The HtrA Protease from Streptococcus pneumoniae Digests Both Denatured Proteins and the Competence-stimulating Peptide

Marco Cassone,Lynn A Spruce,Michael E Sebert,Steven H Seeholzer,Alyssa L Gagne

doi:10.1074/jbc.m112.391482

Marco Cassone, Lynn A Spruce + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m112.391482

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Abstract

The HtrA protease of Streptococcus pneumoniae functions both in a general stress response role and as an error sensor that specifically represses genetic competence when the overall level of biosynthetic errors in cellular proteins is low. However, the mechanism through which HtrA inhibits development of competence has been unknown. We found that HtrA digested the pneumococcal competence-stimulating peptide (CSP) and constituted the primary extracytoplasmic CSP-degrading activity in cultures of S. pneumoniae. Mass spectrometry demonstrated that cleavage predominantly followed residue Phe-8 of the CSP-1 isoform of the peptide within its central hydrophobic patch, and in competition assays, both CSP-1 and CSP-2 interacted with HtrA with similar efficiencies. More generally, analysis of β-casein digestion and of digestion within HtrA itself revealed a preference for substrates with non-polar residues at the P1 site. Consistent with a specificity for exposed hydrophobic residues, competition from native BSA only weakly inhibited digestion of CSP, but denaturation converted BSA into a strong competitive inhibitor of such proteolysis. Together these findings support a model in which digestion of CSP by HtrA is reduced in the presence of other unfolded proteins that serve as alternative targets for degradation. Such competition may provide a mechanism by which HtrA functions in a quality control capacity to monitor the frequency of biosynthetic errors that result in protein misfolding.

Highlights

The pneumococcal HtrA protease represses competence selectively when the frequency of errors in protein synthesis is low
We showed that the HtrA protease of S. pneumoniae was able to digest the peptide pheromone competence-stimulating peptide (CSP), which regulates development of genetic competence
Maximal transformation was reported to be induced by CSP-1 at concentrations of at least nM [42], whereas half-maximal activation of competence signaling by CSP-1 and CSP-2 was seen at 4.4 and nM, respectively [30]

Summary

Background

The pneumococcal HtrA protease represses competence selectively when the frequency of errors in protein synthesis is low. Consistent with a specificity for exposed hydrophobic residues, competition from native BSA only weakly inhibited digestion of CSP, but denaturation converted BSA into a strong competitive inhibitor of such proteolysis Together these findings support a model in which digestion of CSP by HtrA is reduced in the presence of other unfolded proteins that serve as alternative targets for degradation. We considered that CSP might effectively mimic the exposed hydrophobic region of a misfolded protein and thereby give rise to a situation in which digestion of the signaling peptide was in competition with digestion of generic, misfolded proteins Such competition would present a model to explain the loss of competence repression by HtrA that is observed when frequent translational errors generate proteins with intrinsic folding defects.

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION