The general aim of this research is to characterize the mechanisms that mediate intracellular degradation of newly synthesized collagen. The importance of collagen as a structural element and as a modulator of developmental processes is well known, and studies on intracellular degradation of collagen can be expected to provide additional insights into how production of this ubiquitous molecule is regulated. Beyond this, these studies may serve as a paradigm: To the extent that collagen is a representative secretory protein, investigations into its rapid catabolism will elucidate the nature of what may be a common posttranslational modification. Collagen degradation is studied by incubating cultured cells or tissues with (14C)-proline and measuring the relative amount of (14C) hydroxyproline in a low molecular fraction. When stationary phase cultures of human fibroblasts are incubated under conditions which permit maximum hydroxylation of prolyl residues, approximately 15% of all (14C) hydroxyproline is found in low molecular weight fraction: this is termed the basallevel of degradation. The level of degradation increases significantly when culture conditions are manipulated so that the conformation of newly synthesized molecules is abnormal, or when cells are incubated with an agent that raises the intracellular level of cAMP. The increase in degradation above the basal level is termed enhanced degradation. The specific objectives of this research are as follows: (1) To test the hypothesis that enhanced degradation, that is, degradation above the basal level, is mediated by lysosomal proteases. (2) To test the hypothesis that basal degradation is not mediated by lysosomal proteases. (3) To test the hypothesis that basal degradation occurs between the time that newly synthesized collagen molecules leave the rough endoplasmic reticulum and the time they leave the Golgi complex. (4) To test the hypothesis that proteases capable of degrading collagen or collagenous peptides are located in the secretory pathway.
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| Bienkowski, R S; Curran, S F; Berg, R A (1986) Kinetics of intracellular degradation of newly synthesized collagen. Biochemistry 25:2455-9 |
