Regulation of Plasmodium falciparum transcription

恶性疟原虫转录的调控

基本信息

批准号：
8417773
负责人：
Manuel Llinas
金额：
$ 16.24万
依托单位：
PRINCETON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-02-15 至 2013-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8417773
关键词：
Affect Antimalarials Binding Binding Sites Biochemistry Biological Assay Cell Nucleus Cessation of life ChIP-on-chip Clinical Collection Complex Confocal Microscopy Culicidae DNA DNA Binding DNA Binding Domain DNA Microarray Chip DNA-Binding Proteins DNA-Protein Interaction Data Detection Development Disease Drug Targeting Drug resistance Environment Erythrocytes Euchromatin Eukaryota Evolution Exhibits Family Gene Expression Gene Expression Regulation Genes Genetic Genetic Transcription Genome Goals Growth Health Hepatocyte Hour Human In Vitro Individual Infection Intervention Knock-out Libraries Ligands Malaria Measures Methods Modeling Modification Molecular Molecular Biology Nuclear Nuclear Localization Signal Oligonucleotides Organism Parasites Pattern Peptides Phase Plants Plasmids Plasmodium Plasmodium falciparum Protein Binding Proteins Recombinants Regulation Research Role Signal Transduction Specificity Staging Tertiary Protein Structure Time Transcript Transcription Factor AP-2 Alpha Transcriptional Regulation Work base chromatin immunoprecipitation functional genomics in vivo novel novel therapeutic intervention nucleocytoplasmic transport plant growth/development promoter tool transcription factor

项目摘要

DESCRIPTION (provided by applicant): The long-term goals of the proposed work are to understand the molecular mechanisms involved in transcriptional regulation in the malaria-causing parasite Plasmodium falciparum. During the malaria infection, the red blood cell stage of Plasmodium development is responsible for all of the clinical manifestations of this disease. This developmental stage is characterized by a cyclical 48-hour growth and replication phase that must be exquisitely regulated at the transcriptional level to maximize developmental efficiency and fidelity. The mechanisms used by the parasite to achieve this high level of transcriptional control remain unclear, although there is mounting evidence that transcription is strictly regulated in this organism. Our hypothesis is that a recently identified set of putative transcriptional regulators - the Apicomplexan AP2 (ApiAP2) proteins - are a major family of key transcriptional regulators during parasite development. The role of ApiAP2 proteins as transcriptional regulators will be analyzed using biochemistry, molecular biology and functional genomics tools. The first approach will determine the DNA recognition sequences for ApiAP2 proteins in vitro using two complementary methods. One method is a PCR-based selective enrichment of ligands by exponential enrichment (SELEX) using recombinant ApiAP2 proteins and a random library of putative DNA binding sites. Alternatively, a protein binding microarray (PBM) will be used to hybridize ApiAP2 proteins to a comprehensive collection of double stranded oligonucleotides. The second approach will be to establish the in vivo role of ApiAP2 proteins by 1) modulating transcriptional levels of these putative regulators and 2) generating knockout parasite lines. The effects of these genetic modifications will be assayed globally using DNA microarrays to detect changes in gene expression. Next, the binding of ApiAP2 proteins to DNA will be measured in vivo by chromatin immunoprecipitation with DNA microarray detection (ChIP-chip). These results will define the gene sets directly regulated by the ApiAP2 proteins and will be compared to the in vitro binding results in Aim 2 as well as to prior gene expression data to establish a model for stage-specific transcriptional regulation during parasite development. Finally, we plan to establish and experimentally verify a peptide- based signaling motif that will allow us to predict the active nuclear localization of Plasmodial proteins. The ApiAP2 proteins are the first large family of putative transcription factor domains identified in the genome of P. falciparum and hold great promise for antimalarial intervention since there are no mammalian counterparts to these proteins.

描述（由申请人提供）：拟议工作的长期目标是了解引起疟疾的寄生虫疟原虫的转录调节所涉及的分子机制。在疟疾感染期间，疟原虫发育的红细胞阶段造成了该疾病的所有临床表现。这个发育阶段的特征是周期性的48小时生长和复制阶段，必须在转录水平上进行精心调节，以最大程度地提高发育效率和保真度。寄生虫用来实现这种高水平的转录控制的机制尚不清楚，尽管有越来越多的证据表明转录受到该生物体的严格调节。我们的假设是，最近确定的一组推定的转录调节剂 - Apicomplexan AP2（APIAP2）蛋白 - 在寄生虫开发过程中是主要转录调节剂的主要家族。将使用生物化学，分子生物学和功能基因组学工具分析APIAP2蛋白作为转录调节剂的作用。第一种方法将使用两种互补方法在体外确定APIAP2蛋白的DNA识别序列。一种方法是使用重组APIAP2蛋白和推定的DNA结合位点的随机库来通过指数富集（SELEX）对配体的选择性富集。或者，蛋白结合微阵列（PBM）将用于将APIAP2蛋白与全面的双链寡核苷酸融合。第二种方法是通过1）调节这些推定调节剂的转录水平以及2）产生基因敲除寄生虫线来确定APIAP2蛋白的体内作用。这些遗传修饰的影响将使用DNA微阵列在全球范围内进行测定，以检测基因表达的变化。接下来，将通过与DNA微阵列检测（CHIP-CHIP）的染色质免疫沉淀（CHIP-CHIP）在体内测量APIAP2蛋白与DNA的结合。这些结果将定义由APIAP2蛋白直接调节的基因集，并将与AIM 2的体外结合结果以及先前的基因表达数据进行比较，以建立寄生虫发育过程中阶段特异性转录调控的模型。最后，我们计划建立和实验验证基于肽的信号传导基序，这将使我们能够预测质蛋白的主动核定位。 APIAP2蛋白是在恶性疟原虫基因组中鉴定出的假定转录因子结构域的第一个大型家族，并且对抗疟疾干预保持了巨大的希望，因为没有哺乳动物对这些蛋白质的哺乳动物对应物。