Smt3 conjugation in cell biology and development

描述(申请人提供)：泛素样蛋白正在接受 由于他们最近在不同领域中发现的角色而日益受到关注 生物过程。一种这样的蛋白质，命名为Smt3(也称为相扑或 Sentin)，具有广泛的可疑功能，包括 转录调控、核运输和胞质分裂。像泛素一样， Smt3通过异肽键与靶蛋白共价连接 赖氨酸残留物。这些靶标包括转录因子、 核孔复合体，以及隔膜复合体的成分。虽然很多人 已经确定了Smt3-偶联靶标，对Smt3功能的研究 后生动物在很大程度上仅限于细胞培养分析。这项建议 描述了一种生化、细胞生物学和遗传分析相结合的方法。 Smt3-在果蝇中的结合，目的是确定功能 这一过程在发育中的有机体中的作用。 具体的目的是：(1)确定Smt3-偶联的靶点 在果蝇体内表达标记形式的Smt3，然后使用标记 从蛋白质提取物中提纯Smt3结合物；(2)测定结果 Smt3在果蝇细胞培养过程中的干扰作用 蛋白质定位和转录调控；(3)确定 利用遗传学研究Smt3-偶联在完整生物体中的发育作用 和反向遗传方法扰乱Smt3在体内的结合， 在观察到的表型和特定的目标之间建立直接联系的目标 Smt3--结合。 这些研究对人类健康有很多影响。例如，一个 新发现的Smt3结合靶点是早幼粒细胞白血病 蛋白质PML。这种蛋白与Smt3的结合似乎控制了它的 对离散核灶的定位，称为PML小体。最近的实验 提示果蝇的细胞核可能含有类似的Smt3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.