Function of the Essential KHARON1 Protein in Bloodstream Form African Trypanosomes

非洲锥虫血流中必需的 KHARON1 蛋白的功能

基本信息

批准号：
9007963
负责人：
Scott M Landfear
金额：
$ 52.41万
依托单位：
OREGON HEALTH & SCIENCE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-02-16 至 2021-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9007963
关键词：
Address African African Trypanosomiasis Biological Biological Process Biology Biotin Blood Circulation Cell division Cell membrane Cells Cessation of life Complex Cytokinesis Cytoskeleton Data Disease Environment Exhibits Flagella Genes Glucose Transporter Individual Insecta Integral Membrane Protein Ions Kinesin Knowledge Label Laboratories Leishmania Life Cycle Stages Location Mass Spectrum Analysis Mediating Membrane Membrane Proteins Messenger RNA Methodology Methods Mitotic spindle Modeling Molecular Multiple Partners Organelles Organism Parasites Pathogenesis Play Protein Subunits Proteins Proteome RNA Interference Role Signal Transduction Site Staging Structure Surface Trypanosoma Trypanosoma brucei brucei Trypanosoma cruzi Virulence base cell motility cohort interest knock-down macrophage mutant novel prevent protein complex public health relevance research study trafficking

项目摘要

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.

描述（由适用提供）：诸如Brucei锥虫和利什曼原虫等动力质体寄生虫会导致毁灭性疾病，使数百万人受苦。概括。在这些单细胞寄生虫中介导运动性的鞭子样鞭毛在引起寄生虫，血液形式（BF）T。Brucei和Leishmania的amastigotes的阶段起着至关重要的作用。我们的实验室发现了一种新型的细胞骨架相关蛋白Kharon1，该蛋白与Flagllar Axoneme的底部相关，并介导了整合性膜蛋白靶向墨西哥和T. Brucei中的Flagllar膜（FM）。尽管对于所有动力质体寄生虫可能很重要，但由于该模型寄生虫的许多技术优势，该应用将重点关注Kharon1在T. Brucei中的功能。 TBKHARON1 mRNA的敲低对BF锥虫是致命的，它可以防止细胞分裂或细胞因子性，可能是因为它会损害各种整体膜蛋白的鞭毛靶向，从而破坏了鞭毛在裂解形成中所需要的frrow furrow furrow forrow for cytokiness os Cytokiness的启动。该应用程序的一个主要目的是全面确定需要使用定量质谱的FM蛋白的队列，这些FM蛋白需要TBKHARON1才能传输到鞭毛。定义依赖Kharon的FM蛋白质组对于理解这种新型Flagllar贩运机制的生物学功能至关重要。然而，最近对布鲁氏菌的研究表明，寄生虫分裂期间的TBKHARON1也位于有丝分裂主轴中，并且与质膜下方的亚细胞细胞骨架有关。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，并且这些复合物中的蛋白质亚基的子集可能是针对Kharon1的3个已知亚细胞位置的特定于：Flagellar Axoneme的3个已知亚细胞位置：Flagellar Axoneme的基础，有丝分裂的斑点斑点和cyspellicult cyteckelet。因此，第二个主要目的是确定与TBKHARON1相关的其他蛋白质亚基，尤其是搜索可能针对3个亚细胞位置的每个亚基。这些实验将采用最近的方法来识别多亚基蛋白复合物，“生物素接近标记”或“生物剂”的成分。一旦确定了这种其他亚基，将使用条件RNA干扰（RNAI）询问其生物学功能。特别是，如果存在特定地点的哈龙络合物和亚基，RNAi将被应用于如此独特的亚基，以阐明flagelum底部的tbkharon1的潜在不同作用，在有丝分裂中主轴，并在细胞细胞骨架中。总体而言，该项目将定义Kharon1的T. brucei的生物学A分子功能，该蛋白在非洲锥虫和其他动型寄生虫的疾病引起疾病阶段起着至关重要的作用。