Global sumoylation analysis in Trypanosoma brucei

描述（由申请人提供）：动胞体目含有几种具有医学重要性的病原体，包括人类利什曼病、非洲锥虫病和恰加斯病的病原体。这些生物采用一种不寻常的基因表达机制，包括蛋白质编码基因的多顺反子转录，成熟的mRNA是通过一个共同的5‘外显子的反式剪接、剪接的前导子和在3’端添加一个聚(a)尾巴来产生的。到目前为止，还没有证据表明在着丝质体的转录起始水平上调节蛋白质的表达。相反，大多数调控发生在转录后。控制的一个层面是蛋白质的稳定性。蛋白稳定性、定位和周转的分子决定因素尚未在着丝质体中得到表征。了解着丝体寄生虫的蛋白质动力学是很重要的，因为在寄生虫的生命周期中，特定蛋白质的损失和获得是实现宿主之间过渡的关键。这一应用的假设是几百种布鲁氏锥虫蛋白的稳定性、功能和定位将通过与泛素样蛋白修饰剂SUMO的共价附着而被修饰。本提案的目的是：1)通过严格的亲和纯化，然后使用一种称为“MudPIT”的灵敏度极高的质谱法，确定与his8标记的SUMO结合的布氏T.布鲁氏蛋白的身份。2)选择可能参与转录和/或RNA加工的SUMO偶联靶点将受到挑战并进行实验验证。考虑到着丝质体转录后基因调控的重要性，再加上MudPIT和布鲁氏体基因组数据库的可用性，这是一个项目，将产生对寄生虫学家和真核细胞生物学家都感兴趣的结果。公共卫生相关性：人类利什曼病、非洲锥虫病和恰加斯病的病原体通过昆虫媒介在人与人之间传播。病原体的蛋白质组成在昆虫和哺乳动物阶段都是不同的，并且在宿主之间的过渡期间需要迅速改变。该提案旨在了解SUMO如何促进原循环（昆虫）阶段蛋白质的功能，定位和稳定性，长期目标是了解该过程的独特和关键特征，使寄生虫能够在其生命周期中进展。

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.