The Experts below are selected from a list of 171 Experts worldwide ranked by ideXlab platform
D J Morré - One of the best experts on this subject based on the ideXlab platform.
-
Use of dipyridyl-dithio substrates to measure directly the protein disulfide-thiol interchange activity of the auxin stimulated NADH: Protein disulfide Reductase (NADH oxidase) of soybean plasma membranes
Molecular and Cellular Biochemistry, 1999Co-Authors: Mary Luz Gomez-rey, Cora Schramke, Oudam Em, Juliana Lawler, James Hobeck, D J MorréAbstract:Dipyridyl-dithio substrates were cleaved by isolated vesicles of plasma membranes prepared from etiolated hypocotyls of soybean. The cleavage was stimulated by auxins at physiological concentrations. The substrates utilized were principally 2,2′-dithiodippyrine (DTP) and 6,6′-dithiodinicotinic acid (DTNA). The DTP generated 2 moles of 2-pyridinethione whereas the 6,6′-dithiodinicotinic acid generated 2 moles of 6-nicotinylthionine. Both products absorbed at 340 nm. The auxin herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D) stimulated the activity approximately 2-fold to a maximum at about 10 μM. Concentrations of 2,4-D greater than 100 μM inhibited the activity. Indole-3-acetic acid stimulated the activity as well. The growth-inactive auxin, 2,3-dichlorophenoxyacetic acid (2,3-D), was without effect. DTNA cleavage correlated with oxidation of NADH and reduction of protein disulfide bonds reported earlier in terms of location at the external plasma membrane surface, absolute specific activity, pH dependence and auxin specificity. The dipyridyl-dithio substrates provide, for the first time, a direct measure of the disulfide-thiol interchange activity of the protein previously measured only indirectly as an auxin-dependent ability of isolated plasma membrane vesicles to restore activity to scrambled and inactive RNase.
-
A protein disulfide-thiol interchange protein with NADH: protein disulfide Reductase (NADH oxidase) activity as a molecular target for low levels of exposure to organic solvents in plant growth
Human & Experimental Toxicology, 1998Co-Authors: D J MorréAbstract:A number of solvents including ethyl, amyl, butyl, octyl and benzyl alcohols, ethylene glycol, ethyl acetate, acetone, diethyl ether, propylene oxide, rho-dioxane, benzene, xylene, chloroform and carbon tetrachloride stimulate the growth of plants or plant parts at low concentrations and inhibit at high concentrations. These same solvents, at low dilutions, stimulate the activity of a growth-related protein disulfide-thiol interchange protein (TIP) with NADH: protein disulfide Reductase (NADH oxidase) (NOX) activity with plasma membrane vesicles isolated from elongating regions cut from dark grown seedlings of soybeans. Based on these and other findings, we suggest the TIP/NOX protein to be the molecular target of the biological effects of low levels of exposure (hormesis) involved in the stimulation of plant growth.
Arne Holmgren - One of the best experts on this subject based on the ideXlab platform.
-
Activity assays of mammalian thioredoxin and thioredoxin Reductase: Fluorescent disulfide substrates, mechanisms, and use with tissue samples
Analytical Biochemistry, 2013Co-Authors: Sergio J. Montano, Jun Lu, Tomas N. Gustafsson, Arne HolmgrenAbstract:Abstract Thioredoxin (Trx) is a protein disulfide Reductase that, together with nicotinamide adenine dinucleotide phosphate (NADPH) and thioredoxin Reductase (TrxR), controls oxidative stress or redox signaling via thiol redox control. Human cytosolic Trx1 has Cys32 and Cys35 as the active site and three additional cysteine residues (Cys62, Cys69, and Cys73), which by oxidation generates inactive Cys62 to Cys69 two-disulfide Trx. This, combined with TrxR with a broad substrate specificity, complicates assays of mammalian Trx and TrxR. We sought to understand the autoregulation of Trx and TrxR and to generate new methods for quantification of Trx and TrxR. We optimized the synthesis of two fluorescent substrates, di-eosin–glutathione disulfide (Di-E–GSSG) and fluorescein isothiocyanate-labeled insulin (FiTC–insulin), which displayed higher fluorescence on disulfide reduction. Di-E–GSSG showed a very large increase in fluorescence quantum yield but had a relatively low affinity for Trx and was also a weak direct substrate for TrxR, in contrast to GSSG. FiTC–insulin was used to develop highly sensitive assays for TrxR and Trx. Reproducible conditions were developed for reactivation of modified Trx, commonly present in frozen or oxidized samples. Trx in cell extracts and tissue samples, including plasma and serum, were subsequently analyzed, showing highly reproducible results and allowing measurement of trace amounts of Trx.
