Towards the reconstitution and a structure of the Plasmodium vacuolar translocon

恶性疟原虫是疟疾的病原体，每年造成60万人死亡，并导致3亿至5亿人临床疾病。尽管目前有几种药物用于治疗疟疾，但抗药性寄生虫的出现和传播是有效控制疾病的主要威胁。迫切需要针对基本和原始寄生过程的新的抗疟疾疗法。这种微生物在红细胞内生长和在血流中茁壮成长的核心能力是，它能够将大约5%-8%的蛋白质输出到包裹的液泡之外，进入宿主细胞的胞浆。恶性疟原虫在人类红细胞内的细胞内生存依赖于寄生虫蛋白的出口，这些寄生虫蛋白重塑受感染的宿主细胞，以支持其毒力和寄生生活方式。为了做到这一点，寄生虫在包裹的液泡膜上安装了一个大型蛋白质复合体，即出口蛋白转运子(PTEX)，使其能够将这数百种蛋白质输出到红细胞中。PTEX由五种寄生虫蛋白组成：出口蛋白2(EXP2)，它组装成一个跨膜孔来运输货物蛋白；ATPase HSP101，它通过展开货物蛋白并通过EXP2通道提供能量来驱动出口过程；以及PTEX150、PTEX88和TRX2蛋白，它识别、准备和将转位的蛋白运送到“通道-引擎”复合体。必需亚基EXP2、HSP101和PTEX150紧密结合成一个核心复合体。作为许多关键生物学过程的共同门户，PTEX代表着疟原虫生命周期中的一个致命弱点。然而，到目前为止，这种复杂的蛋白质输出机的结构和详细的作用机制仍然不清楚。为此，我们已经从重组源中纯化了TRX2、EXP2和HSP101亚基，并解决了HSP101 ATPase的TRX2亚基和N-末端结构域的晶体结构。通过冷冻电子显微镜和X射线结晶学的结合，我们将追踪ATPase HSP101的结构，它是疟原虫蛋白质运输的关键调节因子。我们还寻求完成PTEX150的重建。由于疟原虫基因组高度的AT密码子偏好性，PTEX150含有几个富含天冬酰胺的重复序列，这些重复序列具有很高的无序和聚集倾向。为了克服这一障碍，我们建议探索使用疟原虫HSP110，一种 伴侣蛋白可以稳定富含天冬酰胺的寄生蛋白，作为共表达伙伴能够和/或改善PTEX150的表达，并将其重组为具有功能的PTEX三元核心。利用酵母或昆虫细胞作为异源表达系统，我们将利用合成的基因在这种特殊的伴侣蛋白存在下共表达PTEX亚单位，并重组更高阶的PTEX复合体。

Plasmodium falciparum, the causative agent of malaria, is responsible for 600,000 deaths every year and causes clinical illness in 300-500 million more. Although several drugs are currently used to treat malaria, the emergence and spread of drug-resistant parasites is a major threat to effective disease control. Novel antimalarial therapies targeting essential and original parasitic processes are urgently needed. Central to the capacity of this microorganism to grow inside red blood cells and to thrive inside the blood stream is its ability to export about 5-8% of its proteins beyond an encasing vacuole and into the cytosol of its host cell. The intracellular survival of Plasmodium falciparum within human red blood cells is dependent on export of parasite proteins that remodel the infected host cell to support its virulence and parasitic lifestyle. To do so, the parasite installs a large protein complex, the Plasmodium Translocon of EXported proteins) (PTEX), in the membrane of the encasing vacuole that enables it to export these hundreds of proteins into the red blood cell. PTEX is composed of five parasite proteins: the EXported Protein 2 (EXP2) that assembles into a trans-membrane pore to conduct the cargo proteins, the ATPase HSP101 that provides the energy driving the export process by unfolding the cargo proteins and threading them through the EXP2 channel and, proteins PTEX150, PTEX88 and TRX2 that recognize, prepare and deliver the translocated proteins to the `channel-engine' complex. The essential subunits EXP2, HSP101 and PTEX150 tightly associate into a core complex. As a common portal for numerous crucial biological processes, PTEX represents an Achilles heel in Plasmodium's life cycle. However, to this day, the structure and detailed mechanism of action of this complex protein export machine remain unknown. To this aim, we have already purified subunits TRX2, EXP2, and HSP101 expressed from recombinant sources and solved the crystal structures of the TRX2 subunit and the N-terminal domain of the HSP101 ATPase. Through the combination of cryo-electron microscopy and X-ray crystallography, we will pursue the structure of the ATPase HSP101, a key regulator of protein trafficking in Plasmodium. We also seek to complete the reconstitution of PTEX150. As a consequence of the high AT codon bias of Plasmodium's genome, PTEX150 contains several asparagine-rich repeats, sequences with a high propensity for disorder and aggregation. To overcome this obstacle, we propose to explore the use of the Plasmodium HSP110, a chaperone shown to stabilize the asparagine-rich parasitic proteins, as a coexpression partner to enable and/or improve expression of PTEX150 and its reconstitution into a functional PTEX ternary core. Using yeast or insect cells as heterologous expression systems, we will use synthetic genes to coexpress PTEX subunits in presence of this specialized chaperone and reconstitute higher order PTEX complexes.