The role of the scaffolding protein WLP1 in maintaining multiprotein complexes of malaria gametocytes

人类疟原虫恶性疟原虫在其质膜上显示出大量的黏附蛋白，这些黏附蛋白在病原体的入侵和传播阶段尤为重要。这些粘附蛋白中的大多数组装成多蛋白复合物（MPCs），如merozoite表面蛋白1粘附复合物和顶膜抗原1/Rhoptry颈复合物，它们位于merozoite表面，对红细胞感染至关重要。另一种被充分研究的MPC与配子体的质膜有关，配子体是寄生虫在人-蚊子传播过程中形成的性前体细胞。配子体特异性粘附复合体由6个lccl结构域（PfCCp）蛋白和富含半胱氨酸的motif蛋白Pfs230和Pf48/45组成，它们在蚊子体内的有性繁殖和寄生虫的进一步发育中起着至关重要的作用。在最近的一项研究中，我们发现了pfccp复合物的一个新的相互作用伙伴，wd40重复蛋白样蛋白PfWLP1。WD40结构域在真核生物中高度保守，在细胞骨架稳定过程中作为促进蛋白质相互作用的支架。由于其重要的功能，特别是对细胞增殖，人类wd40重复蛋白目前在癌症治疗中被探索。在恶性疟原虫中，计算机分析鉴定出80个推测的wd40重复蛋白，其中PfWLP1是疟原虫特有的。PfWLP1在恶性疟原虫配子体中高度表达，与质膜下的微管共定位。该蛋白进一步与基于pfccp的MPCs相关，转基因寄生虫中PfWLP1的缺失导致这些复合物的不稳定性。鉴于我们目前的数据，我们假设PfWLP1作为支架，将外周基于pfccp的MPCs连接到细胞内细胞骨架，从而维持复合物的稳定性。在本研究中，我们旨在通过以下方法揭示PfWLP1在MPC维持中的作用：1)使用基因敲低方法在配子细胞成熟和配子发生过程中功能表征wfwlp1；2)通过BioID方法破译pfwlp1调控的MPCs的组成；3)研究PfWLP1与相应基因敲除和基因敲除系中选定的细胞骨架元件之间的相互依存关系。本研究获得的数据将深入了解维持疟疾配子体完整性所需的结构元件和粘附外周复合物的高度交织网络。此外，该项目将揭示新的脚手架蛋白PfWLP1是疟疾寄生虫传播的关键参与者。

The human malaria parasite Plasmodium falciparum displays a remarkable number of adhesive proteins on its plasma membrane, which are particularly important for the invasion and transmission stages of the pathogen. The majority of these adhesion proteins assemble to multi-protein complexes (MPCs), like the merozoite surface protein 1 adhesion complex and the apical membrane antigen 1/Rhoptry neck-complex, which are located on the merozoite surface and essential for red blood cell infection. Another well-studied MPC associates with the plasma membrane of gametocytes, sexual precursor cells that are formed during human-to-mosquito transmission of the parasite. The gametocyte-specific adhesion complex is composed of the six LCCL-domain (PfCCp) proteins and the cysteine-rich motif proteins Pfs230 and Pf48/45, all of which are ascribed crucial roles for sexual reproduction and further development of the parasite in the mosquito. In a recent study, we identified a novel interaction partner of the PfCCp-based complex, the WD40-repeat protein-like protein PfWLP1. WD40 domains are highly conserved in eukaryotes and act as scaffolds to facilitate protein interactions, among others during cytoskeleton stabilization. Due to their important functions particularly for proliferating cells, human WD40-repeat proteins are currently explored in cancer therapy. In P. falciparum, in-silico analyses identified 80 putative WD40-repeat proteins with PfWLP1 being specific to the genus Plasmodium. PfWLP1 is highly expressed in the P. falciparum gametocytes, where it co-localizes with microtubules underneath the plasma membrane. The protein is further associated with the PfCCp-based MPCs and the loss of PfWLP1 in transgenic parasites results in instability of these complexes. In view of our current data, we hypothesize that PfWLP1 acts as a scaffold linking the peripheral PfCCp-based MPCs to the intracellular cytoskeleton, thereby maintaining the stability of the complex. In this study we aim to uncover the role of PfWLP1 in MPC maintenance by 1) functionally characterizing WLP1 during gametocyte maturation and gametogenesis using gene-knockdown approaches; 2) deciphering the composition of the PfWLP1-regulated MPCs via BioID methods; and 3) investigating the interdependence between PfWLP1 and selected cytoskeletal elements in corresponding gene-knockdown and gene-knockout lines. Data gained by this study will provide in-depth knowledge on the highly interwoven network of structural elements and adhesive peripheral complexes that are needed to maintain the integrity of malaria gametocytes. Moreover, the project will shed light on the novel scaffolding protein PfWLP1 as a key player of malaria parasite transmission.