Sequence requirements for Lon-dependent degradation of the Escherichia coli transcription activator SoxS: identification of the SoxS residues critical to proteolysis and specific inhibition of in vitro degradation by a peptide comprised of the N-terminal 21 amino acid residues.
When Escherichia coli encounter redox-cycling compounds that endogenously generate superoxide, the cell's defense response is initiated by the de novo synthesis of SoxS, which then activates transcription of the genes of the SoxRS regulon. Recently, we showed that after the oxidative stress is relieved, the SoxRS system resets by an active process wherein SoxS synthesis ceases and the intrinsically unstable SoxS protein is rapidly degraded, primarily by Lon protease. Here, we use deletion mutants and a library of alanine-stretch mutants of the entire protein to identify the SoxS features responsible for Lon-dependent proteolysis in vivo. We found that the 17 amino acid residues at the SoxS N terminus play the primary role in protease recognition and that the addition of the N-terminal 21 residues of SoxS to the otherwise stable green fluorescent protein is sufficient to signal the chimera for Lon-dependent degradation. With a minimal in vitro degradation system, we confirm the intrinsic instability of SoxS and the sequence requirements for Lon-dependent degradation. Lastly, we demonstrate that the addition of a peptide comprised of the 21 N-terminal amino acid residues of SoxS is able to inhibit specifically the in vitro proteolysis of SoxS.
Shah
Ishita M
IM
Department of Biological Sciences, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, MD 21250, USA.
Wolf
Richard E
RE
D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D049070:Protease La / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D017384:Sequence Deletion; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D029968:Escherichia coli Proteins; D010446:Peptide Fragments; D015534:Trans-Activators; C070725:SoxS protein, E coli; C483567:Lon protein, E coli; D049070:Protease La; D000409:Alanine