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.
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| Bassuk, J A; Kao, W W; Herzer, P et al. (1989) Prolyl 4-hydroxylase: molecular cloning and the primary structure of the alpha subunit from chicken embryo. Proc Natl Acad Sci U S A 86:7382-6 |
| Bassuk, J A; Berg, R A (1989) Protein disulphide isomerase, a multifunctional endoplasmic reticulum protein. Matrix 9:244-58 |
