Abderrahim Malki

Chaperone properties of Escherichia coli thioredoxin and thioredoxin reductase

Thioredoxin, thioredoxin reductase and NADPH form the thioredoxin system and are the major cellular protein disulphide reductase. We report here that Escherichia coli thioredoxin and thioredoxin reductase interact with unfolded and denatured proteins, in a manner similar to that of molecular chaperones that are involved in protein folding and protein renaturation after stress. Thioredoxin and/or thioredoxin reductase promote the functional folding of citrate synthase and alpha-glucosidase after urea denaturation. They also promote the functional folding of the bacterial galactose receptor, a protein without any cysteines. Furthermore, redox cycling of thioredoxin/thioredoxin reductase in the presence of NADPH and cystine stimulates the renaturation of the galactose receptor, suggesting that the thioredoxin system functions like a redox-powered chaperone machine. Thioredoxin reductase prevents the aggregation of citrate synthase under heat-shock conditions. It forms complexes that are more stable than those formed by thioredoxin with several unfolded proteins such as reduced carboxymethyl alpha-lactalbumin and unfolded bovine pancreatic trypsin inhibitor. These results suggest that the thioredoxin system, in addition to its protein disulphide isomerase activity possesses chaperone-like properties, and that its thioredoxin reductase component plays a major role in this function.

Escherichia coli HdeB Is an Acid Stress Chaperone

We cloned, expressed, and purified the hdeB gene product, which belongs to the hdeAB acid stress operon. We extracted HdeB from bacteria by the osmotic-shock procedure and purified it to homogeneity by ion-exchange chromatography and hydroxyapatite chromatography. Its identity was confirmed by mass spectrometry analysis. HdeB has a molecular mass of 10 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which matches its expected molecular mass. We purified the acid stress chaperone HdeA in parallel in order to compare the two chaperones. The hdeA and hdeB mutants both display reduced viability upon acid stress, and only the HdeA/HdeB expression plasmid can restore their viability to close to the wild-type level, suggesting that both proteins are required for optimal protection of the bacterial periplasm against acid stress. Periplasmic extracts from both mutants aggregate at acidic pH, suggesting that HdeA and HdeB are required for protein solubilization. At pH 2, the aggregation of periplasmic extracts is prevented by the addition of HdeA, as previously reported, but is only slightly reduced by HdeB. At pH 3, however, HdeB is more efficient than HdeA in preventing periplasmic-protein aggregation. The solubilization of several model substrate proteins at acidic pH supports the hypothesis that, in vitro, HdeA plays a major role in protein solubilization at pH 2 and that both proteins are involved in protein solubilization at pH 3. Like HdeA, HdeB exposes hydrophobic surfaces at acidic pH, in accordance with the appearance of its chaperone properties at acidic pH. HdeB, like HdeA, dissociates from dimers at neutral pH into monomers at acidic pHs, but its dissociation is complete at pH 3 whereas that of HdeA is complete at a more acidic pH. Thus, we can conclude that Escherichia coli possesses two acid stress chaperones that prevent periplasmic-protein aggregation at acidic pH.

The E. coli cell surface specifically prevents the initiation of DNA replication at oriC on heminethylated DNA templates

A particular outer membrane fraction previously defined as possessing specific affinity for the hemimethylated form of the origin of replication of the E. coli chromosome (oriC) is shown to inhibit the initiation of DNA synthesis at this site on hemimethylated DNA templates in vitro. The replication of fully methylated or unmethylated DNA templates is not affected. Also, no inhibition is observed if initiation takes place at random sites on the hemimethylated template. The key inactivation step appears to be membrane inhibition of DnaA initiator protein binding to oriC. Remethylation of the membrane-bound hemimethylated DNA results in reactivation. Our results demonstrate direct involvement of the membrane in the control of DNA replication. We propose that association/dissociation of the origin from the cell membrane is one of the control elements governing interinitiation times in E. coli.

Peptidase Activity of the Escherichia coli Hsp31 Chaperone

Hsp31, the Escherichia coli hcha gene product, is a molecular chaperone whose activity is inhibited by ATP at high temperature. Its crystal structure reveals a putative Cys184, His185, and Asp213 catalytic triad similar to that of the Pyrococcus horikoshii protease PH1704, suggesting that it should display a proteolytic activity. A preliminary report has shown that Hsp31 has an exceedingly weak proteolytic activity toward bovine serum albumin and a peptidase activity toward two peptide substrates with small amino acids at their N terminus (alanine or glycine), but the physiological significance of this observation remains unclear. In this study, we report that Hsp31 does not diplay any significant proteolytic activity but has peptidolytic activity. The aminopeptidase cleavage preference of Hsp31 is Ala > Lys > Arg > His, suggesting that Hsp31 is an aminopeptidase of broad specificity. Its aminopeptidase activity is inhibited by the thiol reagent iodoacetamide and is completely abolished in a C185A mutant, which is consistent with Hsp31 being a cysteine peptidase. The aminopeptidase activity of Hsp31 is also inhibited by EDTA and 1,10-phenanthroline, in concordance with the importance of the putative His85, His122, and Glu90 metal-binding site revealed by crystallographic studies. An Hsp31-deficient mutant accumulates more 8–12-mer peptides than its parental strain, and purified Hsp31 can transform these peptides into smaller peptides, suggesting that Hsp31 has an important peptidase function both in vivo and in vitro. Proteins interacting with Hsp31 have been identified by reverse purification of a crude E. coli extract on an Hsp31-affinity column, followed by SDS-polyacrylamide electrophoresis and mass spectrometry. The ClpA component of the ClpAP protease, the chaperone GroEL, elongation factor EF-Tu, and tryptophanase were all found to interact with Hsp31, thus substantiating the role of Hsp31 as both chaperone and peptidase.

