Global sumoylation analysis in Trypanosoma brucei

描述（由申请方提供）：动质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。这些生物体采用不寻常的基因表达机制，其涉及蛋白质编码基因的多顺反子转录，其中成熟mRNA通过共同的5'外显子（剪接的前导序列）的反式剪接和在3'末端添加聚（A）尾而产生。到目前为止，没有证据表明在动质体转录起始水平上调节蛋白质表达。相反，大多数调节发生在转录后。一个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在动质体中还没有得到表征。了解动质体寄生虫中的蛋白质动力学是很重要的，因为特定蛋白质的丢失和获得是寄生虫生命周期中实现宿主之间转换的关键。本申请的假设是，几百个布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的共价连接进行修饰。本研究的目的是：1）确定T.通过严格的亲和纯化，然后通过称为“MudPIT”的极其灵敏的质谱变体，将布鲁氏菌蛋白与His 8标记的SUMO缀合。2)选择可能参与转录和/或RNA加工的SUMO结合靶点，将进行挑战和实验验证。考虑到动质体中转录后基因调控的重要性以及MudPIT和T.这个项目将产生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快速变化。该提案旨在了解SUMO如何有助于蛋白质在前循环（昆虫）阶段的功能，定位和稳定性，长期目标是了解这一过程的独特和关键特征，使寄生虫在其生命周期中取得进展。

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.