-
Thioredoxin 1 is inactivated due to oxidation induced by peroxiredoxin under oxidative stress and reactivated by the glutaredoxin system.
Journal of Biological Chemistry, 2013Co-Authors: Yatao Du, Huihui Zhang, Xu Zhang, Jun Lu, Arne HolmgrenAbstract:The mammalian cytosolic thioredoxin system, comprising thioredoxin (Trx), Trx Reductase, and NADPH, is the major Protein-Disulfide Reductase of the cell and has numerous functions. Besides the active site thiols, human Trx1 contains three non-active site cysteine residues at positions 62, 69, and 73. A two-disulfide form of Trx1, containing an active site disulfide between Cys-32 and Cys-35 and a non-active site disulfide between Cys-62 and Cys-69, is inactive either as a disulfide Reductase or as a substrate for Trx Reductase. This could possibly provide a structural switch affecting Trx1 function during oxidative stress and redox signaling. We found that two-disulfide Trx1 was generated in A549 cells under oxidative stress. In vitro data showed that two-disulfide Trx1 was generated from oxidation of Trx1 catalyzed by peroxiredoxin 1 in the presence of H2O2. The redox Western blot data indicated that the glutaredoxin system protected Trx1 in HeLa cells from oxidation caused by ebselen, a superfast oxidant for Trx1. Our results also showed that physiological concentrations of glutathione, NADPH, and glutathione Reductase reduced the non-active site disulfide in vitro. This reaction was stimulated by glutaredoxin 1 via the so-called monothiol mechanism. In conclusion, reversible oxidation of the non-active site disulfide of Trx1 is suggested to play an important role in redox regulation and cell signaling via temporal inhibition of its Protein-Disulfide Reductase activity for the transmission of oxidative signals under oxidative stress.
-
In vivo redox state of Human thioredoxin and redox shift by the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA)
Free Radical Biology and Medicine, 2012Co-Authors: Johanna Ungerstedt, Huihui Zhang, Yatao Du, Deepika Nair, Arne HolmgrenAbstract:Abstract The cytosolic thioredoxin (Trx1) system is essential for maintaining a reduced intracellular environment, via reduced Trx1 acting as a general protein disulfide Reductase. Trx1 is implicated in cell signaling such as proliferation, DNA synthesis, enzyme activation, cell cycle regulation, transcription, gene activation, and prevention of apoptosis. Human Trx1 contains the active-site cysteines, Cys32 and Cys35, and three additional structural cysteines, Cys62, Cys69, and Cys73, that regulate Trx1 structure and activity via a second disulfide formation, S-glutathionylation or S-nitrosylation. The present study uses an electrophoretic redox Western blot method to analyze the oxidation state of Trx1 in vivo separating the protein-changed isoform following alkylation with iodoacetic acid in 8 M urea. Treatment with the histone deacetylase inhibitor SAHA increased Trx1 inhibitor thioredoxin interacting protein (Txnip) levels, decreased Trx1 activity, and switched the Trx1 oxidation state toward a more oxidized one, as a result of complex formation with Trx1, and increased reactive oxygen species (ROS). SAHA is currently in clinical trials for cancer treatment, and one possible mechanism for its anticancer effect is via effects on the Trx1 system. Determining the exact oxidation state of human cytosolic Trx1 may be useful in developing and evaluating cancer drugs and antioxidant agents.
