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Helicobacter pylori HP0377, a member of the Dsb family, is an untypical multifunctional CcmG that cooperates with dimeric thioldisulfide oxidase HP0231.

Roszczenko P, Grzeszczuk M, Kobierecka P, Wywial E, Urbanowicz P, Wincek P, Nowak E, Jagusztyn-Krynicka EK - BMC Microbiol. (2015)

Bottom Line: Our biochemical analysis indicates that HP0377 is a specific reductase, as it does not reduce insulin.In H. pylori HP0377 is re-reduced by CcdA (HP0265); however in E. coli it remains in the oxidized state as it does not interact with E. coli DsbD.Our in vivo work also suggests that both HP0377, which plays a role in apocytochrome reduction, and HP0378, which is involved in heme transport and its ligation into apocytochrome, provide essential functions in H. pylori.

View Article: PubMed Central - PubMed

Affiliation: Department of Bacterial Genetics, Institute of Microbiology, Faculty of Biology, University of Warsaw, Warsaw, Poland. paula.roszczenko@gmail.com.

ABSTRACT

Background: In the genome of H. pylori 26695, 149 proteins containing the CXXC motif characteristic of thioldisulfide oxidoreductases have been identified to date. However, only two of these proteins have a thioredoxin-like fold (i.e., HP0377 and HP0231) and are periplasm-located. We have previously shown that HP0231 is a dimeric oxidoreductase that catalyzes disulfide bond formation in the periplasm. Although HP0377 was originally described as DsbC homologue, its resolved structure and location of the hp0377 gene in the genome indicate that it is a counterpart of CcmG/DsbE.

Results: The present work shows that HP0377 is present in H. pylori cells only in a reduced form and that absence of the main periplasmic oxidase HP0231 influences its redox state. Our biochemical analysis indicates that HP0377 is a specific reductase, as it does not reduce insulin. However, it possesses disulfide isomerase activity, as it catalyzes the refolding of scrambled RNase. Additionally, although its standard redox potential is -176 mV, it is the first described CcmG protein having an acidic pKa of the N-terminal cysteine of the CXXC motif, similar to E. coli DsbA or E. coli DsbC. The CcmG proteins that play a role in a cytochrome c-maturation, both in system I and system II, are kept in the reduced form by an integral membrane protein DsbD or its analogue, CcdA. In H. pylori HP0377 is re-reduced by CcdA (HP0265); however in E. coli it remains in the oxidized state as it does not interact with E. coli DsbD. Our in vivo work also suggests that both HP0377, which plays a role in apocytochrome reduction, and HP0378, which is involved in heme transport and its ligation into apocytochrome, provide essential functions in H. pylori.

Conclusions: The present data, in combination with the resolved three-dimensional structure of the HP0377, suggest that HP0377 is an unusual, multifunctional CcmG protein.

No MeSH data available.


Related in: MedlinePlus

Isomerase activity assay. The reaction contained 40 μM scrambled RNase in 200 mM potassium phosphate buffer, pH 7.0, 2 mM EDTA, 20 μM DTT, and 9 mM cCMP. The reaction was performed in the absence or presence of 20 μM EcDsbC, 20 μM HP0377. The cleavage of cCMP by refolded RNase was monitored continuously at 296 nm. The changes in the absorbance at 296 nm as a function of time are presented. Three independent experiments were performed
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Fig3: Isomerase activity assay. The reaction contained 40 μM scrambled RNase in 200 mM potassium phosphate buffer, pH 7.0, 2 mM EDTA, 20 μM DTT, and 9 mM cCMP. The reaction was performed in the absence or presence of 20 μM EcDsbC, 20 μM HP0377. The cleavage of cCMP by refolded RNase was monitored continuously at 296 nm. The changes in the absorbance at 296 nm as a function of time are presented. Three independent experiments were performed

