Specific inactivation of prolyl 4-hydroxylase and inhibition of collagen synthesis by oxaproline-containing peptides in cultured human skin fibroblasts.
The crucial role of collagen in fibrotic disorders has prompted attempts to develop drugs that inhibit collagen accumulation. Peptides containing the unphysiological amino acid 5-oxaproline (Opr) have recently been found to act as specific syncatalytic inactivators of pure prolyl 4-hydroxylase, the enzyme that catalyzes the formation of 4-hydroxyproline in collagens. The present study indicates that oxaproline-containing peptides benzyloxycarbonyl-Phe-Opr-Gly-benzyl ester (I) and benzyloxycarbonyl-Phe-Opr-Gly-ethyl ester (II) inactivate prolyl 4-hydroxylase in cultured human skin fibroblasts, peptide I being about twice as potent as peptide II. Inactivation by 50% was observed after culturing with about 20-40 microM concentrations of peptide I for 48 h. The inactivation appears to be specific, as no changes were found in the activities of two other intracellular enzymes of collagen synthesis, lysyl hydroxylase and galactosylhydroxylysyl glucosyltransferase. Synthesis of 4-hydroxyproline by the cells was markedly decreased, and 4-hydroxyproline-deficient procollagen accumulated intracellularly, whereas no changes were found in the incorporation of [14C]leucine into protein after culturing of the cells with a 30 microM concentration of peptide I for 48 h. No changes were seen in the viability of the cells or the release of lactate dehydrogenase from them into the culture medium. No significant changes were found in the steady-state levels of the mRNAs for the pro-alpha 1 chains of type I and type III procollagens or for the alpha and beta subunits of prolyl 4-hyroxylase or fibronectin after culturing with 75 microM peptide I for 48 h. The data indicate that inactivation of cellular prolyl 4-hydroxylase has marked effects on cellular 4-hydroxyproline formation and collagen secretion but no effects on the steady-state levels of mRNAs for type I and III procollagens or the two types of subunit of prolyl 4-hydroxylase.
Karvonen
K
K
Collagen Research Unit, University of Oulu, Finland.
Ala-Kokko
L
L
Pihlajaniemi
T
T
Helaakoski
T
T
Henke
S
S
Günzler
V
V
Kivirikko
K I
KI
Savolainen
E R
ER
D002478:Cells, Cultured; D003094:Collagen / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology; D006909:Hydroxyproline / Q000096:biosynthesis; D007700:Kinetics; D046911:Macromolecular Substances; D009842:Oligopeptides / Q000494:pharmacology*; D011347:Procollagen / Q000378:metabolism; D011393:Procollagen-Proline Dioxygenase / Q000037:antagonists & inhibitors*; D011392:Proline / Q000031:analogs & derivatives* / Q000494:pharmacology; D012333:RNA, Messenger / Q000187:drug effects / Q000235:genetics; D012867:Skin / Q000201:enzymology*
D046911:Macromolecular Substances; D009842:Oligopeptides; D011347:Procollagen; D012333:RNA, Messenger; C057984:5-oxaproline; D003094:Collagen; D011392:Proline; D011393:Procollagen-Proline Dioxygenase; D006909:Hydroxyproline