-
Thioredoxin and thioredoxin Reductase: Current research with special reference to human disease
Biochemical and Biophysical Research Communications, 2010Co-Authors: Arne Holmgren, Jun LuAbstract:Abstract Thioredoxin (Trx) and thioredoxin Reductase (TrxR) plus NADPH, comprising the thioredoxin system, has a large number of functions in DNA synthesis, defense against oxidative stress and apoptosis or redox signaling with reference to many diseases. All three isoenzymes of mammalian TrxR contain an essential selenocysteine residue, which is the target of several drugs in cancer treatment or mercury intoxication. The cytosolic Trx1 acting as the cells’ protein disulfide Reductase is itself reversibly redox regulated via three structural Cys residues. The evolution of mammalian Trx system compared to its prokaryotic counterparts may be an adaptation to the use of hydrogen peroxide and nitric oxide in redox regulation and signal transduction.
-
Regulation of the catalytic activity and structure of human thioredoxin 1 via oxidation and S-nitrosylation of cysteine residues.
Journal of Biological Chemistry, 2008Co-Authors: Seyed Isaac Hashemy, Arne HolmgrenAbstract:Abstract The mammalian cytosolic/nuclear thioredoxin system, comprising thioredoxin (Trx), selenoenzyme thioredoxin Reductase (TrxR), and NADPH, is the major Protein-Disulfide Reductase of the cell and has numerous functions. The active site of reduced Trx comprises Cys32-Gly-Pro-Cys35 thiols that catalyze target disulfide reduction, generating a disulfide. Human Trx1 has also three structural Cys residues in positions 62, 69, and 73 that upon diamide oxidation induce a second Cys62–Cys69 disulfide as well as dimers and multimers. We have discovered that after incubation with H2O2 only monomeric two-disulfide molecules are generated, and they are inactive but able to regain full activity in an autocatalytic process in the presence of NADPH and TrxR. There are conflicting results regarding the effects of S-nitrosylation on Trx antioxidant functions and which residues are involved. We found that S-nitrosoglutathione-mediated S-nitrosylation at physiological pH is critically dependent on the redox state of Trx. Starting from fully reduced human Trx, both Cys69 and Cys73 were nitrosylated, and the active site formed a disulfide; the nitrosylated Trx was not a substrate for TrxR but regained activity after a lag phase consistent with autoactivation. Treatment of a two-disulfide form of Trx1 with S-nitrosoglutathione resulted in nitrosylation of Cys73, which can act as a trans-nitrosylating agent as observed by others to control caspase 3 activity (Mitchell, D. A., and Marletta, M. A. (2005) Nat. Chem. Biol. 1, 154–158). The reversible inhibition of human Trx1 activity by H2O2 and NO donors is suggested to act in cell signaling via temporal control of reduction for the transmission of oxidative and/or nitrosative signals in thiol redox control.
Atsushi Sakamoto - One of the best experts on this subject based on the ideXlab platform.
