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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

Localization of HP0377. Panel a: Immunoblot analysis with subcellular fractions of H. pylori strain 26695 using anti-HP0377 antibody. Lanes: 1 - whole cell lysate; 2 - periplasmic proteins; 3 - protein ladder; 4 - cytoplasmic proteins, 5 - inner membrane proteins; 6 - outer membrane proteins; 7 - membranes protein. Panel b: Immunoblot analysis with subcellular fractions of H. pylori strain 26695 using anti-DsbI antibody. Lanes: 1 - whole cell lysate; 2 - periplasmic proteins; 3 - protein ladder; 4 - inner membrane proteins; 5 - outer membrane proteins; 6 - membrane proteins, 7 - cytoplasmic proteins
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Fig9: Localization of HP0377. Panel a: Immunoblot analysis with subcellular fractions of H. pylori strain 26695 using anti-HP0377 antibody. Lanes: 1 - whole cell lysate; 2 - periplasmic proteins; 3 - protein ladder; 4 - cytoplasmic proteins, 5 - inner membrane proteins; 6 - outer membrane proteins; 7 - membranes protein. Panel b: Immunoblot analysis with subcellular fractions of H. pylori strain 26695 using anti-DsbI antibody. Lanes: 1 - whole cell lysate; 2 - periplasmic proteins; 3 - protein ladder; 4 - inner membrane proteins; 5 - outer membrane proteins; 6 - membrane proteins, 7 - cytoplasmic proteins

Mentions: Most of the CcmG proteins are membrane-anchored via the N-terminal transmembrane domain. In silico analysis (HMM Expasy, TOPCON, Smart programs) did not indicate the presence of the transmembrane N-terminal domain in HP0377, though it confirmed the occurrence of this kind of domain located in the N-terminus of B. subtilis ResA. At the same time, in silico analysis using the LipoP 1.0 server showed that the HP0377 amino-acid sequence contains a putative signal sequence that can be processed by signal peptidase II, which suggested that HP0377 is an inner membrane lipoprotein. To confirm the predicted localization of HP0377 in H. pylori, a subcellular fractionation experiment was carried out. Detection of HP0377 with specific rabbit antibody against rHP0377 revealed that HP0377 is present in the inner membrane proteins compartment (Fig. 9a). As a control for the method of subcellular fractionation of H. pylori used, we traced the cellular location of DsbI (i.e., HP0595), which is an inner-membrane protein (Fig. 9b).Fig. 9


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)

Localization of HP0377. Panel a: Immunoblot analysis with subcellular fractions of H. pylori strain 26695 using anti-HP0377 antibody. Lanes: 1 - whole cell lysate; 2 - periplasmic proteins; 3 - protein ladder; 4 - cytoplasmic proteins, 5 - inner membrane proteins; 6 - outer membrane proteins; 7 - membranes protein. Panel b: Immunoblot analysis with subcellular fractions of H. pylori strain 26695 using anti-DsbI antibody. Lanes: 1 - whole cell lysate; 2 - periplasmic proteins; 3 - protein ladder; 4 - inner membrane proteins; 5 - outer membrane proteins; 6 - membrane proteins, 7 - cytoplasmic proteins
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig9: Localization of HP0377. Panel a: Immunoblot analysis with subcellular fractions of H. pylori strain 26695 using anti-HP0377 antibody. Lanes: 1 - whole cell lysate; 2 - periplasmic proteins; 3 - protein ladder; 4 - cytoplasmic proteins, 5 - inner membrane proteins; 6 - outer membrane proteins; 7 - membranes protein. Panel b: Immunoblot analysis with subcellular fractions of H. pylori strain 26695 using anti-DsbI antibody. Lanes: 1 - whole cell lysate; 2 - periplasmic proteins; 3 - protein ladder; 4 - inner membrane proteins; 5 - outer membrane proteins; 6 - membrane proteins, 7 - cytoplasmic proteins
Mentions: Most of the CcmG proteins are membrane-anchored via the N-terminal transmembrane domain. In silico analysis (HMM Expasy, TOPCON, Smart programs) did not indicate the presence of the transmembrane N-terminal domain in HP0377, though it confirmed the occurrence of this kind of domain located in the N-terminus of B. subtilis ResA. At the same time, in silico analysis using the LipoP 1.0 server showed that the HP0377 amino-acid sequence contains a putative signal sequence that can be processed by signal peptidase II, which suggested that HP0377 is an inner membrane lipoprotein. To confirm the predicted localization of HP0377 in H. pylori, a subcellular fractionation experiment was carried out. Detection of HP0377 with specific rabbit antibody against rHP0377 revealed that HP0377 is present in the inner membrane proteins compartment (Fig. 9a). As a control for the method of subcellular fractionation of H. pylori used, we traced the cellular location of DsbI (i.e., HP0595), which is an inner-membrane protein (Fig. 9b).Fig. 9

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