Mentions: Next we determined the ability of HP0377 to reduce insulin in the presence of DTT. The insulin reduction assay is commonly used to determine whether a protein can function as an oxidoreductase, regardless of its function in the reducing or the oxidizing pathway in vivo. Insulin contains two intramolecular disulfide bonds that connect the A and B chains: reduction of these disulfide bonds causes the precipitation of the B chain, which can be monitored by following the increase of turbidity at 650 nm [38]. As shown in Additional file 2: Figure S2, the reaction lag time with HP0377 in the insulin reduction assay is almost as long as the control reaction (about 60 min), whereas the lag time with EcDsbA is 24 min. Therefore, HP0377 likely cooperates with a specific substrate, which is typical of CcmG proteins. However, these data are in contrast to some previous data [22]. To clarify this inconsistency, and because there is a similarity between the active site of HP0377 (CSYC motif and Thr in cis-Pro loop) and the disulfide isomerase EcDsbC (CPYC motif and Thr in cis-Pro loop), we performed a disulfide isomerase assay by evaluating the ability of HP0377 to reactivate oxidized, scrambled RNase (scRNaseA). ScRNase was prepared as described in the methods section and did not contain free cysteines, as confirmed with Ellman reagent. In this assay, the refolding efficiency of HP0377 was almost as high as EcDsbC (Fig. 3). To get a complete biochemical characterization of HP0377, we also investigated its ability to catalyze the refolding of reduced-unfolded RNaseA. As expected, HP0377 did not show activity in this assay (Additional file 3: Figure S3). As HP0377 revealed high isomerizing activity, and because Dsb proteins involved in the isomerization pathway exist as dimers, we also evaluated the potential oligomerization of HP0377 using two methods (gel filtration and glutaraldehyde crosslinking strategy) (Figs. 4, 5) HP0377 that lacked its own signal sequence (the 25-221 amino acid residues of the native HP0377) and contained a C-terminal 6 His tag was purified from E. coli cytoplasm and used in both assays. We found that exposure of HP0377 to glutaraldehyde, which stabilizes oligomeric proteins by covalent crosslink formation, resulted in generation of a protein with a molecular weight of 48 kDa. This result clearly showed that HP0377 can exist as a dimer. The size exclusion experiment showed that HP0377 eluted as two peaks, one with an estimated mass of 24 kDa, consistent with the size of the monomer, and the second with estimated mass of 48 kDa, consistent with the size of the homodimer. Thus, the presented data allowed us to conclude that at least a portion of HP0377 exists as a dimer.Fig. 3


Helicobacter pylori HP0377, a member of the Dsb family, is an untypical multifunctional CcmG that cooperates with dimeric thioldisulfide oxidase HP0231.

Roszczenko P, Grzeszczuk M, Kobierecka P, Wywial E, Urbanowicz P, Wincek P, Nowak E, Jagusztyn-Krynicka EK - BMC Microbiol. (2015)

Isomerase activity assay. The reaction contained 40 μM scrambled RNase in 200 mM potassium phosphate buffer, pH 7.0, 2 mM EDTA, 20 μM DTT, and 9 mM cCMP. The reaction was performed in the absence or presence of 20 μM EcDsbC, 20 μM HP0377. The cleavage of cCMP by refolded RNase was monitored continuously at 296 nm. The changes in the absorbance at 296 nm as a function of time are presented. Three independent experiments were performed
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4491210&req=5

