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Redox biology of Mycobacterium tuberculosis H37Rv: protein-protein interaction between GlgB and WhiB1 involves exchange of thiol-disulfide

BACKGROUND: Mycobacterium tuberculosis, an intracellular pathogen encounters redox stress throughout its life inside the host. In order to protect itself from the redox onslaughts of host immune system, M. tuberculosis appears to have developed accessory thioredoxin-like proteins which are represent...

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Autores principales: Garg, Saurabh, Alam, Md Suhail, Bajpai, Richa, Kishan, KV Radha, Agrawal, Pushpa
Formato: Texto
Lenguaje:English
Publicado: BioMed Central 2009
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2631452/
https://www.ncbi.nlm.nih.gov/pubmed/19121228
http://dx.doi.org/10.1186/1471-2091-10-1
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author Garg, Saurabh
Alam, Md Suhail
Bajpai, Richa
Kishan, KV Radha
Agrawal, Pushpa
author_facet Garg, Saurabh
Alam, Md Suhail
Bajpai, Richa
Kishan, KV Radha
Agrawal, Pushpa
author_sort Garg, Saurabh
collection PubMed
description BACKGROUND: Mycobacterium tuberculosis, an intracellular pathogen encounters redox stress throughout its life inside the host. In order to protect itself from the redox onslaughts of host immune system, M. tuberculosis appears to have developed accessory thioredoxin-like proteins which are represented by ORFs encoding WhiB-like proteins. We have earlier reported that WhiB1/Rv3219 is a thioredoxin like protein of M. tuberculosis and functions as a protein disulfide reductase. Generally thioredoxins have many substrate proteins. The current study aims to identify the substrate protein(s) of M. tuberculosis WhiB1. RESULTS: Using yeast two-hybrid screen, we identified alpha (1,4)-glucan branching enzyme (GlgB) of M. tuberculosis as a interaction partner of WhiB1. In vitro GST pull down assay confirmed the direct physical interaction between GlgB and WhiB1. Both mass spectrometry data of tryptic digests and in vitro labeling of cysteine residues with 4-acetamido-4' maleimidyl-stilbene-2, 2'-disulfonic acid showed that in GlgB, C(95 )and C(658 )are free but C(193 )and C(617 )form an intra-molecular disulfide bond. WhiB1 has a C(37)XXC(40 )motif thus a C(40)S mutation renders C(37 )to exist as a free thiol to form a hetero-disulfide bond with the cysteine residue of substrate protein. A disulfide mediated binary complex formation between GlgB and WhiB1C(40)S was shown by both in-solution protein-protein interaction and thioredoxin affinity chromatography. Finally, transfer of reducing equivalent from WhiB1 to GlgB disulfide was confirmed by 4-acetamido-4' maleimidyl-stilbene-2, 2'-disulfonic acid trapping by the reduced disulfide of GlgB. Two different thioredoxins, TrxB/Rv1471 and TrxC/Rv3914 of M. tuberculosis could not perform this reaction suggesting that the reduction of GlgB by WhiB1 is specific. CONCLUSION: We conclude that M. tuberculosis GlgB has one intra-molecular disulfide bond which is formed between C(193 )and C(617). WhiB1, a thioredoxin like protein interacts with GlgB and transfers its electrons to the disulfide thus reduces the intra-molecular disulfide bond of GlgB. For the first time, we report that GlgB is one of the in vivo substrate of M. tuberculosis WhiB1.
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spelling pubmed-26314522009-01-28 Redox biology of Mycobacterium tuberculosis H37Rv: protein-protein interaction between GlgB and WhiB1 involves exchange of thiol-disulfide Garg, Saurabh Alam, Md Suhail Bajpai, Richa Kishan, KV Radha Agrawal, Pushpa BMC Biochem Research Article BACKGROUND: Mycobacterium tuberculosis, an intracellular pathogen encounters redox stress throughout its life inside the host. In order to protect itself from the redox onslaughts of host immune system, M. tuberculosis appears to have developed accessory thioredoxin-like proteins which are represented by ORFs encoding WhiB-like proteins. We have earlier reported that WhiB1/Rv3219 is a thioredoxin like protein of M. tuberculosis and functions as a protein disulfide reductase. Generally thioredoxins have many substrate proteins. The current study aims to identify the substrate protein(s) of M. tuberculosis WhiB1. RESULTS: Using yeast two-hybrid screen, we identified alpha (1,4)-glucan branching enzyme (GlgB) of M. tuberculosis as a interaction partner of WhiB1. In vitro GST pull down assay confirmed the direct physical interaction between GlgB and WhiB1. Both mass spectrometry data of tryptic digests and in vitro labeling of cysteine residues with 4-acetamido-4' maleimidyl-stilbene-2, 2'-disulfonic acid showed that in GlgB, C(95 )and C(658 )are free but C(193 )and C(617 )form an intra-molecular disulfide bond. WhiB1 has a C(37)XXC(40 )motif thus a C(40)S mutation renders C(37 )to exist as a free thiol to form a hetero-disulfide bond with the cysteine residue of substrate protein. A disulfide mediated binary complex formation between GlgB and WhiB1C(40)S was shown by both in-solution protein-protein interaction and thioredoxin affinity chromatography. Finally, transfer of reducing equivalent from WhiB1 to GlgB disulfide was confirmed by 4-acetamido-4' maleimidyl-stilbene-2, 2'-disulfonic acid trapping by the reduced disulfide of GlgB. Two different thioredoxins, TrxB/Rv1471 and TrxC/Rv3914 of M. tuberculosis could not perform this reaction suggesting that the reduction of GlgB by WhiB1 is specific. CONCLUSION: We conclude that M. tuberculosis GlgB has one intra-molecular disulfide bond which is formed between C(193 )and C(617). WhiB1, a thioredoxin like protein interacts with GlgB and transfers its electrons to the disulfide thus reduces the intra-molecular disulfide bond of GlgB. For the first time, we report that GlgB is one of the in vivo substrate of M. tuberculosis WhiB1. BioMed Central 2009-01-05 /pmc/articles/PMC2631452/ /pubmed/19121228 http://dx.doi.org/10.1186/1471-2091-10-1 Text en Copyright © 2009 Garg et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( (http://creativecommons.org/licenses/by/2.0) ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Garg, Saurabh
Alam, Md Suhail
Bajpai, Richa
Kishan, KV Radha
Agrawal, Pushpa
Redox biology of Mycobacterium tuberculosis H37Rv: protein-protein interaction between GlgB and WhiB1 involves exchange of thiol-disulfide
title Redox biology of Mycobacterium tuberculosis H37Rv: protein-protein interaction between GlgB and WhiB1 involves exchange of thiol-disulfide
title_full Redox biology of Mycobacterium tuberculosis H37Rv: protein-protein interaction between GlgB and WhiB1 involves exchange of thiol-disulfide
title_fullStr Redox biology of Mycobacterium tuberculosis H37Rv: protein-protein interaction between GlgB and WhiB1 involves exchange of thiol-disulfide
title_full_unstemmed Redox biology of Mycobacterium tuberculosis H37Rv: protein-protein interaction between GlgB and WhiB1 involves exchange of thiol-disulfide
title_short Redox biology of Mycobacterium tuberculosis H37Rv: protein-protein interaction between GlgB and WhiB1 involves exchange of thiol-disulfide
title_sort redox biology of mycobacterium tuberculosis h37rv: protein-protein interaction between glgb and whib1 involves exchange of thiol-disulfide
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2631452/
https://www.ncbi.nlm.nih.gov/pubmed/19121228
http://dx.doi.org/10.1186/1471-2091-10-1
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