Peptidase activity of the E. coli HSP31 chaperone

Hsp31, the Escherichia coli hcha gene product, is a molecular chaperone whose activity is inhibited by ATP at high temperature. Its crystal structure reveals a putative Cys184, His185, and Asp213 catalytic triad similar to that of the Pyrococcus horikoshii protease PH1704, suggesting that it should display a proteolytic activity. A preliminary report has shown that Hsp31 has an exceedingly weak proteolytic activity toward bovine serum albumin and a peptidase activity toward two peptide substrates with small amino acids at their N terminus (alanine or glycine), but the physiological significance of this observation remains unclear. In this study, we report that Hsp31 does not diplay any significant proteolytic activity but has peptidolytic activity. The aminopeptidase cleavage preference of Hsp31 is Ala > Lys > Arg > His, suggesting that Hsp31 is an aminopeptidase of broad specificity. Its aminopeptidase activity is inhibited by the thiol reagent iodoacetamide and is completely abolished in a C185A mutant, which is consistent with Hsp31 being a cysteine peptidase. The aminopeptidase activity of Hsp31 is also inhibited by EDTA and 1,10-phenanthroline, in concordance with the importance of the putative His85, His122, and Glu90 metal-binding site revealed by crystallographic studies. An Hsp31-deficient mutant accumulates more 8–12-mer peptides than its parental strain, and purified Hsp31 can transform these peptides into smaller peptides, suggesting that Hsp31 has an important peptidase function both in vivo and in vitro. Proteins interacting with Hsp31 have been identified by reverse purification of a crude E. coli extract on an Hsp31-affinity column, followed by SDS-polyacrylamide electrophoresis and mass spectrometry. The ClpA component of the ClpAP protease, the chaperone GroEL, elongation factor EF-Tu, and tryptophanase were all found to interact with Hsp31, thus substantiating the role of Hsp31 as both chaperone and peptidase.

Solubilization of protein aggregates by the acid stress chaperones HdeA and HdeB.

The acid stress chaperones HdeA and HdeB of Escherichia coli prevent the aggregation of periplasmic proteins at acidic pH. We show in this report that they also form mixed aggregates with proteins that have failed to be solubilized at acidic pH and allow their subsequent solubilization at neutral pH. HdeA, HdeB, and HdeA and HdeB together display an increasing efficiency for the solubilization of protein aggregates at pH 3. They are less efficient for the solubilization of aggregates at pH 2, whereas HdeB is the most efficient. Increasing amounts of periplasmic proteins draw increasing amounts of chaperone into pellets, suggesting that chaperones co-aggregate with their substrate proteins. We observed a decrease in the size of protein aggregates in the presence of HdeA and HdeB, from very high molecular mass aggregates to 100–5000-kDa species. Moreover, a marked decrease in the exposed hydrophobicity of aggregated proteins in the presence of HdeA and HdeB was revealed by 1,1′-bis(4-anilino)naphtalene-5,5′-disulfonic acid binding experiments. In vivo, during the recovery at neutral pH of acid stressed bacterial cells, HdeA and HdeB allow the solubilization and renaturation of protein aggregates, including those formed by the maltose receptor MalE, the oligopeptide receptor OppA, and the histidine receptor HisJ. Thus, HdeA and HdeB not only help to maintain proteins in a soluble state during acid treatment, as previously reported, but also assist, both in vitro and in vivo, in the solubilization at neutral pH of mixed protein-chaperone aggregates formed at acidic pH, by decreasing the size of protein aggregates and the exposed hydrophobicity of aggregated proteins.

Competition between the replication initiator DnaA and the sequestration factor SeqA for binding to the hemimethylated chromosomal origin of E. coli in vitro.

Following replication initiation, the replication origin (oriC) in Escherichia coli enters a hemimethylated state at Dam methylation sites which are recognized by the SeqA protein. SeqA binds preferentially to hemimethylated GATC sequences of DNA in vitro. SeqA is essential for the synchronous initiation of chromosome replication from oriC copies in vivo.