-
Overexpression of the protein disulfide isomerase AtCYO1 in chloroplasts slows dark-induced senescence in Arabidopsis
BMC Plant Biology, 2018Co-Authors: Jun Tominaga, Yasutoshi Nakahara, Daisuke Horikawa, Ayumi Tanaka, Maki Kondo, Yasuhiro Kamei, Tsuneaki Takami, Wataru Sakamoto, Kazutoshi Unno, Atsushi SakamotoAbstract:Background Chlorophyll breakdown is the most obvious sign of leaf senescence. The chlorophyll catabolism pathway and the associated proteins/genes have been identified in considerable detail by genetic approaches combined with stay-green phenotyping. Arabidopsis CYO1 (AtCYO1), a protein disulfide Reductase/isomerase localized in the thylakoid membrane, is hypothesized to assemble the photosystem by interacting with cysteine residues of the subunits. Results In this study, we report that ectopic overexpression of AtCYO1 in leaves induces a stay-green phenotype during darkness, where oxidative conditions favor catabolism. In AtCYO1ox leaves, Fv/Fm and both chlorophyll a and chlorophyll b content remained high during dark-induced senescence. The thylakoid ultrastructure was preserved for a longer time in AtCYO1ox leaves than in wild type leaves. AtCYO1ox leaves maintained thylakoid chlorophyll-binding proteins associated with both PSII (D1, D2, CP43, CP47, LHCB2, and Cyt f ) and PSI (PSA-A/B), as well as stromal proteins (Rubisco and ferredoxin-NADP+ Reductase). AtCYO1ox did not affect senescence-inducible gene expression for chlorophyll catabolism or accumulation of chlorophyll catabolites. Conclusions Our results suggest that ectopic overexpression of AtCYO1 had a negative impact on the initiation of chlorophyll degradation and proteolysis within chloroplasts. Our findings cast new light on the redox regulation of protein disulfide bonds for the maintenance of functional chloroplasts.
-
Overexpression of the protein disulfide isomerase AtCYO1 in chloroplasts slows dark-induced senescence in Arabidopsis
BMC Plant Biology, 2018Co-Authors: Jun Tominaga, Yasutoshi Nakahara, Daisuke Horikawa, Ayumi Tanaka, Maki Kondo, Yasuhiro Kamei, Tsuneaki Takami, Wataru Sakamoto, Kazutoshi Unno, Atsushi SakamotoAbstract:Chlorophyll breakdown is the most obvious sign of leaf senescence. The chlorophyll catabolism pathway and the associated proteins/genes have been identified in considerable detail by genetic approaches combined with stay-green phenotyping. Arabidopsis CYO1 (AtCYO1), a protein disulfide Reductase/isomerase localized in the thylakoid membrane, is hypothesized to assemble the photosystem by interacting with cysteine residues of the subunits. In this study, we report that ectopic overexpression of AtCYO1 in leaves induces a stay-green phenotype during darkness, where oxidative conditions favor catabolism. In AtCYO1ox leaves, Fv/Fm and both chlorophyll a and chlorophyll b content remained high during dark-induced senescence. The thylakoid ultrastructure was preserved for a longer time in AtCYO1ox leaves than in wild type leaves. AtCYO1ox leaves maintained thylakoid chlorophyll-binding proteins associated with both PSII (D1, D2, CP43, CP47, LHCB2, and Cyt f) and PSI (PSA-A/B), as well as stromal proteins (Rubisco and ferredoxin-NADP+ Reductase). AtCYO1ox did not affect senescence-inducible gene expression for chlorophyll catabolism or accumulation of chlorophyll catabolites. Our results suggest that ectopic overexpression of AtCYO1 had a negative impact on the initiation of chlorophyll degradation and proteolysis within chloroplasts. Our findings cast new light on the redox regulation of protein disulfide bonds for the maintenance of functional chloroplasts.
