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

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

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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.

项目摘要疟疾是最广泛的原生动物引起的人类感染，是由属寄生虫引起的疟原虫，恶性疟原虫是人类最严重的疾病形式。尽管目前有几种药物用于治疗疟疾，但疟疾寄生虫迅速对当前所有可用前线药物的抗药性产生了阻力。这表明有紧急的确定新的有效抗疟疾药物的新药物目标的迫切需要。 DNA到RNA（基因表达的第一步）的转录是通过RNA聚合酶（RNAP）在中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是一个经过验证的抗生素和抗病毒开发的药物靶标。体外纯化RNAP的生化表征除结构研究外，还起着重要作用来定义我们对结构和功能的理解 rnap。纯化的RNAP还促进了大规模的药物筛查和药物的表征候选铅化合物和RNAP的结构研究揭示了药物作用的机制为以及通过在硅筛选中加速的药物设计。疟原虫中的RNA合成发生在三个细胞器中（核，线粒体和非 - 光合体形成型），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apicoplast rNAP。最终目标这项研究将是将所有内源性核RNAP与恶性疟原虫细胞分离，并确定3D 这些酶通过单粒子冷冻电子显微镜（Cryo-EM）的结构。这些高分辨率结构将阐明疟原虫中转录的机理，并为针对恶性疟原虫RNAP的新抗疟药的设计。最后，这些结构还将提供新的在真核生物的寄生虫适应过程中，深入了解RNAP的演变。在此提案中，我们试图建立研究恶性疟原虫RNAP的结构和功能的方法 II，负责mRNA，SNRNA和调节RNAS转录。 AIM 1将使用CRISPR/CAS9 基因组编辑以生成表达亲和力标记最大亚基的恶性疟原虫的稳定细胞系（RPB1） RNAP II。 AIM 2将使用A中建立一种从恶性疟原虫提取物的RNAP II的纯化方法色谱技术的组合将通过体外转录测定法来测试RNAP II 活动。在AIM 3中，我们将通过Cryo-EM确定RNAP II的原子分辨率3D结构。提议工作将为研究疟原虫所有三个核RNAP的结构和功能铺平道路。