Characterization of the Escherichia coli YedU protein as a molecular chaperone.

We have cloned, purified to homogeneity, and characterized as a molecular chaperone the Escherichia coli YedU protein. The purified protein shows a single band at 31 kDa on SDS-polyacrylamide gels and forms dimers in solution. Like other chaperones, YedU interacts with unfolded and denatured proteins. It promotes the functional folding of citrate synthase and alpha-glucosidase after urea denaturation and prevents the aggregation of citrate synthase under heat shock conditions. YedU forms complexes with the permanently unfolded protein, reduced carboxymethyl alpha-lactalbumin. In contrast to DnaK/Hsp70, ATP does not stimulate YedU-dependent citrate synthase renaturation and does not affect the interaction between YedU and unfolded proteins, and YedU does not display any peptide-stimulated ATPase activity. We conclude that YedU is a novel chaperone which functions independently of an ATP/ADP cycle.

Protein Aggregation in a Mutant Deficient in YajL, the Bacterial Homolog of the Parkinsonism-associated Protein DJ-1

YajL is the closest prokaryotic homolog of the parkinsonism-associated protein DJ-1 (40% sequence identity and similar three-dimensional structure), a protein of unknown function involved in the cellular response to oxidative stress. We report here that a yajL mutant of Escherichia coli displays an increased sensitivity to oxidative stress. It also exhibits a protein aggregation phenotype in aerobiosis, but not in anaerobiosis or in aerobic cells overexpressing superoxide dismutase, suggesting that protein aggregation depends on the presence of reactive oxygen species produced by respiratory chains. The protein aggregation phenotype of the yajL mutant, which can be rescued by the wild-type yajL gene, but not by the corresponding cysteine 106 mutant allele, is similar to that of multiple mutants deficient in superoxide dismutases and catalases, although intracellular hydrogen peroxide levels were not increased in the yajL mutant, suggesting that protein aggregation in this strain does not result from a hydrogen peroxide detoxification defect. Aggregation-prone proteins included 17 ribosomal proteins, the ATP synthase β subunit, flagellin, and the outer membrane proteins OmpA and PAL; all of them are part of multiprotein complexes, suggesting that YajL might be involved in optimal expression of these complexes, especially during oxidative stress. YajL stimulated the renaturation of urea-unfolded citrate synthase and the solubilization of the urea-unfolded ribosomal proteins S1 and L3 and was more efficient as a chaperone in its oxidized form than in its reduced form. The mRNA levels of several aggregated proteins of the yajL mutant were severely affected, suggesting that YajL also acts at the level of gene expression. These two functions of YajL might explain the protein aggregation phenotype of the yajL mutant.

YajL, Prokaryotic Homolog of Parkinsonism-associated Protein DJ-1, Functions as a Covalent Chaperone for Thiol Proteome

Background: A novel function for YajL, the prokaryotic homolog of the Parkinsonism-associated protein DJ-1. Results: YajL and DJ-1 form mixed disulfides with members of the thiol proteome. Conclusion: This covalent chaperone function supports their role in oxidative stress protection. Significance: There is an exciting encounter between the crucial cysteine 106 of these covalent chaperones and the oxidized cysteines of their substrates. YajL is the closest Escherichia coli homolog of the Parkinsonism-associated protein DJ-1, a multifunctional oxidative stress response protein whose biochemical function remains unclear. We recently reported the aggregation of proteins in a yajL mutant in an oxidative stress-dependent manner and that YajL exhibits chaperone activity. Here, we show that YajL displays covalent chaperone and weak protein oxidoreductase activities that are dependent on its exposed cysteine 106. It catalyzes reduced RNase oxidation and scrambled RNase isomerization and insulin reduction and forms mixed disulfides with many cellular proteins upon oxidative stress. The formation of mixed disulfides was detected by immunoblotting bacterial extracts with anti-YajL antibodies under nonreducing conditions. Disulfides were purified from bacterial extracts on a YajL affinity column, separated by nonreducing-reducing SDS-PAGE, and identified by mass spectrometry. Covalent YajL substrates included ribosomal proteins, aminoacyl-tRNA synthetases, chaperones, catalases, peroxidases, and other proteins containing cysteines essential for catalysis or FeS cluster binding, such as glyceraldehyde-3-phosphate dehydrogenase, aldehyde dehydrogenase, aconitase, and FeS cluster-containing subunits of respiratory chains. In addition, we show that DJ-1 also forms mixed disulfides with cytoplasmic proteins upon oxidative stress. These results shed light on the oxidative stress-dependent chaperone function of YajL and identify YajL substrates involved in translation, stress protection, protein solubilization, and metabolism. They reveal a crucial role for cysteine 106 and suggest that DJ-1 also functions as a covalent chaperone. These findings are consistent with several defects observed in yajL or DJ-1 mutants, including translational defects, protein aggregation, oxidative stress sensitivity, and metabolic deficiencies.