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

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 (DegP _red ), which represents its protease-active conformation. Besides removing damaged proteins, DegP _red 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 DegP _red . Because in vitro , the lack of DSB sensitizes DegP to lower substrate levels, we suggest that in vivo DegP _red 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.