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，并使3 -5亿人患上临床疾病。虽然目前有几种药物用于治疗疟疾，但耐药寄生虫的出现和传播是对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型抗疟疾疗法。这种微生物在红细胞内生长和在血液中茁壮成长的能力的核心是它能够将大约5-8%的蛋白质输出到包裹液泡外，进入宿主细胞的细胞质。恶性疟原虫在人红细胞内的细胞内存活依赖于寄生虫蛋白的输出，这些蛋白重塑被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，寄生虫在包裹液泡的膜上安装了一个大的蛋白质复合物，即输出蛋白的疟原虫转位子（PTEX），使它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）组装成跨膜孔来传导货物蛋白，atp酶HSP101通过展开货物蛋白并将其穿过EXP2通道来提供驱动出口过程的能量，蛋白质PTEX150、PTEX88和TRX2识别、准备并将易位蛋白运送到“通道引擎”复合物。基本亚基EXP2、HSP101和PTEX150紧密结合成一个核心复合物。作为许多关键生物过程的共同门户，PTEX代表了疟原虫生命周期中的阿喀琉斯之踵。然而，直到今天，这种复杂的蛋白质输出机器的结构和详细的作用机制仍然未知。为此，我们已经纯化了重组源表达的TRX2、EXP2和HSP101亚基，并解析了TRX2亚基的晶体结构和HSP101 atp酶的n端结构域。通过低温电子显微镜和x射线晶体学的结合，我们将追求atp酶HSP101的结构，这是疟原虫蛋白质运输的关键调节因子。我们还寻求完成PTEX150的重构。由于疟原虫基因组的AT密码子高度偏倚，PTEX150包含几个富含天冬酰胺的重复序列，这些序列具有高度的无序和聚集倾向。为了克服这一障碍，我们建议探索利用HSP110， a

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.