Smt3-conjugation in cell biology and development

描述（由申请人提供）：泛素样蛋白正在接受 越来越多的关注，由于他们最近发现的作用，在不同的 生物过程。一种这样的蛋白质，命名为Smt 3（也称为Sumo或Smt 3）， Sentrin），具有广泛的可疑功能，包括在 转录调控、核转运和胞质分裂。像泛素一样， smt 3通过异肽键与靶蛋白共价连接， 赖氨酸残基。这些靶点包括转录因子，转录因子的组成部分， 核孔复合物和隔蛋白复合物的组分。虽然许多 Smt 3-缀合靶点已经被鉴定，Smt 3功能的研究在 后生动物在很大程度上限于细胞培养分析。这项建议 描述了一个组合的生化，细胞生物学，和遗传分析， 果蝇中Smt 3-缀合，目标是确定Smt 3-缀合的功能性。 这一过程在生物体发育中的作用。 具体目标是：（1）确定Smt 3-缀合的靶点， 通过在体内表达Smt 3的标记形式，然后使用这些标记， 从蛋白质提取物中纯化Smt 3-缀合物;（2）测定Smt 3-缀合物的结果。 在培养的果蝇细胞中干扰Smt 3结合， 蛋白质定位和转录调控;（3）确定 Smt 3结合在完整生物体中的发育作用 和反向遗传方法干扰Smt 3-结合在体内，与 目标是在观察到的表型和特定靶点之间建立直接联系， Smt 3-共轭。 这些研究对人类健康有许多影响。例如一 最近发现的Smt 3结合的靶点是早幼粒细胞白血病 PML蛋白。这种蛋白质与Smt 3的结合似乎控制了其 定位于称为PML小体的离散核灶。最近的实验 表明果蝇细胞核可能含有类似的Smt 3依赖灶。

DESCRIPTION (provided by applicant): Ubiquitin-like proteins are receiving increasing attention due to their recently discovered roles in diverse biological processes. One such protein, named Smt3 (also known as Sumo or Sentrin), has a wide range of suspected functions, including functions in transcriptional regulation, nuclear transport, and cytokinesis. Like ubiquitin, Smt3 becomes covalently attached to target proteins via isopeptide linkages to lysine residues. These targets include transcription factors, components of the nuclear pore complex, and components of the septin complex. While many Smt3-conjugation targets have been identified, studies of Smt3 function in metazoans has largely been limited to cell culture analysis. This proposal describes a combined biochemical, cell biological, and genetic analysis of Smt3-conjugation in Drosophila, with the goal being to determine the functional roles of this process in the developing organism. The specific aims are to: (1) Identify targets of Smt3-conjugation in Drosophila by expressing tagged forms of Smt3 in vivo and then using the tags to purify Smt3-conjugates from protein extracts; (2) Determine the results of perturbing Smt3-conjugation in cultured Drosophila cells on such processes as protein localization and transcriptional regulation; (3) Determine the developmental roles of Smt3-conjugation in the intact organism by using genetic and reverse genetic approaches to perturb Smt3-conjugation in vivo, with the goal of making direct links between observed phenotypes and specific targets of Smt3-conjugation. These studies have many implications for human health. For example, one recently discovered target of Smt3-conjugation is the promyelocytic leukemia protein PML. The conjugation of this protein to Smt3 appears to control its localization to discrete nuclear foci termed PML bodies. Recent experiments suggest that Drosophila nuclei may contain similar Smt3-dependent foci.