The enzyme farnesyl diphosphate synthase (FPPS) is positioned in the intersection of different sterol biosynthesis pathways such as those producing isoprenoids, dolichols and ergosterol. FPPS is ubiquitous in eukaryotes and is inhibited by nitrogen-containing bisphosphonates (N-BP). N-BP activity and the mechanisms of cell death as well as damage to the ultrastructure due to N-BP has not yet been investigated in Leishmania infantum and Giardia. Thus, we evaluated the effect of N-BP on cell viability and ultrastructure and then performed structural modelling and phylogenetic analysis on the FPPS enzymes of Leishmania and Giardia. We performed multiple sequence alignment with MAFFT, phylogenetic analysis with MEGA7, and 3D structural modelling for FPPS with Modeller 9.18 and on I-Tasser server. We performed concentration curves with N-BP in Leishmania promastigotes and Giardia trophozoites to estimate the IC50via the MTS/PMS viability method. The ultrastructure was evaluated by transmission electron microscopy, and the mechanism of cell death by flow cytometry. The nitrogen-containing bisphosphonate risedronate had stronger anti-proliferative activity in Leishmania compared to other N-BPs with an IC50 of 13.8 µM, followed by ibandronate and alendronate with IC50 values of 85.1 µM and 112.2 µM, respectively. The effect of N-BPs was much lower on trophozoites of Giardia than Leishmania (IC50 of 311 µM for risedronate). Giardia treated with N-BP displayed concentric membranes around the nucleus and nuclear pyknosis. Leishmania had mitochondrial swelling, myelin figures, double membranes, and plasma membrane blebbing. The same population labelled with annexin-V and 7-AAD had a loss of membrane potential (TMRE), indicative of apoptosis. Multiple sequence alignments and structural alignments of FPPS proteins showed that Giardia and Leishmania FPPS display low amino acid identity but possess the conserved aspartate-rich motifs. Giardia and Leishmania FPPS enzymes are phylogenetically distant but display conserved protein signatures. The N-BPs effect on FPPS was more pronounced in Leishmania than Giardia. This might be due to general differences in metabolism and differences in the FPPS catalytic site.

中文翻译：

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关于法呢基二磷酸合酶（FPPS）的结构和双膦酸盐对利什曼原虫和贾第鞭毛虫的增殖和超微结构的活性的见解

法呢基二磷酸合酶（FPPS）位于不同的固醇生物合成途径（例如产生类异戊二烯，三元醇和麦角固醇的途径）的交叉点。FPPS在真核生物中无处不在，并被含氮双膦酸盐（N-BP）抑制。尚未在婴儿利什曼原虫和贾第鞭毛虫中研究N-BP活性和细胞死亡机制以及由于N-BP造成的超微结构破坏。因此，我们评估了N-BP对细胞活力和超微结构的影响，然后对利什曼原虫和贾第鞭毛虫的FPPS酶进行了结构建模和系统发育分析。我们使用MAFFT进行了多序列比对，使用MEGA7进行了系统发育分析，以及使用Modeller 9.18和I-Tasser服务器进行FPPS的3D结构建模。我们用利什曼原虫前鞭毛体和贾第鞭毛虫滋养体中的N-BP进行了浓度曲线，以通过MTS / PMS生存力方法估算IC50。通过透射电子显微镜评估超微结构，并通过流式细胞术评估细胞死亡的机制。与其他N-BP相比，含氮的双膦酸利塞膦酸盐在利什曼原虫中具有更强的抗增殖活性，IC50为13.8 µM，其次是伊班膦酸盐和阿仑膦酸盐，IC50值分别为85.1 µM和112.2 µM。N-BPs对贾第鞭毛虫的滋养体的作用要比利什曼原虫低得多（利塞膦酸盐的IC50为311 µM）。用N-BP处理的贾第鞭毛虫在核周围有同心膜，并且核固缩。利什曼原虫具有线粒体肿胀，髓鞘形态，双膜和质膜起泡。用膜联蛋白-V和7-AAD标记的同一群体的膜电位（TMRE）丧失，表明细胞凋亡。FPPS蛋白的多个序列比对和结构比对显示，贾第鞭毛虫和利什曼原虫FPPS显示出低氨基酸同一性，但具有保守的富含天冬氨酸的基序。贾第鞭毛虫和利什曼原虫FPPS酶在系统发育上距离遥远，但显示出保守的蛋白质特征。在利什曼原虫中，N-BPs对FPPS的影响比贾第虫更为明显。这可能是由于代谢的一般差异和FPPS催化位点的差异。FPPS蛋白的多个序列比对和结构比对显示，贾第鞭毛虫和利什曼原虫FPPS显示出低氨基酸同一性，但具有保守的富含天冬氨酸的基序。贾第鞭毛虫和利什曼原虫FPPS酶在系统发育上距离遥远，但显示出保守的蛋白质特征。在利什曼原虫中，N-BPs对FPPS的影响比贾第虫更为明显。这可能是由于代谢的一般差异和FPPS催化位点的差异。FPPS蛋白的多个序列比对和结构比对显示，贾第鞭毛虫和利什曼原虫FPPS显示出低氨基酸同一性，但具有保守的富含天冬氨酸的基序。贾第鞭毛虫和利什曼原虫FPPS酶在系统发育上距离遥远，但显示出保守的蛋白质特征。在利什曼原虫中，N-BPs对FPPS的影响比贾第虫更为明显。这可能是由于代谢的一般差异和FPPS催化位点的差异。