Identifying Critical Interactions in the Unique Trypanosoma brucei 5S Ribonucleoprotein Complex and their Role in Ribosome Biogenesis

鉴定独特的布氏锥虫 5S 核糖核蛋白复合物中的关键相互作用及其在核糖体生物发生中的作用

• 批准号: 9394359
• 负责人: Daniel Jaremko
• 金额: $ 2.94万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-19 至 2021-07-18
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9394359
• 关键词: Adverse effects; Affect; African; African Trypanosomiasis; Binding; Biogenesis; Cell Line; Cell Survival; Cell physiology; Cells; Chagas Disease; Co-Immunoprecipitations; Complex; Country; Cryoelectron Microscopy; Development; Drug Targeting; Environment; Family; Fractionation; Future; Growth; Homologous Gene; Homologous Protein; Human; In Vitro; Individual; Interruption; Laboratories; Lead; Leishmaniasis; Life; Location; Maps; Membrane; Molecular Conformation; Northern Blotting; Organism; Parasites; Pathogenicity; Pharmaceutical Preparations; Phenotype; Polyribosomes; Population; Populations at Risk; Process; Proteins; Quantitative Reverse Transcriptase PCR; RNA; RNA Interference; RNA-Protein Interaction; Resolution; Ribonuclease H; Ribonucleoproteins; Ribosomal RNA; Ribosomes; Role; Structure; Surface Plasmon Resonance; Translations; Trypanosoma; Trypanosoma brucei brucei; Trypanosoma cruzi; Trypanosomatina; Western Blotting; Work; Yeasts; burden of illness; combat; drug development; human disease; in vivo; insight; knock-down; member; novel; novel therapeutics; pathogen; protein protein interaction; small molecule

ABSTRACT Trypanosoma brucei is a eukaryotic parasite responsible for the disease Human African Trypanosomiasis (HAT), which has an at-risk population of 70 million. HAT is nearly always fatal without treatment, but current medications are expensive, difficult to administer, and rife with adverse side effects. Therefore, there is a need for the development of new drugs. One approach to which is targeting processes that are both essential for parasite survival and pathogen-specific. Ribosome biogenesis is the process of assembling numerous protein and ribosomal RNA components into mature, functional ribosomes. One critical process of ribosome biogenesis is the formation and incorporation of the 5S ribonucleoprotein (RNP) complex into developing 60S ribosomal subunits. Recent high resolution cryo-electron microscopy structures have identified key inter- and intra-subunit interactions between members of the 5S RNP complex and the 60S subunit, which lead to conformational shifts in protein and RNA components of the ribosome. Therefore, the incorporation of the 5S RNP acts as a crucial regulatory checkpoint, with disruption of the process resulting in cessation of ribosome maturation at the level of the 60S subunit. Given that functional ribosomes are required for survival, this makes the assembly and incorporation of the 5S RNP complex a promising target for future drug development. Work in our laboratory has identified the trypanosome-specific proteins P34/P37 as a unique and essential part of the T. brucei 5S RNP. We have also shown direct and unique in vitro interactions between P34/P37 and the protein L5 and 5S rRNA, two well-studied components of the 5S RNP. Recently, we identified T. brucei homologues of the proteins L11, Rpf2 and Rrs1, which are involved in maturation and incorporation of the 5S RNP in yeast. We hypothesize that these homologues form a network of interactions between homologous and unique components of the 5S RNP complex, and are crucial for its formation and incorporation. The specific aims that will be the focus of this project are the following: 1) To characterize the interactions between the T. brucei homologues of L11, Rpf2/Rrs1 and other homologous and unique components of the 5S RNP complex using in vitro studies. 2) Determine the in vivo importance of L11 and Rpf2/Rrs1 in ribosome biogenesis using RNAi knock- down cell lines. HAT remains a significant disease burden on populations in many sub-Saharan African countries, in part due to the many problems associated with current treatments. Using small molecules to target the unique proteins P34/P37 and their essential role as members of the 5S RNP complex is a potential path to developing new treatments to combat HAT. Therefore, characterizing the network of interactions occurring between members of the 5S RNP complex in T. brucei will set a future path toward the development of small molecule chemotherapeutics as novel treatments for HAT.

抽象的 布氏锥虫是一种真核寄生虫，导致人类非洲锥虫病 (HAT)， 其中有 7000 万高危人口。如果不进行治疗，HAT 几乎总是致命的，但目前 药物价格昂贵、难以管理且充满不良副作用。因此，需要 用于新药的开发。一种方法是针对对于以下方面都至关重要的流程： 寄生虫的生存和病原体特异性。核糖体生物合成是组装大量蛋白质的过程 和核糖体RNA成分转化为成熟的、有功能的核糖体。核糖体生物发生的一个关键过程 是 5S 核糖核蛋白 (RNP) 复合物的形成和掺入正在发育的 60S 核糖体 亚单位。最近的高分辨率冷冻电子显微镜结构已经确定了关键的亚基间和亚基内 5S RNP 复合体成员与 60S 亚基之间的相互作用，导致构象变化 存在于核糖体的蛋白质和RNA成分中。因此，5S RNP 的纳入至关重要。 监管检查点，该过程的中断导致核糖体成熟停止在 60S亚基。鉴于功能性核糖体是生存所必需的，这使得组装和 5S RNP 复合物的掺入是未来药物开发的有希望的目标。我们实验室的工作有 鉴定出锥虫特异性蛋白 P34/P37 是 T. brucei 5S RNP 的独特且重要的部分。 我们还展示了 P34/P37 与蛋白质 L5 和 5S rRNA 之间直接且独特的体外相互作用， 5S RNP 的两个经过深入研究的组件。最近，我们鉴定了 T. brucei 蛋白 L11 的同源物， Rpf2 和 Rrs1，参与酵母中 5S RNP 的成熟和掺入。我们假设 这些同源物形成了同源和独特成分之间的相互作用网络 5S RNP 复合体，对其形成和合并至关重要。具体目标将是 该项目的重点如下： 1) 表征 L11、Rpf2/Rrs1 和其他 T. brucei 同源物之间的相互作用 使用体外研究研究 5S RNP 复合物的同源且独特的成分。 2) 使用 RNAi 敲入确定 L11 和 Rpf2/Rrs1 在核糖体生物合成中的体内重要性 下调细胞系。 HAT 仍然是许多撒哈拉以南非洲国家人口的重大疾病负担，部分原因是 与当前治疗相关的许多问题。使用小分子靶向独特的蛋白质 P34/P37 及其作为 5S RNP 复合体成员的重要作用是开发新的潜在途径 对抗 HAT 的治疗方法。因此，表征之间发生的相互作用网络 T. brucei 5S RNP 综合体的成员将为小型化的未来发展制定道路 分子化疗作为 HAT 的新型治疗方法。