FACILITATED PROTEIN AGGREGATION - EFFECTS OF CALCIUM ON THE CHAPERONEAND ANTI-CHAPERONE ACTIVITY OF PROTEIN DISULFIDE-ISOMERASE

Protein disulfide-isomerase (PDI) catalyzes the formation and isomeriz ation of disulfides during oxidative protein folding in the eukaryotic endoplasmic reticulum. At high concentrations, it also serves as a ch aperone and inhibits aggregation. However, at lower concentrations, PD I can display the unusual ability to facilitate aggregation, termed an ti-chaperone activity (Puig, A., and Gilbert, H. F. (1994) J. Biol. Ch em. 269, 7764-7771). Under reducing conditions (10 mM dithiothreitol) and at a low concentration (0.1-0.3 mu M) relative to the unfolded pro tein substrate, PDI facilitates aggregation of alcohol dehydrogenase ( 11 mu M) that has been denatured thermally or chemically. But at highe r concentrations (>0.8 mu M), PDI inhibits aggregation under the same conditions, With denatured citrate synthase, PDI does not facilitate a ggregation, but higher concentrations do inhibit aggregation. Anti-cha perone behavior is associated with the appearance of both PDI and subs trate proteins in insoluble complexes, while chaperone behavior result s in the formation of large (>500 kDa) but soluble complexes that cont ain both proteins. Physiological concentrations of calcium and magnesi um specifically increase the apparent rate of PDI dependent aggregatio n and shift the chaperone activity to higher PDI concentrations. Howev er, calcium has no effect on the K-m or V-max for PDI-catalyzed oxidat ive folding, suggesting that the interactions that lead to chaperone/a nti-chaperone behavior are distinct from those required for catalytic activity. To account for this unusual behavior of a folding catalyst, a model with analogy to classic immunoprecipitation is proposed; multi valent interactions between PDI and a partially aggregated protein sti mulate further aggregate formation by non-covalently cross-linking sma ller aggregates. However, at high ratios of PDI to substrate, cross-li nking may be inhibited by saturation of the sites with PDI. The effect s of PDI concentration on substrate aggregation and the modulation of the behavior by physiological levels of calcium may have implications for the involvement of PDI in protein folding, aggregation, and retent ion in the endoplasmic reticulum.