Oxidatively denaturated proteins are degraded by an ATP-independent proteolytic pathway in escherichia coli

Abstract

E. coli contains a soluble proteolytic pathway which can recognize and degrade oxidatively denatured protein fragments, and which may act as a “secondary antioxidant defense.” We now provide evidence that this proteolytic pathway is distinct from the previously described ATP-dependent, and protease “La”-dependent, pathway which may degrade other abnormal proteins. Cells (K12) which are depleted of ATP, by arsenate treatment or anaerobic incubation (after growth on succinate), exhibited proteolytic response to oxidative stress which were indistinguishable from those observed cells with normal ATP levels. Further more, the proteolytic responses to oxidative damage by menadione or H 2O 2 were almost identical in the isogenic strains RM312 (a K12 derivative) and RM1385 (a lon deletion mutant of RM312). Since the lon (or capR) gene codes for the ATP-dependent protease “La,” these results indicate that neither ATP nor protease “La” are required for the degradation of oxidatively denatured proteins. We next prepared cell-free extracts of K12, RM312, and RM1385 and tested the activity of their soluble proteases against proteins (albumin, hemoglobin, superoxide dismutase, catalase) which been oxidatively denatured (in vitro) by exposure to ·OH, ·OH + O 2 − (+O 2), H 2O 2, or ascorbate plus iron. The breakdown of oxidatively denatured proteins was several-fold higher than that of untreated proteins in extracts from all three strains, and ATP did not stimulate degradation. Incubation of extracts at 45°C, which inactivates protease “La,” actually stimulated the degradation of oxidatively denatured proteins. Although Ca 2+ had little effect on proteolysis, serine reagents, transition metal chelators, and hemin effectively inhibited the degradation of oxidatively denatured proteins in both intact and cell-free extracts. Degradation of oxidatively denatured proteins in cell-free extracts was maximal at pH 7.8 and was unaffected by dialysis of the extracts against membranes with molecular weight cutoffs as high as 50,000. Our results indicate the presence of a neutral, ATP- and calcium- independent proteolytic pathway in the E. coli cytosol, which contains serine- and metallo- proteases (with molecular weights greater than 50,000), and which preferentially degrades oxidatively denatured proteins.