REGULATION OF PROLYL HYDROXYLASE

脯氨酰羟化酶的调节

• 批准号: 3156114
• 负责人: RICHARD A BERG
• 金额: $ 19.64万
• 依托单位: UNIV OF MED/DENT NJ-R W JOHNSON MED SCH
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-04-01 至 1995-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3156114
• 关键词: ascorbate; chick embryo; collagen; enzyme structure; fibroblasts; gene expression; genetic regulatory element; messenger RNA; molecular cloning; posttranslational modifications; protein biosynthesis; protein structure function; tissue /cell culture; transcription factor; transforming growth factors; unspecific monooxygenase

The regulation of collagen has clear health-related implications because a number of human diseases or dysfunctions such as rheumatoid arthritis, pulmonary fibrosis, cirrhosis, atherosclerosis, acute and chronic inflammation, Ehler's Danlos syndromes, Osteogenesis Imperfecta and wound healing are associated with a change in collagen metabolism. The overall objective of this study is to understand how collagen production is regulated in fibroblasts and how its regulation is coordinated with regulation of prolyl hydroxylase and protein disulfide isomerase, enzymes that catalyze essential posttranslational modifications of collagen. Although low levels of several posttranslational modifying enzymes cause nonlethal connective tissue disorders, the synthesis of hydroxyproline is essential for collagen production and fiber formation. The rate of collagen production, the activity of prolyl hydroxylase and the activity of protein disulfide isomerase are intimately related. For example, if prolyl hydroxylase activity is low, underhydroxylated collagen is produced which is subject to enhanced intracellular degradation. Prolyl hydroxylase is a tetrameric glycoprotein, alpha2beta2, where alpha and beta are nonidentical polypeptides of molecular weights 64 kd and 60 kd. We have recently cloned and sequenced a cDNA for most of the alpha subunit of chick prolyl hydroxylase. From our data on the cDNA and from amino acid sequencing we have determined the primary structure for the chick alpha subunit. It has been shown that the beta subunit of prolyl hydroxylase is identical to protein disulfide isomerase (PDI), thyroid hormone binding protein, P55, and glycosylation site binding protein (GSSP). This unusual protein is the major resident protein of the endoplasmic reticulum. Half of the prolyl hydroxylase in cultured fibroblasts contains tetrameres containing alpha' subunits which differ from alpha in having two high mannose oligosaccharides per polypeptide instead of one as in alpha. When prolyl hydroxylase is synthesized only new alpha and alpha' are translated; beta is derived from a per-existing pool of beta-like cross-reacting protein (CRP) that is identical to protein disulfide isomerase. Elucidating the structure and regulation of the alpha subunit will provide the basis for the design of antifibrotic drugs. An understanding of the expression and regulation of prolyl hydroxylase will no doubt advance the development of therapy for fibrotic diseases such as scleroderma for which, despite decades of work, there exists no effective therapy today. The aims of the present proposal are: (1) to isolate and sequence the gene for the alpha/alpha' subunit of chick prolyl hydroxylase; (2) to measure mRNA levels for alpha and beta subunits and compare these levels with the levels of mRNA for the type I collagen alpha1 and alpha2 chains to see if these polypeptides are coordinately regulated; (3) to measure the transcription rates of alpha and beta; (4) to clone the 5' regulatory elements for alpha and beta subunits and (5) to examine their functions in expression assays and determine how they function in regulation of gene expression.

胶原蛋白的调节具有明显的健康相关影响，因为 人类疾病或功能障碍的数量，如类风湿性关节炎， 急性和慢性肺纤维化、肝硬变、动脉粥样硬化 炎症、Ehler‘s Danlos综合征、成骨不全与创伤 愈合与胶原蛋白代谢的改变有关。整体而言 这项研究的目的是了解胶原蛋白的产生是如何 成纤维细胞中的调控及其与 Pro羟基酶和蛋白质二硫键异构酶的调节 它催化了胶原蛋白的翻译后修饰。 尽管低水平的几种翻译后修饰酶能引起 非致命性结缔组织疾病，羟脯氨酸的合成是 对胶原蛋白的产生和纤维的形成至关重要。这一速度 胶原蛋白的产生、脯氨酸羟基酶的活性和胶原的活性 蛋白质二硫键异构酶关系密切。例如，如果Prolyl 羟基酶活性低，会产生羟化不足的胶原蛋白 受到细胞内降解增强的影响。 Pro羟基酶是一种四聚体糖蛋白，alpha2beta2，其中 和β是分子量为kd和60的不同多肽 KD。我们最近克隆并测序了大部分α基因的cdna。 鸡脯氨酸羟基酶的亚基。从我们关于cDNA的数据和从 氨基酸测序我们已经确定了该蛋白的一级结构 鸡的阿尔法亚单位。已有研究表明，脯氨酸的β亚基 羟基酶与蛋白质二硫键异构酶(PDI)、甲状腺 激素结合蛋白、P55和糖基化结合蛋白 (GSSP)。这种不寻常的蛋白质是主要的驻留蛋白质 内质网。培养物中一半的脯氨酸羟基酶 成纤维细胞含有含有不同α‘亚基的四聚体 在每个多肽中含有两个高甘露糖低聚糖 而不是阿尔法中的一个。当仅合成脯氨酸羟基酶时 转换新的Alpha和Alpha‘；Beta派生自已有的 与蛋白质相同的类β交叉反应蛋白(CRP)池 二硫键异构酶。 阐明α亚基的结构和调节将提供 为抗肝纤维化药物的设计奠定了基础。对这一概念的理解 脯氨酸羟基酶的表达和调控无疑将促进 硬皮病等纤维性疾病的治疗进展， 尽管进行了几十年的研究，但目前还没有有效的治疗方法。 本提案的目的是：(1)分离和测序该基因 用于鸡的α/α‘亚基；(2)测量 α和β亚基的mRNA水平，并将这些水平与 I型胶原α1和α2链的mRNA水平 这些多肽是协同调节的；(3)测量 α和β转录速率；(4)克隆5‘调控基因 α和β亚基的元素和(5)检查它们的功能 表达分析并确定它们在基因调控中的作用 表情。