Fig3: Isomerase activity assay. The reaction contained 40 μM scrambled RNase in 200 mM potassium phosphate buffer, pH 7.0, 2 mM EDTA, 20 μM DTT, and 9 mM cCMP. The reaction was performed in the absence or presence of 20 μM EcDsbC, 20 μM HP0377. The cleavage of cCMP by refolded RNase was monitored continuously at 296 nm. The changes in the absorbance at 296 nm as a function of time are presented. Three independent experiments were performed
Mentions: Next we determined the ability of HP0377 to reduce insulin in the presence of DTT. The insulin reduction assay is commonly used to determine whether a protein can function as an oxidoreductase, regardless of its function in the reducing or the oxidizing pathway in vivo. Insulin contains two intramolecular disulfide bonds that connect the A and B chains: reduction of these disulfide bonds causes the precipitation of the B chain, which can be monitored by following the increase of turbidity at 650 nm [38]. As shown in Additional file 2: Figure S2, the reaction lag time with HP0377 in the insulin reduction assay is almost as long as the control reaction (about 60 min), whereas the lag time with EcDsbA is 24 min. Therefore, HP0377 likely cooperates with a specific substrate, which is typical of CcmG proteins. However, these data are in contrast to some previous data [22]. To clarify this inconsistency, and because there is a similarity between the active site of HP0377 (CSYC motif and Thr in cis-Pro loop) and the disulfide isomerase EcDsbC (CPYC motif and Thr in cis-Pro loop), we performed a disulfide isomerase assay by evaluating the ability of HP0377 to reactivate oxidized, scrambled RNase (scRNaseA). ScRNase was prepared as described in the methods section and did not contain free cysteines, as confirmed with Ellman reagent. In this assay, the refolding efficiency of HP0377 was almost as high as EcDsbC (Fig. 3). To get a complete biochemical characterization of HP0377, we also investigated its ability to catalyze the refolding of reduced-unfolded RNaseA. As expected, HP0377 did not show activity in this assay (Additional file 3: Figure S3). As HP0377 revealed high isomerizing activity, and because Dsb proteins involved in the isomerization pathway exist as dimers, we also evaluated the potential oligomerization of HP0377 using two methods (gel filtration and glutaraldehyde crosslinking strategy) (Figs. 4, 5) HP0377 that lacked its own signal sequence (the 25-221 amino acid residues of the native HP0377) and contained a C-terminal 6 His tag was purified from E. coli cytoplasm and used in both assays. We found that exposure of HP0377 to glutaraldehyde, which stabilizes oligomeric proteins by covalent crosslink formation, resulted in generation of a protein with a molecular weight of 48 kDa. This result clearly showed that HP0377 can exist as a dimer. The size exclusion experiment showed that HP0377 eluted as two peaks, one with an estimated mass of 24 kDa, consistent with the size of the monomer, and the second with estimated mass of 48 kDa, consistent with the size of the homodimer. Thus, the presented data allowed us to conclude that at least a portion of HP0377 exists as a dimer.Fig. 3

Bottom Line: Our biochemical analysis indicates that HP0377 is a specific reductase, as it does not reduce insulin.In H. pylori HP0377 is re-reduced by CcdA (HP0265); however in E. coli it remains in the oxidized state as it does not interact with E. coli DsbD.Our in vivo work also suggests that both HP0377, which plays a role in apocytochrome reduction, and HP0378, which is involved in heme transport and its ligation into apocytochrome, provide essential functions in H. pylori.

View Article: PubMed Central - PubMed

Affiliation: Department of Bacterial Genetics, Institute of Microbiology, Faculty of Biology, University of Warsaw, Warsaw, Poland. paula.roszczenko@gmail.com.

ABSTRACT

Background: In the genome of H. pylori 26695, 149 proteins containing the CXXC motif characteristic of thioldisulfide oxidoreductases have been identified to date. However, only two of these proteins have a thioredoxin-like fold (i.e., HP0377 and HP0231) and are periplasm-located. We have previously shown that HP0231 is a dimeric oxidoreductase that catalyzes disulfide bond formation in the periplasm. Although HP0377 was originally described as DsbC homologue, its resolved structure and location of the hp0377 gene in the genome indicate that it is a counterpart of CcmG/DsbE.

Results: The present work shows that HP0377 is present in H. pylori cells only in a reduced form and that absence of the main periplasmic oxidase HP0231 influences its redox state. Our biochemical analysis indicates that HP0377 is a specific reductase, as it does not reduce insulin. However, it possesses disulfide isomerase activity, as it catalyzes the refolding of scrambled RNase. Additionally, although its standard redox potential is -176 mV, it is the first described CcmG protein having an acidic pKa of the N-terminal cysteine of the CXXC motif, similar to E. coli DsbA or E. coli DsbC. The CcmG proteins that play a role in a cytochrome c-maturation, both in system I and system II, are kept in the reduced form by an integral membrane protein DsbD or its analogue, CcdA. In H. pylori HP0377 is re-reduced by CcdA (HP0265); however in E. coli it remains in the oxidized state as it does not interact with E. coli DsbD. Our in vivo work also suggests that both HP0377, which plays a role in apocytochrome reduction, and HP0378, which is involved in heme transport and its ligation into apocytochrome, provide essential functions in H. pylori.

Conclusions: The present data, in combination with the resolved three-dimensional structure of the HP0377, suggest that HP0377 is an unusual, multifunctional CcmG protein.

No MeSH data available.


Related in: MedlinePlus