Global sumoylation analysis in Trypanosoma brucei

布氏锥虫的整体 SUMO 化分析

基本信息

批准号：
7659346
负责人：
DAVID A CAMPBELL
金额：
$ 23.1万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
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：
2009-05-05 至 2011-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7659346
关键词：
Affect Affinity Chromatography African Trypanosomiasis Binding Biological Assay Cell Line Cellular biology Chagas Disease Co-Immunoprecipitations Code Communities Complex Coupled Data Set Engineering Epitopes Equipment Eukaryota Eukaryotic Cell Exons Future Gene Expression Gene Expression Regulation Genes Genetic Transcription Genome Gills Goals Hand Histidine Human Human Resources Insect Vectors Insecta Kinetoplastida Leishmaniasis Link Mass Spectrum Analysis Medical Messenger RNA Molecular Mutagenesis Nature Nickel Organism Parasites Parasitology Pathway interactions Peptides Persons Play Poly(A) Tail Polyadenylation Post-Transcriptional Regulation Post-Translational Protein Processing Process Protein Analysis Protein Dynamics Proteins Proteomics Publishing RNA RNA Interference RNA Polymerase II RNA Processing Reagent Regulation Research Small Ubiquitin-Related Modifier Proteins Spliced Leader Sequences Staging Structure Sumoylation Pathway System Techniques Technology Trans-Splicing Transcript Transcription Initiation Trypanosoma brucei brucei Trypsin Ubiquitin Ubiquitin Like Proteins Ubiquitin family Validation Variant Whole Organism Analysis Yeasts base cell growth genome database interest pathogen polypeptide promoter protein expression public health relevance success tool vector

项目摘要

DESCRIPTION (provided by applicant): The Order Kinetoplastida contains several pathogens of medical importance, including the causative agents of human leishmaniasis, African trypanosomiasis and Chagas disease. These organisms employ an unusual mechanism of gene expression that involves polycistronic transcription of protein-coding genes, with mature mRNA being generated via trans-splicing of a common 5' exon, the spliced leader, and addition of a poly(A) tail at the 3' end. To date there is no evidence for regulation of protein expression at the level of transcription initiation in kinetoplastids. In contrast, most of the regulation occurs post-transcriptionally. One level of control is the stability of the protein. The molecular determinants of protein stability, localization and turnover have not been characterized in kinetoplastids. It is important to understand protein dynamics in the kinetoplastid parasites because the loss and gain of specific proteins is key to fulfilling the transition between hosts in the parasite lifecycle. The hypothesis underlying this application is that the stability, function, and localization of a few hundred Trypanosoma brucei proteins will be modified by covalent attachment with the ubiquitin-like protein modifier SUMO. The Aims of this proposal are: 1) To determine the identity of T. brucei proteins that are conjugated with His8-tagged SUMO by stringent affinity purification followed by an exquisitely sensitive variant of mass spectrometry referred to as 'MudPIT'. 2) Select targets of SUMO conjugation that are likely involved in transcription and/or RNA processing will be challenged and validated experimentally. Given the importance of post-transcriptional gene regulation in kinetoplastids coupled with the availability of MudPIT and the T. brucei genome database, this is a project that will yield results of interest to parasitologists and eukaryotic cell biologists alike. PUBLIC HEALTH RELEVANCE: The causative agents of human leishmaniasis, African trypanosomiasis and Chagas disease are transmitted from person to person via insect vectors. The pathogens' protein compositions are distinct in both insect and mammalian stages and needs to change rapidly during the transition between hosts. This proposal seeks to understand how SUMO contributes to the function, localization, and stability of proteins in the procyclic (insect) stage, with the long-term goal to understand the unique and critical features of this process that allow the parasite to progress through its lifecycle.

描述（由申请人提供）：动力学的命令包含几种医学重要性的病原体，包括人类利什曼病，非洲锥虫病和chagas病的病原体。这些生物采用了涉及蛋白质编码基因的多重分发性转录的异常机制，其成熟的mRNA是通过对普通5'外显子（剪接的领导者的反式）进行的，并在3'端添加poly（a）的尾巴。迄今为止，还没有证据表明在动作质体中转录起始水平上调节蛋白质表达。相比之下，大多数调节在转录后发生。控制水平是蛋白质的稳定性。蛋白质稳定性，定位和周转的分子决定因素尚未在动力质体中表征。重要的是要了解动力质体寄生虫中的蛋白质动力学，因为特定蛋白质的损失和增益是实现寄生虫生命周期中宿主之间过渡的关键。该应用的基础假设是，将通过与泛素样蛋白修饰剂SUMO共价附着来修改几百个锥虫蛋白的稳定性，功能和定位。该提案的目的是：1）确定布鲁氏抗体蛋白的身份，这些蛋白质与His8标签的Sumo通过严格的亲和力纯化结合，然后是称为“ Mudpit”的质谱法的精美敏感变体。 2）可能与转录和/或RNA处理有关的Sumo共轭的选择目标将受到挑战和实验验证。鉴于转录后基因调节在动力质体中的重要性以及Mudpit和Brucei基因组基因组数据库的可用性，这是一个项目，该项目将为寄生虫学家和真核细胞生物学家提供感兴趣的结果。公共卫生相关性：人类利什曼病，非洲锥虫病和查加斯病的病因是通过昆虫向量传播的。病原体的蛋白质成分在昆虫和哺乳动物阶段都不同，并且在宿主之间的过渡过程中需要迅速变化。该提案旨在了解相扑如何促进蛋白质在Procyclic（昆虫）阶段的功能，定位和稳定性，其长期目标是了解该过程的独特和关键特征，从而使寄生虫能够通过其生命周期发展。