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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

Models representing the role of H. pylori HP0377 in cytochrome c biogenesis and in oxidized protein folding. Following synthesis, apocytochrome c is translocated across the membrane by the Sec protein complex, and there it is oxidized by HP0231 (periplasmic dimeric oxidoreductase). Subsequently, its CXXCH motif is reduced by HP0377, and it is ligated to heme. Likely, as observed for other microorganisms, some molecules of the apocytochrome c are not oxidized and are directly ligated with heme (HP0231-independent pathway). Lack of HP0231 prevents HP0377 reduction by HP0265. A small portion of HP0377 is present in a dimeric form and potentially plays a role in the Dsb isomerization/reduction pathway. So far, the substrates of this pathway remains unknown
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Fig11: Models representing the role of H. pylori HP0377 in cytochrome c biogenesis and in oxidized protein folding. Following synthesis, apocytochrome c is translocated across the membrane by the Sec protein complex, and there it is oxidized by HP0231 (periplasmic dimeric oxidoreductase). Subsequently, its CXXCH motif is reduced by HP0377, and it is ligated to heme. Likely, as observed for other microorganisms, some molecules of the apocytochrome c are not oxidized and are directly ligated with heme (HP0231-independent pathway). Lack of HP0231 prevents HP0377 reduction by HP0265. A small portion of HP0377 is present in a dimeric form and potentially plays a role in the Dsb isomerization/reduction pathway. So far, the substrates of this pathway remains unknown

Mentions: HP0377 is a thioredoxin-fold protein containing the CSYC motif, which indicates that it functions as a disulfide oxidoreductase. Although there is no evidence that HP0377 is involved in cytochrome c assembly in vivo, that is the likely case because its resolved structure is similar to that of other CcmG proteins, and because it is able to reduce the oxidized form of apocytochrome c in vitro [23]. Additionally, it is co-transcribed with the ccsBA (hp0378) gene that is involved in heme transport and its ligation to apocytochrome c [20, 21]. In this work we have showed that HP0377 is present in vivo in the reduced form, which is a characteristic feature of thiol oxidoreductases being reductants. The HP0377 redox state in vivo is consistent with its redox potential determined by us (-171 mV) and by others (-180 mV), which classifies HP0377 as a mild reductant. This value is in good accord with the standard redox potential determined for the most gram-negative CcmG proteins, such as EcCcmG (-178 mV), BjCcmG (-217 mV) or PaCcmG (-213 mV) [51–53]. Additionally, we have shown that HP0377, like other CcmGs, does not reduce insulin. This finding is consistent with a generally accepted view that CcmG proteins are specific thiol-oxidoreductases involved in only the cytochrome c maturation pathway. So far, the only known exception to this rule is TlpA from B. japonicum. This protein is a reductant for the copper metallochapherone Scol, but it also acts in the cytochrome maturation process and catalyzes the insulin reduction [54]. The TlpA structure, when compared to other CcmGs, revealed some unusual properties that potentially substantiate its activity in the insulin reduction assay [55, 56]. Although HP0377 is inactive in the insulin reduction assay, it confers a disulfide isomerase activity almost as high as that of EcDsbC. This atypical HP0377 attribute is in accord with its capability to generate a dimeric form, as shown by size exclusion and glutaradehyde crosslinking methods. It should be noted, in contrast to our data, that Yoon et al. reported that HP0377 is monomeric. As we and Yoon et al. used almost identical recombinant HP0377 proteins, the inconsistency may result from various experimental methods employed [22]. Furthermore, the pKa of the N-terminal cysteine of the CXXC motif of HP0377 appears to be similar to those observed for EcDsbC or EcDsbA but not to those determined for most CcmGs. This observation suggests that the activity of HP0377 in H. pylori, which possesses only two proteins having a TRX fold with the CXXC motif (i.e., HP0231 and HP0377), is distinct from that described for the classical CcmGs. Taking into account that there is no classical DsbC protein in the H. pylori proteome and the dimeric HP0231 does not catalyze the recovery of active RNase from scrambled RNase (unpublished data), we concluded that HP0377, in contrast to most CcmGs that are involved in only the cytochrome c biogenesis process, is at least a bifunctional reductase. Figure 11 presents a model of HP0377 functioning. As the gel filtration experiment indicated that HP0377 exists as a mixture of monomeric and dimeric forms, its dual function may be regulated by the mutual ratio between the two forms. This suggestion is also supported by our interesting finding that HP0377 is essential for cell viability. Additionally, the in vivo experiments provide some intriguing data. We have noticed that the in vivo redox state of HP0377 is conditioned by the presence of HP0231 or HP0595. Previous experiments on the function of HP0231 showed that it is a periplasmic oxidase, and that HP0595 is partially responsible for HP0231 reoxidation. Therefore, they create a redox pair playing a role in introduction of disulfide bonds [31]. As H. pylori does not encode a classical DsbA, HP0231 may oxidize apocytochrome c just after its transport across the inner membrane. By analogy to the function of B. subtilis ResA, we suggest that lack of oxidized apocytochrome c in hp0231 mutated cells prevents HP0377 from reacting with its redox partner (i.e., HP0265), so a part of HP0377 remains in the oxidized form [57]. However this hypothesis requires verification because HP0377, as indicated by biochemical characterization, may also play a role in processes other than the cytochrome c maturation process. Overproduction of the periplasmic thiol oxidases, HP0231 and HP0595, results in changes to the mutual ratio of the reduced and oxidized forms of HP0377. As in the hp0231 mutated cells, only part of HP0377 is present in a reduced form. Since the presence of this form is necessary for HP0377 to fulfill its function, we decided to compare the cytochrome c activity in hp0231 mutated cells to that observed in wt cells, using the TMPD-oxidation assay. TMPD (N,N,N′,N′-Tetramethyl-p-phenylenediamine) is an artificial substrate of cytochrome c oxidase [58]. We did not observe any differences in bacterial cell pigmentation (data not shown), indicating that when the main periplasmic oxidase, i.e., HP0231, is absent, full HP0377 activity is probably dispensable for cytochrome c biogenesis.Fig. 11


