Biophysical studies on the interaction of antizyme and ornithine decarboxylase

抗酶与鸟氨酸脱羧酶相互作用的生物物理学研究

• 批准号: 7593451
• 负责人: Herbert Tabor
• 金额: $ 25.41万
• 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/7593451
• 关键词: Amines; Amino Acid Sequence; Anabolism; Biochemical; Bypass; Cells; Complex; Crystallization; Differentiation and Growth; Enzymes; Escherichia coli; Evolution; Life; Organism; Ornithine Decarboxylase; Peptide Sequence Determination; Physiological; Plasmids; Polyamines; Property; Protein Overexpression; Proteins; Purpose; Putrescine; Saccharomyces cerevisiae; Spermidine; Spermine; Structure; System; Techniques; Terminator Codon; Translations; Ubiquitination; Variant; Yeasts; in vivo; interest; light scattering; multicatalytic endopeptidase complex; mutant; ornithine decarboxylase antizyme

The polyamines, putrescine, spermidine, and spermine, are major polybasic compounds in all living cells. These amines are important for many systems related to growth and differentiation. For many years we have been studying how these polyamines are synthesized, how their biosynthesis and degradation are regulated, their physiologic functions, how they act in vivo, and the structure of the various biosynthetic enzymes. For this purpose we have constructed null mutants in each of the biosynthetic steps in both Escherichia coli and Saccharomyces cerevisiae, and have prepared overexpression systems for the biosynthetic enzymes. Our overall studies have aimed at the use of these mutants to elucidate the physiological functions of the polyamines. Our current studies are concerned with the interaction of yeast antizyme and yeast ornithine decarboxylase. This is a most unusual regulatory sustem. Antizyme ineracts with ornithine decarboxylase, resulting in inhibition of its activity. Antizyme also acts to target the ornithine decarboxylase protein to the proteasome for degradation; this degradation is unusual in not involving ubiquitination. Biosynthesis of antizyme is most unusual in requiring a frameshift in translation, in order to bypass a stop codon; this frameshift only occurs in the presence of increased spermdine. This unusual requirement for a frameshift has been conserved throughout evolution, even though there is considerable variation in the protein sequences in different organisms. In our current studies we have constructed a plasmids for both ornithine decarboxylase and for antizyme (with the in-frame sequence) , and have purified both yeast ornithine decarboxylase and yeast antizyme to homogeneity both for attempts at crystallization and for biophysical studies on the interactions of he two proteins. Preliminary studies have already been carried out with light scattering techniquesto find out the bioophysical properties of these two enzymes separately as a background for subsequent studies on their interactions. Preliminary characterization with CD spectra were also performed in order to study the secondary structure of these two proteins both individually and when resent as a complex. We have also continued attempts to crystallize the two proteins, especially since there is no x-ray structure available for the full lenghth yeast antizyme protein.

多胺，腐胺，亚精胺和精胺，是所有活细胞中的主要多元化合物。这些胺对于与生长和分化相关的许多系统是重要的。多年来，我们一直在研究这些多胺是如何合成的，它们的生物合成和降解是如何调节的，它们的生理功能，它们如何在体内发挥作用，以及各种生物合成酶的结构。为此目的，我们已经构建了空突变体在大肠杆菌和酿酒酵母的生物合成步骤中的每一个，并已准备过表达系统的生物合成酶。我们的总体研究旨在利用这些突变体来阐明多胺的生理功能。 我们目前的研究涉及酵母抗酶和酵母鸟氨酸脱羧酶的相互作用。这是一个极不寻常的管理制度。抗酶与鸟氨酸脱羧酶相互作用，导致其活性抑制。 抗酶还作用于将鸟氨酸脱羧酶蛋白靶向蛋白酶体进行降解;这种降解不涉及泛素化是不寻常的。 抗酶的生物合成最不寻常的是需要翻译中的移码，以绕过终止密码子;这种移码仅发生在精子蛋白增加的情况下。尽管不同生物体的蛋白质序列存在相当大的差异，但这种不寻常的移码要求在整个进化过程中一直是保守的。 在我们目前的研究中，我们已经构建了一个质粒的鸟氨酸脱羧酶和抗酶（与框内序列），并已纯化酵母鸟氨酸脱羧酶和酵母抗酶的同质性都在结晶和生物物理研究的尝试，他两种蛋白质的相互作用。初步的研究已经进行了与光散射techniquesto找出这两种酶的生物物理性质分别作为背景，为后续的研究它们之间的相互作用。还进行了初步表征与CD光谱，以研究这两种蛋白质的二级结构，无论是单独的，当重新作为一个复合物。我们还继续尝试结晶这两种蛋白质，特别是因为没有完整长度的酵母抗酶蛋白的X射线结构。