-
Rice CYO1, an ortholog of Arabidopsis thaliana cotyledon chloroplast biogenesis factor AtCYO1, is expressed in leaves and involved in photosynthetic performance
Journal of Plant Physiology, 2016Co-Authors: Jun Tominaga, Yasutoshi Nakahara, Daisuke Horikawa, Tsuneaki Takami, Wataru Sakamoto, Atsushi Sakamoto, Haruka Mizutani, Hiroshi ShimadaAbstract:Abstract In the dicotyledonous plant Arabidopsis thaliana , the cotyledon chloroplast biogenesis factor AtCYO1 is crucial for the biogenesis of cotyledon chloroplasts. Arabidopsis mutants lacking AtCYO1 have pale cotyledons but develop normal mature leaves. In the monocotyledonous plant Oryza sativa , the gene OsCYO1 has high sequence identity to AtCYO1 , but its function is unknown. We examined the role of OsCYO1 in O. sativa . We first confirmed that transformation with OsCYO1 could recover the phenotype of the Arabidopsis cyo1 mutant. Similar to AtCYO1, recombinant OsCYO1 has protein disulfide Reductase (PDR) activity, which increased as a function of dieosin glutathione disulfide concentration with an apparent K m of 3.2 μM and K cat of 0.53 min −1 . The PDR activity was reduced when NADPH or NADH was used as an electron donor; however, PDR activity was observed with OsCYO1 and glutathione, suggesting that glutathione may serve as a reducing agent for OsCYO1 in vivo . In O. sativa , the OsCYO1 transcript level was higher in leaves compared with the coleoptile, which is the first leaf-like organ that forms during rice embryogenesis. Many OsCYO1 mutant lines defective in RNA interference had green leaves, however, three mutant lines had not only albino coleoptile but also albino leaves. Those having green leaves reduced photosynthetic performance in leaves. Our results demonstrate that OsCYO1 is enzymatically equivalent to AtCYO1 but that the physiological role of OsCYO1 in monocotyledonous plants may differ from that of AtCYO1 in dicotyledonous plants.
Jun Tominaga - One of the best experts on this subject based on the ideXlab platform.
-
Overexpression of the protein disulfide isomerase AtCYO1 in chloroplasts slows dark-induced senescence in Arabidopsis
BMC Plant Biology, 2018Co-Authors: Jun Tominaga, Yasutoshi Nakahara, Daisuke Horikawa, Ayumi Tanaka, Maki Kondo, Yasuhiro Kamei, Tsuneaki Takami, Wataru Sakamoto, Kazutoshi Unno, Atsushi SakamotoAbstract:Background Chlorophyll breakdown is the most obvious sign of leaf senescence. The chlorophyll catabolism pathway and the associated proteins/genes have been identified in considerable detail by genetic approaches combined with stay-green phenotyping. Arabidopsis CYO1 (AtCYO1), a protein disulfide Reductase/isomerase localized in the thylakoid membrane, is hypothesized to assemble the photosystem by interacting with cysteine residues of the subunits. Results In this study, we report that ectopic overexpression of AtCYO1 in leaves induces a stay-green phenotype during darkness, where oxidative conditions favor catabolism. In AtCYO1ox leaves, Fv/Fm and both chlorophyll a and chlorophyll b content remained high during dark-induced senescence. The thylakoid ultrastructure was preserved for a longer time in AtCYO1ox leaves than in wild type leaves. AtCYO1ox leaves maintained thylakoid chlorophyll-binding proteins associated with both PSII (D1, D2, CP43, CP47, LHCB2, and Cyt f ) and PSI (PSA-A/B), as well as stromal proteins (Rubisco and ferredoxin-NADP+ Reductase). AtCYO1ox did not affect senescence-inducible gene expression for chlorophyll catabolism or accumulation of chlorophyll catabolites. Conclusions Our results suggest that ectopic overexpression of AtCYO1 had a negative impact on the initiation of chlorophyll degradation and proteolysis within chloroplasts. Our findings cast new light on the redox regulation of protein disulfide bonds for the maintenance of functional chloroplasts.
