Recombinant Deg/HtrA proteases from Synechocystis sp. PCC 6803 differ in substrate specificity, biochemical characteristics and mechanism

Abstract

Cyanobacteria require efficient protein-quality-control mechanisms to survive under dynamic, often stressful, environmental conditions. It was reported that three serine proteases, HtrA (high temperature requirement A), HhoA (HtrA homologue A) and HhoB (HtrA homologue B), are important for survival of Synechocystis sp. PCC 6803 under high light and temperature stresses and might have redundant physiological functions. In the present paper, we show that all three proteases can degrade unfolded model substrates, but differ with respect to cleavage sites, temperature and pH optima. For recombinant HhoA, and to a lesser extent for HtrA, we observed an interesting shift in the pH optimum from slightly acidic to alkaline in the presence of Mg2+ and Ca2+ ions. All three proteases formed different homo-oligomeric complexes with and without substrate, implying mechanistic differences in comparison with each other and with the well-studied Escherichia coli orthologues DegP (degradation of periplasmic proteins P) and DegS. Deletion of the PDZ domain decreased, but did not abolish, the proteolytic activity of all three proteases, and prevented substrate-induced formation of complexes higher than trimers by HtrA and HhoA. In summary, biochemical characterization of HtrA, HhoA and HhoB lays the foundation for a better understanding of their overlapping, but not completely redundant, stress-resistance functions in Synechocystis sp. PCC 6803.

Crystal Structure of the Yeast Metacaspase Yca1

“…The structures were determined by molecular replacement using PHASER (24) with the atomic coordinates of GSU0716 (Protein Data Bank (PDB) code 3BIJ (www.pdb.org)) as the initial search model. Model building and structure refinement were performed using COOT (25) and Phenix (26), respectively.…”

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

“…11) that are characterized by a serine protease domain followed by one or more PDZ domains. The substrate specificity of HtrA proteases ranges from that of DegS in Escherichia coli, which is highly selective for digestion of RseA as a key step in initiating the envelope-stress response (12), to that of proteases such as DegP in E. coli (13) and DegQ in Legionella (14) and homologues in Synechocystis (15), which are broadly active against unfolded proteins in which hydrophobic regions are exposed to solvent. In addition to such endogenous substrates, HtrA proteases have recently been characterized from the pathogens Helicobacter pylori (16) and Campylobacter jejuni (17) that cleave host proteins to promote bacterial migration across epi-thelial barriers.…”

Degradation of PsbO by the Deg Protease HhoA Is Thioredoxin Dependent

“…All three recombinant proteases exhibited proteolytic activity, as had previously been demonstrated [50]. To investigate if redox-dependent degradation of PsbO could be observed in Synechocystis , we performed an in vitro proteolytic assay using a PSII-enriched fraction isolated from the HT3 strain as the source of PsbO.…”

“…Cloning and purification of recombinant HtrA and HhoB proteases followed a similar strategy [50]. Transformed E. coli cells were grown in LB liquid medium containing 100 µg ml −1 ampicillin at 19°C for 10 h, and expression of recombinant proteases was induced by the addition of 0.1 m M isopropyl-1-thio-D-galactoside (IPTG).…”