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)

Models representing the role of H. pylori HP0377 in cytochrome c biogenesis and in oxidized protein folding. Following synthesis, apocytochrome c is translocated across the membrane by the Sec protein complex, and there it is oxidized by HP0231 (periplasmic dimeric oxidoreductase). Subsequently, its CXXCH motif is reduced by HP0377, and it is ligated to heme. Likely, as observed for other microorganisms, some molecules of the apocytochrome c are not oxidized and are directly ligated with heme (HP0231-independent pathway). Lack of HP0231 prevents HP0377 reduction by HP0265. A small portion of HP0377 is present in a dimeric form and potentially plays a role in the Dsb isomerization/reduction pathway. So far, the substrates of this pathway remains unknown
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig11: Models representing the role of H. pylori HP0377 in cytochrome c biogenesis and in oxidized protein folding. Following synthesis, apocytochrome c is translocated across the membrane by the Sec protein complex, and there it is oxidized by HP0231 (periplasmic dimeric oxidoreductase). Subsequently, its CXXCH motif is reduced by HP0377, and it is ligated to heme. Likely, as observed for other microorganisms, some molecules of the apocytochrome c are not oxidized and are directly ligated with heme (HP0231-independent pathway). Lack of HP0231 prevents HP0377 reduction by HP0265. A small portion of HP0377 is present in a dimeric form and potentially plays a role in the Dsb isomerization/reduction pathway. So far, the substrates of this pathway remains unknown
Mentions: HP0377 is a thioredoxin-fold protein containing the CSYC motif, which indicates that it functions as a disulfide oxidoreductase. Although there is no evidence that HP0377 is involved in cytochrome c assembly in vivo, that is the likely case because its resolved structure is similar to that of other CcmG proteins, and because it is able to reduce the oxidized form of apocytochrome c in vitro [23]. Additionally, it is co-transcribed with the ccsBA (hp0378) gene that is involved in heme transport and its ligation to apocytochrome c [20, 21]. In this work we have showed that HP0377 is present in vivo in the reduced form, which is a characteristic feature of thiol oxidoreductases being reductants. The HP0377 redox state in vivo is consistent with its redox potential determined by us (-171 mV) and by others (-180 mV), which classifies HP0377 as a mild reductant. This value is in good accord with the standard redox potential determined for the most gram-negative CcmG proteins, such as EcCcmG (-178 mV), BjCcmG (-217 mV) or PaCcmG (-213 mV) [51–53]. Additionally, we have shown that HP0377, like other CcmGs, does not reduce insulin. This finding is consistent with a generally accepted view that CcmG proteins are specific thiol-oxidoreductases involved in only the cytochrome c maturation pathway. So far, the only known exception to this rule is TlpA from B. japonicum. This protein is a reductant for the copper metallochapherone Scol, but it also acts in the cytochrome maturation process and catalyzes the insulin reduction [54]. The TlpA structure, when compared to other CcmGs, revealed some unusual properties that potentially substantiate its activity in the insulin reduction assay [55, 56]. Although HP0377 is inactive in the insulin reduction assay, it