-
Overexpression of the protein disulfide isomerase AtCYO1 in chloroplasts slows dark-induced senescence in Arabidopsis
BMC Plant Biology, 2018Co-Authors: Jun Tominaga, Yasutoshi Nakahara, Daisuke Horikawa, Ayumi Tanaka, Maki Kondo, Yasuhiro Kamei, Tsuneaki Takami, Wataru Sakamoto, Kazutoshi Unno, Atsushi SakamotoAbstract:Chlorophyll breakdown is the most obvious sign of leaf senescence. The chlorophyll catabolism pathway and the associated proteins/genes have been identified in considerable detail by genetic approaches combined with stay-green phenotyping. Arabidopsis CYO1 (AtCYO1), a protein disulfide Reductase/isomerase localized in the thylakoid membrane, is hypothesized to assemble the photosystem by interacting with cysteine residues of the subunits. In this study, we report that ectopic overexpression of AtCYO1 in leaves induces a stay-green phenotype during darkness, where oxidative conditions favor catabolism. In AtCYO1ox leaves, Fv/Fm and both chlorophyll a and chlorophyll b content remained high during dark-induced senescence. The thylakoid ultrastructure was preserved for a longer time in AtCYO1ox leaves than in wild type leaves. AtCYO1ox leaves maintained thylakoid chlorophyll-binding proteins associated with both PSII (D1, D2, CP43, CP47, LHCB2, and Cyt f) and PSI (PSA-A/B), as well as stromal proteins (Rubisco and ferredoxin-NADP+ Reductase). AtCYO1ox did not affect senescence-inducible gene expression for chlorophyll catabolism or accumulation of chlorophyll catabolites. Our results suggest that ectopic overexpression of AtCYO1 had a negative impact on the initiation of chlorophyll degradation and proteolysis within chloroplasts. Our findings cast new light on the redox regulation of protein disulfide bonds for the maintenance of functional chloroplasts.
-
Rice CYO1, an ortholog of Arabidopsis thaliana cotyledon chloroplast biogenesis factor AtCYO1, is expressed in leaves and involved in photosynthetic performance
Journal of Plant Physiology, 2016Co-Authors: Jun Tominaga, Yasutoshi Nakahara, Daisuke Horikawa, Tsuneaki Takami, Wataru Sakamoto, Atsushi Sakamoto, Haruka Mizutani, Hiroshi ShimadaAbstract:Abstract In the dicotyledonous plant Arabidopsis thaliana , the cotyledon chloroplast biogenesis factor AtCYO1 is crucial for the biogenesis of cotyledon chloroplasts. Arabidopsis mutants lacking AtCYO1 have pale cotyledons but develop normal mature leaves. In the monocotyledonous plant Oryza sativa , the gene OsCYO1 has high sequence identity to AtCYO1 , but its function is unknown. We examined the role of OsCYO1 in O. sativa . We first confirmed that transformation with OsCYO1 could recover the phenotype of the Arabidopsis cyo1 mutant. Similar to AtCYO1, recombinant OsCYO1 has protein disulfide Reductase (PDR) activity, which increased as a function of dieosin glutathione disulfide concentration with an apparent K m of 3.2 μM and K cat of 0.53 min −1 . The PDR activity was reduced when NADPH or NADH was used as an electron donor; however, PDR activity was observed with OsCYO1 and glutathione, suggesting that glutathione may serve as a reducing agent for OsCYO1 in vivo . In O. sativa , the OsCYO1 transcript level was higher in leaves compared with the coleoptile, which is the first leaf-like organ that forms during rice embryogenesis. Many OsCYO1 mutant lines defective in RNA interference had green leaves, however, three mutant lines had not only albino coleoptile but also albino leaves. Those having green leaves reduced photosynthetic performance in leaves. Our results demonstrate that OsCYO1 is enzymatically equivalent to AtCYO1 but that the physiological role of OsCYO1 in monocotyledonous plants may differ from that of AtCYO1 in dicotyledonous plants.
Pushpa Agrawal - One of the best experts on this subject based on the ideXlab platform.
