HYPUSINE FORMATION--BIOCHEMISTRY AND FUNCTION

前体素的形成--生物化学和功能

• 批准号: 3193931
• 负责人: KUANG Yu CHEN
• 金额: $ 19.19万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-04-01 至 1995-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3193931
• 关键词: Neurospora; aminoacid biosynthesis; antibody formation; cell differentiation; cell growth regulation; complementary DNA; enzyme substrate; laboratory mouse; lysine; molecular cloning; neuroblastoma; nicotinamide adenine dinucleotide; polyamines; protein biosynthesis; protein purification; putrescine; spermidine; spermine

Polyamines (spermidine and spermine) and putrescine are essential for cell growth and function. To understand the molecular basis of the action of polyamines in the regulation of growth and differentiation, it is important to focus on specific polyamine-dependent biochemical events. Hypusine formation in an 18kDa cellular protein is by far the most specific polyamine-dependent biological process discovered. This reaction represents a unique post-translational modification in which the aminobutyl moiety of spermidine is transferred to the epsilon-amino group of a lysine residue of an 18kDa protein (catalyzed by E1). This modification appears to be highly conserved, present in fungus as well as all mammalian cells. Our previous studies indicate that hypusine formation can be stimulated by serum and is diminished during mouse neuroblastoma differentiation. The specificity, ubiquity, and its association with cell proliferation suggest that hypusine formation may be of fundamental importance in cell growth regulation. It is possible that some of the important physiological functions of polyamines are mediated via hypusine formation in the 18kDa protein. We have recently found that NAD+ at 0.1 to 1 mM dramatically stimulates hypusine formation in cytosolic lysates isolated from mouse neuroblastoma cells. We also found that the substrate protein in Neurospora crassa has an apparent molecular weight of 21kDa and that neuroblastoma E1 can use the 21kDa as a substrate. These findings allow us to develop both purification and assay procedures for E1 and 18kDa protein. In this proposal, we will focus on the purification and characterization of E1 and 18kDa. Purified proteins will be used to develop enzymatic assay procedure, to produce antibodies, and to isolate cDNA clones for E1 and 18kDa. The enzymatic assay and various probes generated will be used to study the regulation of these proteins in mouse neuroblastoma cells during growth and differentiation. We have previously shown that the differentiation of mouse neuroblastoma cells is accompanied by a significant decrease of spermidine content. Since hypusine formation is solely spermidine dependent, it is possible that regulation of hypusine formation may have a role in mediating the differentiation of mouse neuroblastoma cells.

多胺（精脒和精胺）和腐胺是细胞生长所必需的。 生长和功能。 为了了解这种作用的分子基础 多胺在调节生长和分化中， 专注于特定的多胺依赖的生化事件。 羟腐 18 kDa细胞蛋白质的形成是迄今为止最特异的 多胺依赖的生物过程被发现。 该反应 代表一种独特的翻译后修饰， 亚精胺的一部分被转移到赖氨酸的ε-氨基上， 18 kDa蛋白质的残基（由E1催化）。 这一修改似乎 是高度保守的，存在于真菌和所有哺乳动物细胞中。 我们以前的研究表明，羟腐胺赖氨酸的形成可以刺激 在小鼠神经母细胞瘤分化过程中减少。 的 特异性、普遍性及其与细胞增殖相关性表明 羟腐胺赖氨酸的形成可能在细胞生长中具有根本的重要性 调控 有可能一些重要的生理因素 多胺的功能通过18 kDa的羟腐胺赖氨酸形成介导， 蛋白 我们最近发现，NAD+在0.1至1 mM时， 刺激从小鼠分离的细胞溶质裂解物中羟腐胺赖氨酸形成 神经母细胞瘤细胞 我们还发现， 粗糙脉孢菌具有21 kDa的表观分子量， 神经母细胞瘤E1可以利用21 kDa作为底物。 这些发现让我们 建立E1和18 kDa蛋白的纯化和测定方法。 在本提案中，我们将重点关注的纯化和表征 E1和18 kDa。 纯化的蛋白质将用于开发酶测定法 程序，以产生抗体，并分离E1和 18kDa。 酶测定和产生的各种探针将用于 研究这些蛋白质在小鼠神经母细胞瘤细胞中的调节， 生长和分化。 我们之前已经证明， 小鼠神经母细胞瘤细胞的分化伴随着 亚精胺含量显著降低。 由于羟腐胺的形成是 仅依赖亚精胺，羟腐胺赖氨酸调节 形成可能在介导小鼠的分化中起作用 神经母细胞瘤细胞