confers a disulfide isomerase activity almost as high as that of EcDsbC. This atypical HP0377 attribute is in accord with its capability to generate a dimeric form, as shown by size exclusion and glutaradehyde crosslinking methods. It should be noted, in contrast to our data, that Yoon et al. reported that HP0377 is monomeric. As we and Yoon et al. used almost identical recombinant HP0377 proteins, the inconsistency may result from various experimental methods employed [22]. Furthermore, the pKa of the N-terminal cysteine of the CXXC motif of HP0377 appears to be similar to those observed for EcDsbC or EcDsbA but not to those determined for most CcmGs. This observation suggests that the activity of HP0377 in H. pylori, which possesses only two proteins having a TRX fold with the CXXC motif (i.e., HP0231 and HP0377), is distinct from that described for the classical CcmGs. Taking into account that there is no classical DsbC protein in the H. pylori proteome and the dimeric HP0231 does not catalyze the recovery of active RNase from scrambled RNase (unpublished data), we concluded that HP0377, in contrast to most CcmGs that are involved in only the cytochrome c biogenesis process, is at least a bifunctional reductase. Figure 11 presents a model of HP0377 functioning. As the gel filtration experiment indicated that HP0377 exists as a mixture of monomeric and dimeric forms, its dual function may be regulated by the mutual ratio between the two forms. This suggestion is also supported by our interesting finding that HP0377 is essential for cell viability. Additionally, the in vivo experiments provide some intriguing data. We have noticed that the in vivo redox state of HP0377 is conditioned by the presence of HP0231 or HP0595. Previous experiments on the function of HP0231 showed that it is a periplasmic oxidase, and that HP0595 is partially responsible for HP0231 reoxidation. Therefore, they create a redox pair playing a role in introduction of disulfide bonds [31]. As H. pylori does not encode a classical DsbA, HP0231 may oxidize apocytochrome c just after its transport across the inner membrane. By analogy to the function of B. subtilis ResA, we suggest that lack of oxidized apocytochrome c in hp0231 mutated cells prevents HP0377 from reacting with its redox partner (i.e., HP0265), so a part of HP0377 remains in the oxidized form [57]. However this hypothesis requires verification because HP0377, as indicated by biochemical characterization, may also play a role in processes other than the cytochrome c maturation process. Overproduction of the periplasmic thiol oxidases, HP0231 and HP0595, results in changes to the mutual ratio of the reduced and oxidized forms of HP0377. As in the hp0231 mutated cells, only part of HP0377 is present in a reduced form. Since the presence of this form is necessary for HP0377 to fulfill its function, we decided to compare the cytochrome c activity in hp0231 mutated cells to that observed in wt cells, using the TMPD-oxidation assay. TMPD (N,N,N′,N′-Tetramethyl-p-phenylenediamine) is an artificial substrate of cytochrome c oxidase [58]. We did not observe any differences in bacterial cell pigmentation (data not shown), indicating that when the main periplasmic oxidase, i.e., HP0231, is absent, full HP0377 activity is probably dispensable for cytochrome c biogenesis.Fig. 11

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