Function of the Essential KHARON1 Protein in Bloodstream Form African Trypanosomes

 描述（由申请人提供）：动体寄生虫，如布氏锥虫和利什曼原虫，引起毁灭性疾病，折磨数百万人。摘要在这些单细胞寄生虫中介导运动的鞭状鞭毛在两种寄生虫的致病阶段中起着关键作用，血流形式（BF）T。布氏杆菌和利什曼原虫无鞭毛体。我们的实验室发现了一种新的细胞膜相关蛋白KHARON1，它与鞭毛轴丝的基部相关，并介导了两种L. mexicana和T.布鲁塞。虽然可能对所有动质体寄生虫都很重要，但本申请将集中在T.由于这种模式寄生虫的许多技术优势，TbKharon1 mRNA的敲低对BF锥虫是致命的，在那里它阻止细胞分裂或胞质分裂，可能是因为它损害了各种完整膜蛋白的鞭毛靶向，从而破坏了鞭毛在启动胞质分裂所需的th切割沟形成中的既定作用。本申请的一个主要目的是使用定量质谱法全面鉴定需要TbKHARON1运输到鞭毛的FM蛋白质组。确定KHARON依赖的FM蛋白质组将是至关重要的了解这种新的鞭毛运输机械的生物学功能。然而，最近的研究与T。Brucei等人已经确定TbKHARON1也位于寄生虫分裂期间的有丝分裂纺锤体中，并且与质膜下的膜下细胞骨架相关。这些不同的位置表明，TbKHARON1可能在寄生虫的生物学中发挥多种功能。初步数据还表明，KHARON1可能只是一个或几个多蛋白KHARON复合物的一个组成部分，这些复合物中的蛋白亚基的子集可能是特定的3个已知的KHARON1亚细胞位置：鞭毛轴丝的基础，有丝分裂纺锤体和膜下细胞骨架。因此，第二个主要目的是鉴定与TbKHARON1相关的其他蛋白亚基，特别是寻找可能对3个亚细胞基因座中的每一个特异的亚基。这些实验将采用最近的方法来鉴定多亚基蛋白质复合物的组分，即“生物素邻近标记”或“BioID”。一旦这些额外的亚基被鉴定，它们的生物学功能将使用条件性RNA干扰（RNAi）进行询问。特别地，如果存在位点特异性KHARON复合物和亚基，则RNAi将被应用于这些独特的亚基，以阐明TbKHARON 1在鞭毛基部、在有丝分裂中的潜在不同作用。 纺锤体和表膜下细胞骨架。总的来说，本项目将确定T. KHARON1是一种对非洲锥虫和其他动质体寄生虫的致病阶段的生存能力起重要作用的蛋白质。

 DESCRIPTION (provided by applicant): Kinetoplastid parasites such as Trypanosoma brucei and Leishmania cause devastating diseases that afflict millions of people. Summary. The whip-like flagellum that mediates motility in these single-cell parasites plays a critical role in the disease causing stages of both parasites, bloodstream form (BF) T. brucei and amastigotes of Leishmania. Our laboratory has discovered a novel cytoskeletal-associated protein, KHARON1, which is associated with the base of the flagellar axoneme and mediates targeting of integral membrane proteins to the flagellar membrane (FM) in both L. mexicana and T. brucei. Although likely to be important for all kinetoplastid parasites, this application will focus on the functionof KHARON1 in T. brucei due to the many technical advantages of this model parasite. Knockdown of TbKharon1 mRNA is lethal to BF trypanosomes, where it prevents cell division or cytokinesis, probably because it impairs flagellar targeting of various integral membrane proteins and thus disrupts the well-established role of the flagellum in initiating formation of th cleavage furrow that is required for cytokinesis. One major aim of this application will be to comprehensively identify the cohort of FM proteins that require TbKHARON1 to traffic to the flagellum using quantitative mass spectrometry. Defining the KHARON-dependent FM proteome will be critical for understanding the biological function of this novel flagellar trafficking machinery. However, recent studies with T. brucei have established that TbKHARON1 is also located in the mitotic spindle during parasite division and is associated with the subpellicular cytoskeleton beneath the plasma membrane. These distinct locations suggest that TbKHARON1 may play multiple functions in the biology of the parasite. Preliminary data also indicate that KHARON1 is likely only one component of one or several multiprotein KHARON Complexes and that a subset of the protein subunits in these complexes may be specific for each of the 3 known subcellular locations of KHARON1: the base of the flagellar axoneme, the mitotic spindle, and the subpellicular cytoskeleton. Hence, a second major aim will be to identify other protein subunits that associate with TbKHARON1 and in particular to search for subunits that may be specific for each of the 3 subcellular loci. These experiments will employ a recent methodology for identifying the components of multisubunit protein complexes, `biotin proximity labeling' or `BioID'. Once such additional subunits have been identified, their biological function will be interrogated using conditional RNA interference (RNAi). In particular, if site-specific KHARON Complexes and subunits exist, RNAi will be applied to such unique subunits to illuminate the potentially distinct roles of TbKHARON1 at the base of the flagellum, in the mitotic spindle, and in the subpellicular cytoskeleton. Overall, this project will define the biological an molecular functions in T. brucei of KHARON1, a protein that plays essential roles for viability of the disease-causing stage of African trypanosomes and other kinetoplastid parasites.