Plasmodium falciparum is a unicellular protozoan parasite and causative agent of a severe form of malaria in humans, accounting for very high worldwide fatality rates. At the molecular level, survival of the parasite within the human host is mediated by P. falciparum heat shock proteins (PfHsps) that provide protection during febrile episodes. The ATP-dependent chaperone activity of Hsp70 relies on the co-chaperone J domain protein (JDP), with which it forms a chaperone-co-chaperone complex. The exported P. falciparum JDP (PfJDP), PFA0660w, has been shown to stimulate the ATPase activity of the exported chaperone, PfHsp70-x. Furthermore, PFA0660w has been shown to associate with another exported PfJDP, PFE0055c, and PfHsp70-x in J-dots, highly mobile structures found in the infected erythrocyte cytosol. Therefore, the present study aims to conduct a structural and functional characterization of the full-length exported PfJDP, PFE0055c. Recombinant PFE0055c was successfully expressed and purified and found to stimulate the basal ATPase activity of PfHsp70-x to a greater extent than PFA0660w but, like PFA0660w, did not significantly stimulate the basal ATPase activity of human Hsp70. Small-molecule inhibition assays were conducted to determine the effect of known inhibitors of JDPs (chalcone, C86) and Hsp70 (benzothiazole rhodacyanines, JG231 and JG98) on the basal and PFE0055c-stimulated ATPase activity of PfHsp70-x. In this study, JG231 and JG98 were found to inhibit both the basal and PFE0055c-stimulated ATPase activity of PfHsp70-x. C86 only inhibited the PFE0055c-stimulated ATPase activity of PfHsp70-x, consistent with PFE0055c binding to PfHsp70-x through its J domain. This research has provided further insight into the molecular basis of the interaction between these exported plasmodial chaperones, which could inform future antimalarial drug discovery studies.

恶性疟原虫是一种单细胞原生动物寄生虫，是人类严重疟疾的病原体，在世界范围内造成了非常高的死亡率。在分子水平上，寄生虫在人类宿主内的存活是由恶性疟原虫热休克蛋白 (PfHsps) 介导的，该蛋白在发热发作期间提供保护。Hsp70 的 ATP 依赖性伴侣活性依赖于共同伴侣 J 结构域蛋白 (JDP)，与它形成伴侣-共同伴侣复合物。出口的恶性疟原虫JDP (PfJDP)，PFA0660w，已被证明可刺激输出的伴侣蛋白 PfHsp70-x 的 ATP 酶活性。此外，PFA0660w 已被证明与另一种输出的 PfJDP、PFE0055c 和 PfHsp70-x 相关联，这些 J-dots 是在受感染的红细胞胞质溶胶中发现的高度可移动的结构。因此，本研究旨在对全长输出的 PfJDP PFE0055c 进行结构和功能表征。重组 PFE0055c 被成功表达和纯化，发现对 PfHsp70-x 的基础 ATPase 活性的刺激程度大于 PFA0660w，但与 PFA0660w 一样，没有显着刺激人 Hsp70 的基础 ATPase 活性。进行小分子抑制测定以确定 JDPs（查耳酮，C86）和 Hsp70（苯并噻唑红花青素，JG231 和 JG98）对 PfHsp70-x 的基础和 PFE0055c 刺激的 ATP 酶活性的影响。在这项研究中，发现 JG231 和 JG98 抑制 PfHsp70-x 的基础和 PFE0055c 刺激的 ATP 酶活性。C86 仅抑制 PFE0055c 刺激的 PfHsp70-x 的 ATP 酶活性，这与 PFE0055c 通过其 J 结构域与 PfHsp70-x 结合一致。这项研究进一步深入了解了这些输出的疟原虫伴侣之间相互作用的分子基础，这可以为未来的抗疟药物发现研究提供信息。