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.

摘要 布氏锥虫是引起人类非洲锥虫病（HAT）的真核寄生虫， 有七千万人处于危险之中。HAT在不治疗的情况下几乎总是致命的，但目前 药物价格昂贵、难以管理，并且普遍存在不良副作用。因此需要 用于新药的研发其中一种方法是针对既重要又重要的过程， 寄生虫生存和病原体特异性。核糖体生物合成是大量蛋白质组装的过程 和核糖体RNA组分转化为成熟的功能性核糖体。核糖体生物合成的一个关键过程 是5S核糖核蛋白（RNP）复合物的形成和掺入发育中的60 S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复合物和60 S亚基之间的相互作用，导致构象变化 核糖体的蛋白质和RNA成分。因此，5S RNP的加入是至关重要的 调节检查点，该过程的中断导致核糖体成熟在 60 S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靶点。我们实验室的工作 P34/P37是锥虫特异性蛋白，是锥虫的一个独特的和重要的组成部分。布氏5S RNP. 我们还显示了P34/P37与蛋白质L5和5S rRNA之间的直接和独特的体外相互作用， 5S RNP的两个经过充分研究的组成部分。最近，我们确定了T。蛋白质L11的布氏同源物， Rpf 2和Rrs 1，它们参与酵母中5S RNP的成熟和掺入。我们假设 这些同源物形成了一个网络的相互作用之间的同源和独特的组成部分， 5S RNP复合物，并对其形成和掺入至关重要。具体目标将是 该项目的重点如下： 1)为了表征T. L11、Rpf 2/Rrs 1和其他布鲁氏菌同源物 同源和独特的成分的5S RNP复合物使用在体外研究。 2)使用RNAi敲除技术确定L11和Rpf 2/Rrs 1在核糖体生物合成中的体内重要性 down cell细胞lines. 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。利用小分子来靶向独特的蛋白质 P34/P37及其作为5S RNP复合物成员的重要作用是开发新的 治疗对抗HAT。因此，描述发生在 T.文莱将开辟一条发展小工业的未来道路 分子化学疗法作为HAT的新治疗。