-
Redox biology of Mycobacterium tuberculosis H37Rv: protein-protein interaction between GlgB and WhiB1 involves exchange of thiol-disulfide
BMC Biochemistry, 2009Co-Authors: Saurabh K. Garg, Suhail Alam, K.v. Radha Kishan, Richa Bajpai, Pushpa AgrawalAbstract: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. 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, C95 and C658 are free but C193 and C617 form an intra-molecular disulfide bond. WhiB1 has a C37XXC40 motif thus a C40S mutation renders C37 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 WhiB1C40S 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. We conclude that M. tuberculosis GlgB has one intra-molecular disulfide bond which is formed between C193 and C617. 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.
-
Redox biology of Mycobacterium tuberculosis H37Rv: protein-protein interaction between GlgB and WhiB1 involves exchange of thiol-disulfide
BMC Biochemistry, 2009Co-Authors: Saurabh Garg, K.v. Radha Kishan, Richa Bajpai, Md Suhail Alam, Pushpa AgrawalAbstract: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^37XXC^40 motif thus a C^40S 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^40S 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.
-
Matrix-assisted refolding and redox properties of WhiB3/Rv3416 of Mycobacterium tuberculosis H37Rv
Protein Expression and Purification, 2008Co-Authors: Suhail Alam, Pushpa AgrawalAbstract:Abstract Redox stress is one of the major challenges faced by Mycobacterium tuberculosis during early infection and latency. The mechanism of sensing and adaptation to altered redox conditions is poorly understood. whiB family of Mtb is emerging as an important class of stress responsive genes. WhiB3/Rv3416 has been shown to be important for pathogenesis in animal model and was recently shown to co-ordinate a Fe–S cluster. Here, we report a simple, rapid and efficient matrix-assisted refolding method and important redox properties of WhiB3. Similar to other WhiB proteins, WhiB3 also has four conserved cysteine residues, where two of them are present in a CXXC motif. The Fe–S cluster of WhiB3 remained bound in the presence of strong protein denaturant. Upon cluster removal due to oxidation, the four cysteine residues which are ligands of Fe–S cluster, formed two intra-molecular disulfide bridges where one of them is possibly between the cysteines of CXXC motif, an important feature of several thiol-disulfide oxido-Reductases. Far-UV CD spectroscopy revealed the presence of both α-helices and β-strands in apo WhiB3. The secondary structural elements of apo WhiB3 were found resistant for thermal denaturation. The results demonstrated that apo WhiB3 functions as a protein disulfide Reductase similar to thioredoxins. The importance of WhiB3 in redox sensing and its possible role in mycobacterial physiology has been discussed.
-
Characterization of Mycobacterium tuberculosis WhiB1/Rv3219 as a protein disulfide Reductase
Protein Expression and Purification, 2006Co-Authors: Saurabh K. Garg, Suhail Alam, Vishal Soni, K.v. Radha Kishan, Pushpa AgrawalAbstract:Abstract WhiB family of protein is emerging as one of the most fascinating group and is implicated in stress response as well as pathogenesis via their involvement in diverse cellular processes. Surprisingly, available in vivo data indicate an organism specific physiological role for each of these proteins. The WhiB proteins have four conserved cysteine residues where two of them are present in a C–X–X–C motif. In thioredoxins and similar proteins, this motif works as an active site and confers thiol-disulfide oxidoReductase activity to the protein. The recombinant WhiB1/Rv3219 was purified in a single step from Escherichia coli using Ni 2+ –NTA affinity chromatography and was found to exist as a homodimer. Mass spectrometry of WhiB1 shows that the four cysteine residues form two intramolecular disulfide bonds. Using intrinsic tryptophan fluorescence as a measure of redox state, the redox potential of WhiB1 was calculated as −236 ± 2 mV, which corresponds to the redox potential of many cytoplasmic thioredoxin-like proteins. WhiB1 catalyzed the reduction of insulin disulfide thus clearly demonstrating that it functions as a protein disulfide Reductase. Present study for the first time suggests that WhiB1 may be a part of the redox network of Mycobacterium tuberculosis through its involvement in thiol-disulfide exchange with other cellular proteins.
