Targeting the trypanosomal preribosomal complex

靶向锥虫前核糖体复合物

• 批准号: 9026682
• 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/9026682
• 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; Pharmaceutical Preparations; Process; 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 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在发生核糖体组装的核仁中转录28S、18S和5.8S的35S rRNA前体。通常只有5S rRNA被RNA聚合酶III在核质中转录，并与核糖体蛋白L5一起进入核仁。在唯一具有良好特性的真核系统酿酒酵母中，Rpf2和Rrs1两个组装因子以及核糖体蛋白L11需要将早期的前乳体(5S rRNA和L5)带到组装的核糖体中。我们实验室已经确定了锥虫体内最早的胸前颗粒，它包含保守的组分5S rRNA和L5以及锥体特有的蛋白P34/P37。我们已经确定了这种新型三分子复合体的关键决定因素，以及我们假设对其结构和功能至关重要的复合体的独特特征。我们还表明，L5和P34/P37对于功能性核糖体的形成是必不可少的，因此，布鲁氏毛滴虫的生存能力也是如此。在这项建议中，我们将识别和表征胸前颗粒的组装成分，并利用我们的知识来确定化疗方法的潜在靶点。我们的假设是，基本的胸前复合体各组分之间的相互作用将为化疗破坏布鲁氏毛滴虫核糖体组装提供有效的靶点。该项目的具体目标是：1.确定使5S rRNA复合体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.确定核糖体前复合体成分之间的相互作用网络，并检查中断如何影响细胞内复合体的功能。3.发展FRET方法来研究RNA-蛋白质相互作用、体内蛋白质-蛋白质相互作用以及筛选小干扰分子。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针对病原体核糖体组装的药物的开发成为可能。许多现有的治疗细菌感染的药物都专门结合到细菌核糖体的功能相关部位。前核糖体复合体是这一提议的焦点，对锥虫核糖体的组装和功能以及寄生虫的生存至关重要。复合体中的蛋白质要么是锥虫所特有的(P34/P37)，要么包含与宿主明显不同的特征(L5)。我们的工作还将继续提供新的见解，这些见解将影响真核核糖体生物发生的更广泛领域。此外，该复合体的独特功能将使我们能够定义和利用这一靶点进行化疗开发。