Distinct modes of redox-mediated DegP activation govern its role as a general protease

Abstract

DegP, a bifunctional chaperone-protease central to maintaining envelope proteostasis in Gram-negative bacteria, serves as a paradigm for understanding protein quality control mechanisms. Decades of research, mainly using synthetic peptides/non-native substrates that do not contain cysteines, have established the allosteric mode of DegP activation, where substrate binding remodels inactive DegP into its active conformations. In enteric bacteria, as DegP typically harbours a disulfide bond (DSB) in its protease domain, its redox-mediated activation has also been proposed. However, the cellular factors driving this activation mode and its physiological relevance have remained elusive. Here, we investigated this aspect in Escherichia coli primarily using long-chain fatty acids and alkaline pH as stressors. We show that, under these conditions, DegP considerably accumulates in its thiol form (DegPred), which represents its protease-active conformation. Besides removing damaged proteins, DegPred also induces the Cpx envelope stress response in all tested conditions. Notably, two distinct mechanisms promote redox-mediated DegP activation in a temporal and stress-specific manner: a compromised DSB-forming machinery fails to oxidize DegP, and thiol-containing substrates allosterically convert DegP into DegPred. Because in vitro, the lack of DSB sensitizes DegP to lower substrate levels, we suggest that in vivo DegPred is primed for a robust response to stresses. Finally, the diverse nature of the molecular players that activate Cpx in our tested stress conditions leads us to propose that the DSB of DegP, by acting as a redox sensor, enables DegP to integrate a wide range of inducing cues and thus work as a general protease. Significance StatementThe proteolytic activity of DegP is highly implicated in bacterial survival and virulence. Notably, DegP from the Enterobacteriaceae family of Gram-negative bacteria typically harbours a disulfide bond (DSB) in its protease domain. Because pathogens face redox stress during infection and antibiotic exposure, understanding how redox perturbants modulate DegP function is necessary for exploiting it as a target for therapeutic intervention strategies. Here, using E. coli as the model bacterium, we show that the DSB of DegP acts as a redox sensor under various inducing cues, thereby positioning DegP as a versatile, broad-spectrum protease. The present study opens avenues for investigating whether a similar redox-mediated control of DegP activation influences the pathogenesis of enteric bacteria.