Characterization of the Autocleavage Process of the Escherichia coli HtrA Protein: Implications for its Physiological Role

Abstract

The Escherichia coli HtrA protein is a periplasmic protease/chaperone that is upregulated under stress conditions. The protease and chaperone activities of HtrA eliminate or refold damaged and unfolded proteins in the bacterial periplasm that are generated upon stress conditions. In the absence of substrates, HtrA oligomerizes into a hexameric cage, but binding of misfolded proteins transforms the hexamers into bigger 12-mer and 24-mer cages that encapsulate the substrates for degradation or refolding. HtrA also undergoes partial degradation as a consequence of self-cleavage of the mature protein, producing short-HtrA protein (s-HtrA). The aim of this study was to examine the physiological role of this self-cleavage process. We found that the only requirement for self-cleavage of HtrA into s-HtrA in vitro was the hydrolysis of protein substrates. In fact, peptides resulting from the hydrolysis of the protein substrates were sufficient to induce autocleavage. However, the continuous presence of full-length substrate delayed the process. In addition, we observed that the hexameric cage structure is required for autocleavage and that s-HtrA accumulates only late in the degradation reaction. These results suggest that self-cleavage occurs when HtrA reassembles back into the resting hexameric structure and peptides resulting from substrate hydrolysis are allosterically stimulating the HtrA proteolytic activity. Our data support a model in which the physiological role of the self-cleavage process is to eliminate the excess of HtrA once the stress conditions cease.

Different Contributions of HtrA Protease and Chaperone Activities to Campylobacter jejuni Stress Tolerance and Physiology

“…Furthermore, when purified wild-type HtrA was incubated alone at either 37°C or 45°C, a steady increase in the amount of the short HtrA fragment was observed over the course of 3 h (data not shown). These observations imply that the short HtrA fragment is an autocleavage product, a feature also observed for the E. coli HtrA ortholog (27,62). The amounts of full-length HtrA in the wild type and the htrA(S197A) mutant were identical, as measured by densitometry; however, the wild type contained a substantial (Fig.…”

Agrobacterium tumefaciens ExoR Controls Acid Response Genes and Impacts Exopolysaccharide Synthesis, Horizontal Gene Transfer, and Virulence Gene Expression

“…Because ExoR has multiple predicted protein-protein interaction domains-a tetratricopeptide repeat (TPR) domain and two Sel1 domains-there exists the distinct possibility that it has multiple interacting partners in the periplasm and exerts control over other regulatory pathways. The connection between low pH and ExoR regulation is clear, but some genes in the ExoR regulon are influenced by other environmental signals such as low oxygen concentration (sinR [64] and ntrX [66]) and temperature (heat shock and cold shock genes [67][68][69]). This raises the question, does ExoR control responses to a variety of environmental signals?…”

A Periplasmic LolA Derivative with a Lethal Disulfide Bond Activates the Cpx Stress Response System

“…(E) Cell lysates were prepared at 2 h after the addition of arabinose, and equivalent amounts were analyzed by Western blotting with anti-FLAG peptide and anti-DegP antisera. Bands (asterisks) migrating faster than that of DegP most likely represent its self-cleaved forms (8). The amounts of intact DegP were densitometrically determined and expressed as percentages, taking the amount of DegP in the absence of arabinose as 100.…”