary and tertiary structure prediction revealed a PX domain on the N-terminal half and a Bin/amphiphysin/Rvs (BAR) domain on the C-terminal half of this protein, with these features classifying it in the SNX-BAR subfamily of SNXs. We named the LdBPK_352470.1 gene product LdSNXi, as it is the first SNX identified in Leishmania (L.) donovani. Its expression was confirmed in L. donovani promastigotes under different cell cycle phases, and it was shown to be secreted in the extracellular medium. Using an in vitro lipid binding assay, it was demonstrated that recombinant (r) LdSNXi (rGST-LdSNXi) tagged with glutathione-S-transferase (GST) binds to the PtdIns3P and PtdIns4P PIs. Using a specific a-LdSNXi antibody and immunofluorescence confocal microscopy, the intracellular localization of endogenous LdSNXi was analyzed in L. donovani promastigotes and axenic amastigotes. Additionally, rLdSNXi tagged with enhanced green fluorescent protein (rLdSNXi-EGFP) was heterologously expressed in transfected HeLa cells and its localization was examined. All observed localizations suggest functions compatible with the postulated SNX identity of LdSNXi. Sequence, structure, and evolutionary analysis revealed high homology between LdSNXi and the human SNX2, while the investigation of protein-protein interactions based on STRING (v.11.5) predicted putative molecular partners of LdSNXi in Leishmania.