THE ROLE OF THE NUCLEAR ENVELOPE IN INTRACELLULAR PROTEIN SORTING

核膜在细胞内蛋白质分选中的作用

• 批准号: 3917374
• 负责人: J A HANOVER
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3917374
• 关键词: antigens; cell growth regulation; cell nucleus; chemical conjugate; cytoplasm; fluorescent dye /probe; glucocorticoids; glycoproteins; glycosylation; hormone receptor; laboratory rat; lectin; membrane permeability; membrane proteins; microinjections; monoclonal antibody; mutant; nuclear membrane; phycobilin; pore forming protein; protein biosynthesis; protein sequence; protein transport; simian virus 40; surface antigens; synthetic peptide; tissue /cell culture; virus antigen; virus protein

The proper sorting of proteins to the cell nucleus has been shown to play a key role in the regulation of cell growth and development. When chemically coupled to the large fluorescent protein B-phycoerythrin, synthetic peptides derived from the amino acid sequence of the SV40 Large T antigen specifically target the protein conjugate to the nucleus. Transport of such conjugates across the nuclear envelope was demonstrated after microinjection into cultured cells or in an in vitro import assay using rat liver nuclei. Transport was time, temperature and energy dependent; only conjugates containing the localization sequence were properly transported. Monoclonal antibodies have been generated which bind specifically to this sequence. These antibodies bind to intact T antigen, and other nuclear proteins, suggesting that the sequence is normally exposed on the outer surface of molecules to be transported into the nucleus. A nuclear localization signal has also been identified in the primary structure of the human glucocorticoid receptor. A synthetic peptide of 10 residues derived from the sequence of this receptor are sufficient to direct transport of large molecules into the nucleus. Attempts are being made to interrupt nuclear targeting of the glucocorticoid receptor. The nuclear pore complex traverses the nuclear envelope and mediates uptake to the nucleus. The laboratory has demonstrated that proteins bearing cytoplasmically oriented, O-linked G1cNAc are components of the nuclear pore complex. The nuclear pore glycoproteins can be selectively labelled using the actin wheat germ agglutinin. This lectin reversibly blocks both import into the nucleus and the export of RNA from the nucleus to the cytoplasm. These findings raise the exciting possibility that cytoplasmic glycosylation may be involved in the assembly or functioning of the nuclear pore. Monoclonal antibodies have been raised against these nuclear pore glycoproteins thus allowing their purification. Sequence information from the major nuclear pore protein was obtained and this protein has been molecularly cloned. The structure and function of the nuclear pore protein is currently being probed using recombinant DNA technology.

已证明蛋白质对细胞核的正确排序 在细胞生长调节中发挥关键作用 发展。 当与大荧光化学偶联时 蛋白质 B-藻红蛋白，从氨基衍生的合成肽 SV40 大 T 抗原的酸序列专门针对 蛋白质与细胞核结合。 此类缀合物的运输 显微注射后证明了穿过核膜 进入培养细胞或使用大鼠肝脏进行体外导入测定 原子核。 运输取决于时间、温度和能量；仅有的 含有定位序列的缀合物被正确地 运输。 已产生结合的单克隆抗体 专门针对这个序列。 这些抗体结合完整的 T 抗原和其他核蛋白，表明该序列 通常暴露在分子的外表面 输送到细胞核中。 核定位信号有 在人体的初级结构中也被确定 糖皮质激素受体。 10 个残基的合成肽 衍生自该受体的序列足以指导 将大分子输送到细胞核中。 正在尝试 旨在中断糖皮质激素受体的核靶向。 核孔复合体穿过核膜并 介导细胞核的摄取。 该实验室已证明 带有细胞质导向的 O-连接 G1cNAc 的蛋白质是 核孔复合体的组成部分。 核孔 可以使用肌动蛋白小麦选择性标记糖蛋白 细菌凝集素。 这种凝集素可逆地阻止两者导入 细胞核以及RNA从细胞核到细胞核的输出 细胞质。 这些发现提出了令人兴奋的可能性： 细胞质糖基化可能参与组装或 核孔的功能。 单克隆抗体已 针对这些核孔糖蛋白，从而允许它们 纯化。 来自主核孔的序列信息 获得了蛋白质，并且该蛋白质已被分子克隆。 目前核孔蛋白的结构和功能 正在使用重组 DNA 技术进行探测。