Inhibition of Lon protease by bacterial lipopolisaccharide (LPS) though inhibition of ATPase

Nahoko Sugiyama,Noriko Minami,Yoshiyuki Ishii,Fumio Amano

doi:10.4236/abb.2013.44077

Abstract

Lon protease, an ATP-dependent protease in Escherichia coli, degrades abnormal proteins and regulates several important cellular functions. Here we show novel inhibitory effects of lipopolysaccharide (LPS) on Lon protease activities. LPS inhibited the peptidase, protease, and ATPase activities of Lon; and a dose-response study showed that LPS at low doses more effectively inhibited the ATPase activity than the peptidase one, suggesting different susceptibility to LPS of these activities associated with Lon. Structure-activity relationship studies revealed that ReLPS, detoxified LPS, and mono-phosphoryl as well as diphosphoryl lipid A, also showed similar inhibition, suggesting that neither O-antigen polysaccharide nor O-acyl chain, but rather phosphate groups in the lipid A domain, seem to have been responsible for the inhibitory effects. Besides, LPS was co-precipitated with Lon by an anti-Lon antibody, showing the direct binding of LPS to Lon. These results suggest that LPS bound to Lon and inhibited the protease activity of Lon by inhibiting its ATPase activity. These results also seem to be another example of a negatively charged phosphate group in membrane components of Escherichia coli being involved in the regulation of protease activity of Lon through binding to Lon and inhibiting its ATPase activity, as in the case of cardiolipin.

Highlights

Proteolysis is an important function in cellular processes, regulating stress responses and quality control of proteins by eliminating defective or abnormal proteins which might otherwise lead to defective cell metabolism and other malfunctions
We examined whether LPS would inhibit the protease activity of Lon by measuring the degradation of casein
In order to know whether the inhibitory effects of LPS on the peptidase, protease, and ATPase activities of Lon were due to phosphate groups associated with the lipid A moiety, we examined the effects of LPS analogs ReLPS, lacking the O-antigen polysaccharide, and detoxified LPS, lacking the O-acyl chains, but having lipid A with diphosphate at its 4, 4’-positions

Summary

Introduction

Proteolysis is an important function in cellular processes, regulating stress responses and quality control of proteins by eliminating defective or abnormal proteins which might otherwise lead to defective cell metabolism and other malfunctions. All of them have a similar structure and are high molecular weight complexes with distinct activity sites, an ATPase and a catalytic domain They require ATP hydrolysis before degradation of the substrate, which is necessary to unfold the substrate proteins before importing them suitably to the catalytic domain of the protease. Cardiolipin, a major component of E. coli inner membrane phospholipids and having a negative charge, binds to Lon and inhibits both its ATPase and protease activities [11] These results in the previous studies imply regulatory roles of negatively charged phosphate groups of such molecules on the activity of Lon, the precise mechanisms underlying these interactions at the molecular basis remain unknown

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Conclusion