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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 dsbD mutant E. coli strain transformed with pUWM399 carrying hp0377. 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 using antibodies against HP0377 was performed. Each lane contains proteins isolated from the same amount of bacteria
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Fig10: Redox state of HP0377 in wt and dsbD mutant E. coli strain transformed with pUWM399 carrying hp0377. 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 using antibodies against HP0377 was performed. Each lane contains proteins isolated from the same amount of bacteria

Mentions: To clarify the lack of complementation of the EcdsbC mutation by HP0377, we determined its redox state in E. coli cells. In both the E. coli wild type and in dsbD mutated cells, HP0377 exists in an oxidized form, in contrast to what was observed in H. pylori, where it was present in a reduced form (Fig. 10). These results suggest that HP0377 does not cooperate with E. coli DsbD.Fig. 10


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 dsbD mutant E. coli strain transformed with pUWM399 carrying hp0377. 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 using 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

Fig10: Redox state of HP0377 in wt and dsbD mutant E. coli strain transformed with pUWM399 carrying hp0377. 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 using antibodies against HP0377 was performed. Each lane contains proteins isolated from the same amount of bacteria
Mentions: To clarify the lack of complementation of the EcdsbC mutation by HP0377, we determined its redox state in E. coli cells. In both the E. coli wild type and in dsbD mutated cells, HP0377 exists in an oxidized form, in contrast to what was observed in H. pylori, where it was present in a reduced form (Fig. 10). These results suggest that HP0377 does not cooperate with E. coli DsbD.Fig. 10

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