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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

Redox state of HP0377 in wt and mutants: hp0231::cat, dsbI::aph and complemented strains of H. pylori strain N6. Bacterial cultures were treated with 10 % TCA, followed by alkylation with AMS. Cellular proteins including the reduced (red; DTT treated, modified with AMS) and the oxidized (ox; non-modified with AMS) controls were separated by 14 % SDS-PAGE under non-reducing conditions, and Western blot analysis antibodies against HP0377 was performed. Each lane contains proteins isolated from the same amount of bacteria
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Fig1: Redox state of HP0377 in wt and mutants: hp0231::cat, dsbI::aph and complemented strains of H. pylori strain N6. Bacterial cultures were treated with 10 % TCA, followed by alkylation with AMS. Cellular proteins including the reduced (red; DTT treated, modified with AMS) and the oxidized (ox; non-modified with AMS) controls were separated by 14 % SDS-PAGE under non-reducing conditions, and Western blot analysis antibodies against HP0377 was performed. Each lane contains proteins isolated from the same amount of bacteria

Mentions: Thus, we also determined the redox status of the HP0377 in H. pylori lacking HP0231 or DsbI (HP0595). Both proteins are active in the Dsb oxidative pathway. As described earlier, HP0231 introduces disulfide bonds and DsbI is partially responsible for HP0231 re-oxidation [31]. Our results showed that a significant portion of HP0377 is present in the oxidized form in both the hp0231 and dsbI mutated cells (Fig. 1). Also the overproduction of HP0231 or DsbI from a moderate copy number plasmid disturbs the redox homeostasis and results in the presence of HP0377 in both reduced and oxidized forms (Fig. 1). To clarify the link between HP0231 and cytochrome c biogenesis, we decided to check whether apocytochrome c is a substrate of HP0231. We found that HP0231 was able to oxidize the reduced apocytochrome c in vitro (Fig. 2)Fig. 1


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)

Redox state of HP0377 in wt and mutants: hp0231::cat, dsbI::aph and complemented strains of H. pylori strain N6. Bacterial cultures were treated with 10 % TCA, followed by alkylation with AMS. Cellular proteins including the reduced (red; DTT treated, modified with AMS) and the oxidized (ox; non-modified with AMS) controls were separated by 14 % SDS-PAGE under non-reducing conditions, and Western blot analysis antibodies against HP0377 was performed. Each lane contains proteins isolated from the same amount of bacteria
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig1: Redox state of HP0377 in wt and mutants: hp0231::cat, dsbI::aph and complemented strains of H. pylori strain N6. Bacterial cultures were treated with 10 % TCA, followed by alkylation with AMS. Cellular proteins including the reduced (red; DTT treated, modified with AMS) and the oxidized (ox; non-modified with AMS) controls were separated by 14 % SDS-PAGE under non-reducing conditions, and Western blot analysis antibodies against HP0377 was performed. Each lane contains proteins isolated from the same amount of bacteria
Mentions: Thus, we also determined the redox status of the HP0377 in H. pylori lacking HP0231 or DsbI (HP0595). Both proteins are active in the Dsb oxidative pathway. As described earlier, HP0231 introduces disulfide bonds and DsbI is partially responsible for HP0231 re-oxidation [31]. Our results showed that a significant portion of HP0377 is present in the oxidized form in both the hp0231 and dsbI mutated cells (Fig. 1). Also the overproduction of HP0231 or DsbI from a moderate copy number plasmid disturbs the redox homeostasis and results in the presence of HP0377 in both reduced and oxidized forms (Fig. 1). To clarify the link between HP0231 and cytochrome c biogenesis, we decided to check whether apocytochrome c is a substrate of HP0231. We found that HP0231 was able to oxidize the reduced apocytochrome c in vitro (Fig. 2)Fig. 1

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