Proline Analogues Inhibit Human Skin Fibroblast Growth and Collagen Production in Culture

Abstract

Several structural analogues of proline have been shown to be incorporated into proteins in place of proline. As a consequence, the proliferation of cells in culture and the extracellular deposition of collagen in animal systems are reduced. In this study, the effects of two proline analogues, cis-4-hydroxy-L-proline and L-azetidine-2-carboxylic acid, on the growth parameters and procollagen production by cultured normal human skin fibroblasts were examined. The results indicated that incubation of the cells with the analogues reduced the rate of fibroblast proliferation and lowered the plating efficiency. Further experiments demonstrated that fibroblasts in the presence of L-azetidine-2-carboxylic acid synthesized procollagen polypeptides which were not in a triple-helical conformation, as judged by limited pepsin proteolysis. Also, a significantly increased fraction of the newly synthesized collagenous peptides was in a dialyzable form, suggesting increased degradation of the nonhelical chains. The rate of translation of collagenous polypeptides and the preprocollagen messenger RNA activity in the cells were not affected by the analogues. The proline analogues thus appear to inhibit the production of procollagen on the posttranslational level by preventing the polypeptides from folding into a stable triple-helical conformation. The nonhelical polypeptides are then readily susceptible to proteolysis leading to reduced deposition of extracellular collagen fibers. Similar experiments were also performed with fibroblasts cultured from patients with active progressive systemic sclerosis. Quantitatively and qualitatively comparable inhibition of procollagen production by L-azetidine-2-carboxylic acid was noted with scleroderma cells as with control fibroblast cultures. The results suggest, therefore, that proline analogues may, in the future, prove useful in limiting excessive collagen deposition in scleroderma and other forms of dermal fibrosis.

Overexpression of Decorin by Rat Arterial Smooth Muscle Cells Enhances Contraction of Type I Collagen In Vitro

“…CGC assays were performed in the presence of L-DHP, a proline analog that impairs the secretion of collagen 22 as a consequence of its inability to be hydroxylated by prolyl-4-hydroxylase. 23 Compared with controls lacking L-DHP, the contraction of collagen was inhibited significantly by L-DHP at concentrations of 1 to 2 mmol/L ( Figure 4B). Furthermore, cells in which collagen synthesis was inhibited lacked cellular projections and were not surrounded by collagenous bundles, resembling LXSN control cells (data not shown).…”

Proline Reverses the Abnormal Phenotypes of Colletotrichum trifolii Associated with Expression of Endogenous Constitutively Active Ras

“…The following amino acids were used at the indicated concentrations in 0.2% peptone: aspartic acid-monosodium salt (0.8 mM), threonine-free acid (0.3 mM), serine-free acid (0.5 mM), glutamic acid-monosodium salt (1.3 mM), proline-free acid (1.6 mM), glycine-HCl (4 mM), alanine-free acid (2 mM), cysteine-HCl (30 M), valinefree acid (40 M), methionine-free acid (0.2 mM), isoleucine-free acid (0.2 mM), leucine-free acid (0.5 mM), tyrosine-HCl (70 M), phenylalanine-free acid (0.2 mM), histidine-HCl (50 M), lysine-HCl (0.5 mM), arginine-HCl (0.6 mM), and tryptophan-free acid (30 M). To further investigate the role of proline in reversing aberrant Ras phenotypes, the following proline analogs (22) were tested; D-proline, 2-azetidinecarboxylic acid (AZC), thiazolidine-2-carboxylic acid, and thiazolidine-4-carboxylic acid (thioproline) (all from Sigma-Aldrich). All analogs were used at the same molar concentration (1.6 mM) used for proline.…”

Transport of Pharmacologically Active Proline Derivatives by the Human Proton-Coupled Amino Acid Transporter hPAT1

“…Shortly after, Prockop's group also showed that cis-4-hydroxy-L-proline (CHLP) is incorporated into protocollagen and other proteins in place of L-proline and that the resulting unassembled or malfolded procollagen is not extruded into the extracellular matrix at a normal rate (Rosenbloom and Prockop, 1971;Uitto et al, 1975). An intracellular accumulation of polypeptides not folded into a stable triple-helical conformation was observed that is not tolerated by cells (Kao and Prockop, 1977;Tan et al, 1983). Recently, it was shown that the retention of procollagen within the endoplasmatic reticulum is mediated by prolyl 4-hydroxylase (Walmsley et al, 1999).…”