Targeting the trypanosomal preribosomal complex

靶向锥虫前核糖体复合物

• 批准号: 9280621
• 负责人: Noreen Williams
• 金额: $ 28.62万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-18 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9280621
• 关键词: Affect; Affinity Chromatography; African Trypanosomiasis; Bacterial Infections; Binding; Biogenesis; Biological Assay; Cell Line; Cell Nucleolus; Cells; Chagas Disease; Characteristics; Collaborations; Complex; Computer Simulation; Defect; Development; Disease; Drug Targeting; Fluorescence Resonance Energy Transfer; Genetic Transcription; Goals; Homologous Gene; In Vitro; Knowledge; Laboratories; Leishmania; Leishmaniasis; Molecular; Mutation; Nucleoplasm; Organism; Parasites; Pharmacotherapy; Protein Biosynthesis; Proteins; RNA; RNA Interference; RNA Polymerase I; RNA Polymerase III; RNA-Protein Interaction; Ribosomal Proteins; Ribosomal RNA; Ribosomes; Role; Site; Structure; System; Trypanosoma; Trypanosoma brucei brucei; Trypanosoma cruzi; Work; drug development; high throughput screening; in vivo; insight; member; novel; particle; pathogen; protein function; protein protein interaction; public health relevance; rRNA Precursor; scaffold; screening

 DESCRIPTION (provided by applicant): Ribosomes are comprised of both rRNAs, which perform the catalytic function of protein synthesis, and a large number of proteins that form the structural scaffold. Transcription of the 35S rRNA precursor of 28S, 18S, and 5.8S by RNA polymerase I take place in the nucleolus where ribosomal assembly occurs. Only 5S rRNA is usually transcribed in the nucleoplasm by RNA polymerase III and brought into the nucleolus together with ribosomal protein L5. In the only well characterized eukaryotic system, Saccharomyes cerevisiae, two assembly factors, Rpf2 and Rrs1 as well as ribosomal protein, L11, are required to bring the early preribosomal (5S rRNA and L5) to the assembling ribosome. Our laboratory has defined the earliest preribosomal particle in trypanosomes which contains the conserved components 5S rRNA and L5 as well as the trypanosome-specific proteins, P34/P37. We have identified key determinants of this novel tri-molecular complex as well as unique features of the complex that we hypothesize are essential to its structure and function. We have also shown that both L5 and P34/P37 are essential to the formation of functional ribosomes and therefore, the viability of T. brucei. In this proposal we will identify and characterize the assembly components of the preribosomal particle and use our knowledge to define potential targets for chemotherapeutic approaches. Our hypothesis is that the interactions between the components of the essential preribosomal complex will provide valid targets for chemotherapeutic disruption of ribosomal assembly in T. brucei. The specific aims of the project are to: 1. Determine the critical proteins that enable the association of the 5S rRNA-containing complex with the ribosome and their function in ribosome biogenesis. 2. Define the interaction network among the pre-ribosomal complex components and examine how the disruption affects the function of the complex within the cell. 3. Develop FRET approaches to study RNA-protein interaction, in vivo protein-protein interactions, and screen for small interfering molecules. Although the ribosome is highly conserved, subtle differences between the host and pathogen have enabled the development of drugs specifically targeting pathogen ribosome assembly. Many existing drugs for the treatment of bacterial infections specifically bind to the functionally relevant sites of the bacterial ribosome. The pre-ribosomal complex that is the focus of this proposal is essential to trypanosome ribosome assembly and function and to the survival of the parasite. Proteins within the complex are either unique to trypanosomes (P34/P37) or contain features clearly distinct from those of the host (L5). Our work will also continue to provide new insights that will impact the broader field of eukaryotic ribosomal biogenesis. Moreover, the unique features of the complex will allow us to define and exploit this target for chemotherapeutic development.

 描述（申请人提供）：核糖体由执行蛋白质合成催化功能的rRNA和形成结构支架的大量蛋白质组成。RNA聚合酶I对28 S、18 S和5.8S的35 S rRNA前体的转录发生在核糖体组装发生的核仁中。只有5S rRNA通常在核质中被RNA聚合酶III转录，并与核糖体蛋白L5一起被带入核仁。在唯一一个被充分表征的真核系统中，酿酒酵母，需要两个组装因子Rpf 2和Rrs 1以及核糖体蛋白L11将早期前核糖体（5S rRNA和L5）带到组装的核糖体。我们的实验室已经确定了锥虫中最早的前核糖体颗粒，其包含保守的组分5S rRNA和L5以及锥虫特异性蛋白P34/P37。我们已经确定了这种新型三分子复合物的关键决定因素以及我们假设对其结构和功能至关重要的复合物的独特功能。我们还发现，L5和P34/P37对功能性核糖体的形成至关重要，因此，T.布鲁塞。在这个提议中，我们将确定和表征前核糖体颗粒的组装组分，并利用我们的知识来定义化疗方法的潜在靶点。我们的假设是，基本前核糖体复合物组分之间的相互作用将为T.布鲁塞。该项目的具体目标是：1.确定使含5S rRNA复合物与核糖体结合的关键蛋白质及其在核糖体生物发生中的功能。2.定义前核糖体复合物组分之间的相互作用网络，并检查破坏如何影响细胞内复合物的功能。3.开发FRET方法来研究RNA-蛋白质相互作用，体内蛋白质-蛋白质相互作用，并筛选小干扰分子。虽然核糖体是高度保守的，但宿主和病原体之间的细微差异使得能够开发特异性靶向病原体核糖体组装的药物。许多现有的用于治疗细菌感染的药物特异性结合到细菌核糖体的功能相关位点。前核糖体复合物是该建议的焦点，它对锥虫核糖体的组装和功能以及寄生虫的生存至关重要。复合物中的蛋白质要么是锥虫所特有的（P34/P37），要么含有与宿主明显不同的特征（L5）。我们的工作也将继续提供新的见解，这将影响更广泛的真核生物核糖体生物发生领域。此外，该复合物的独特功能将使我们能够定义和利用该靶点进行化疗开发。