REGULATION OF PROLYL HYDROXYLASE

胶原蛋白的调节具有明确的健康相关影响，因为 许多人类疾病或功能障碍如类风湿性关节炎， 肺纤维化、肝硬化、动脉粥样硬化、急性和慢性 炎症、Ehler’s Danlos综合征、成骨不全和创伤 愈合与胶原代谢的变化有关。 整体 本研究的目的是了解胶原蛋白的产生是如何 在成纤维细胞中调节，以及其调节如何与 脯氨酰羟化酶和蛋白质二硫键异构酶的调节，酶 催化胶原蛋白的翻译后修饰。 尽管低水平的几种翻译后修饰酶会导致 非致死性结缔组织疾病，羟脯氨酸的合成是 对胶原蛋白生成和纤维形成至关重要。 率 胶原蛋白的产生，脯氨酰羟化酶的活性和 蛋白质二硫键异构酶密切相关。 例如，如果脯氨酰 羟化酶活性低，产生未充分羟基化的胶原， 受到增强的细胞内降解。 脯氨酰羟化酶是一种四聚体糖蛋白，α 2 β 2，其中α 和β是分子量分别为64 kd和60 kd的不同多肽 kd. 我们最近克隆并测序了大部分α-半乳糖苷酶基因的cDNA， 鸡脯氨酰羟化酶亚基。 从我们的cDNA数据和 氨基酸测序，我们已经确定了一级结构的 鸡α亚单位 已经表明脯氨酰的β亚基 羟化酶与蛋白质二硫键异构酶（PDI）相同，甲状腺羟化酶 激素结合蛋白、P55和糖基化位点结合蛋白 （GSSP）。 这种不寻常的蛋白质是主要的居民蛋白质的 内质网 培养的脯氨酰羟化酶 成纤维细胞含有四聚体，所述四聚体含有α '亚基， 从每个多肽具有两个高甘露糖寡糖的α 而不是alpha中的一个。 当脯氨酰羟化酶只合成 新的alpha和alpha'被翻译; beta是从先前存在的 与蛋白质相同的β样交叉反应蛋白（CRP）池 二硫键异构酶 阐明α亚基的结构和调节将提供 抗纤维化药物设计的基础。 的理解 脯氨酰羟化酶的表达和调节无疑将促进 纤维化疾病如硬皮病的治疗方法的发展， 尽管经过几十年的努力，今天仍然没有有效的治疗方法。 本研究的目的是：（1）分离和测序该基因 对于鸡脯氨酰羟化酶的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α 1和α 2链的mRNA水平，以观察 这些多肽是协同调节的;（3）测量 α和β的转录速率;（4）克隆5'调控基因， α和β亚基的元件和（5）检查它们在 表达分析，并确定它们如何在基因调控中发挥作用， 表情

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.