Structure of Malaria Parasite RNA polymerase

疟疾寄生虫 RNA 聚合酶的结构

基本信息

批准号：
10433276
负责人：
Manuel Llinas
金额：
$ 23.73万
依托单位：
PENNSYLVANIA STATE UNIVERSITY, THE
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-01-21 至 2023-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10433276
关键词：
AIDS/HIV problem Accounting Affinity Affinity Chromatography Antibiotics Antimalarials Asparagine Aspartate Bacteria Bacteriophages Binding Biochemical Biological Assay Biological Process COVID-19 treatment CRISPR/Cas technology Cell Nucleus Cells Cessation of life Child Clostridium difficile Code Cryoelectron Microscopy DNA DNA-Directed RNA Polymerase Development Diarrhea Disease Drug Design Drug Screening Drug Targeting Enzymes Eukaryota Evolution Future Gene Expression Genetic Transcription Genome Genomic DNA Goals Human In Vitro Infection Intervention Joints Laboratories Life Maintenance Malaria Messenger RNA Methods Mitochondrial RNA Nuclear Nuclear Extract Nuclear RNA Organelles Parasites Persons Pharmaceutical Preparations Plasmodium Plasmodium falciparum Play Pregnant Women Production RNA RNA Polymerase I RNA Polymerase II RNA Polymerase III RNA chemical synthesis Research Research Project Grants Resistance development Resolution Rifampin Role Small Nuclear RNA Specimen Stretching Structure System Techniques Testing Tuberculosis Untranslated RNA Vaccines Virus Virus Replication Work Zidovudine affinity labeling antiviral drug development arginyllysine base design drug action drug candidate enzyme structure genetic manipulation genome editing in silico in vitro activity inhibitor insight lead candidate new therapeutic target novel particle pathogen protein structure public health relevance remdesivir scaffold screening stable cell line three dimensional structure

项目摘要

Project Summary Malaria, the most wide-scale protozoan-induced infection of humans, is caused by parasites from the genus Plasmodium, with Plasmodium falciparum being responsible for the most severe form of disease in humans. Although there are several drugs that are currently used to treat malaria, malaria parasites have rapidly developed resistance to all currently available frontline drugs. This demonstrates that there is an urgent and critical need to identify novel drug targets for the development of new and effective anti-malarial drugs. Transcription of DNA into RNA, the first step of gene expression, is carried out by RNA polymerase (RNAP) in all life forms and viruses. Due to its essential role in life maintenance and virus replication, RNAP is a proven drug target for antibiotic and antiviral developments. The biochemical characterization of purified RNAP in vitro along with structural studies have played essential roles to define our understanding of the structure and function of RNAP. Purified RNAP has also facilitated large-scale drug screening and the characterization of drug candidate lead compounds, and structural studies of RNAPs have revealed the mechanism of drug action as well as accelerated drug design by in silico screening. RNA synthesis in Plasmodium parasites occurs in three organelles (nucleus, mitochondrion and non- photosynthetic apicoplast) and is carried out by five RNAPs including three nuclear RNAPs (RNAP I, RNAP II and RNAP III), bacteriophage-type mitochondrial RNAP and bacterial-type apicoplast RNAP. The ultimate goal of this research will be to isolate all endogenous nuclear RNAPs from P. falciparum cells and determine the 3D structures of these enzymes by single-particle cryo-electron microscopy (cryo-EM). These high-resolution structures will elucidate the mechanism of transcription in Plasmodium and create valuable platforms for the design of new antimalarial drugs targeting P. falciparum RNAPs. Finally, these structures will also provide new insight into the evolution of RNAPs during the course of parasitic adaptation in eukaryotes. In this proposal, we seek to establish methods to investigate the structure and function of P. falciparum RNAP II, which is responsible for mRNAs, snRNAs and regulatory RNAs transcription. Aim 1 will use CRISPR/Cas9 genome editing to generate a stable cell line of P. falciparum expressing an affinity-labeled largest subunit (Rpb1) of RNAP II. Aim 2 will establish a purification method of RNAP II from P. falciparum nuclear extract using a combination of chromatographic techniques that will be tracked by an in vitro transcription assay to test RNAP II activity. In Aim 3, we will determine the atomic-resolution 3D structure of RNAP II by cryo-EM. The proposed work will pave the way for investigating the structure and function of all three nuclear RNAPs from Plasmodium.

项目概要疟疾是最广泛的原生动物引起的人类感染，由该属寄生虫引起疟原虫，其中恶性疟原虫是导致人类最严重疾病的原因。尽管目前有多种药物用于治疗疟疾，但疟疾寄生虫已迅速对目前所有可用的一线药物产生了耐药性。这表明有一个紧迫且迫切需要确定新的药物靶标，以开发新的有效的抗疟疾药物。 DNA转录为RNA，是基因表达的第一步，是由RNA聚合酶（RNAP）进行的所有生命形式和病毒。由于其在生命维持和病毒复制中的重要作用，RNAP 已被证明是一种抗生素和抗病毒药物开发的药物靶标。纯化RNAP的体外生化表征与结构研究一起在定义我们对结构和功能的理解方面发挥了重要作用 RNAP。纯化的 RNAP 还促进了大规模药物筛选和药物表征候选先导化合物和 RNAP 的结构研究揭示了药物作用机制：以及通过计算机筛选加速药物设计。疟原虫寄生虫中的 RNA 合成发生在三种细胞器（细胞核、线粒体和非细胞器）中。光合顶端质体），由五个 RNAP 进行，包括三个核 RNAP（RNAP I、RNAP II）和 RNAP III)、噬菌体型线粒体 RNAP 和细菌型顶质体 RNAP。最终目标这项研究的目的是从恶性疟原虫细胞中分离出所有内源性核 RNAP，并确定 3D 通过单颗粒冷冻电子显微镜（cryo-EM）观察这些酶的结构。这些高分辨率结构将阐明疟原虫的转录机制，并为疟原虫创建有价值的平台针对恶性疟原虫 RNAP 的新型抗疟药物的设计。最后，这些结构还将提供新的深入了解真核生物寄生适应过程中 RNAP 的进化。在本提案中，我们寻求建立研究恶性疟原虫 RNAP 结构和功能的方法 II，负责mRNA、snRNA和调节RNA的转录。目标 1 将使用 CRISPR/Cas9 基因组编辑生成稳定的恶性疟原虫细胞系，表达亲和标记的最大亚基 (Rpb1) RNAP II。目标 2 将建立一种从恶性疟原虫核提取物中纯化 RNAP II 的方法，使用色谱技术的组合，将通过体外转录测定来跟踪以测试 RNAP II 活动。在目标 3 中，我们将通过冷冻电镜确定 RNAP II 的原子分辨率 3D 结构。拟议的这项工作将为研究疟原虫所有三种核 RNAP 的结构和功能铺平道路。