scholarly journals Thioredoxin-like2/2-Cys peroxiredoxin redox cascade supports oxidative thiol modulation in chloroplasts

2018 ◽  
Vol 115 (35) ◽  
pp. E8296-E8304 ◽  
Author(s):  
Keisuke Yoshida ◽  
Ayaka Hara ◽  
Kazunori Sugiura ◽  
Yuki Fukaya ◽  
Toru Hisabori
Keyword(s):  
Redox Regulation ◽  
High Efficiency ◽  
Thioredoxin Reductase ◽  
Reducing Power ◽  
Delayed Response ◽  
Physiological Relevance ◽  
Direct Discrimination ◽  
Chloroplast Stroma

Thiol-based redox regulation is central to adjusting chloroplast functions under varying light conditions. A redox cascade via the ferredoxin-thioredoxin reductase (FTR)/thioredoxin (Trx) pathway has been well recognized to mediate the light-responsive reductive control of target proteins; however, the molecular basis for reoxidizing its targets in the dark remains unidentified. Here, we report a mechanism of oxidative thiol modulation in chloroplasts. We biochemically characterized a chloroplast stroma-localized atypical Trx from Arabidopsis, designated as Trx-like2 (TrxL2). TrxL2 had redox-active properties with an unusually less negative redox potential. By an affinity chromatography-based method, TrxL2 was shown to interact with a range of chloroplast redox-regulated proteins. The direct discrimination of thiol status indicated that TrxL2 can efficiently oxidize, but not reduce, these proteins. A notable exception was found in 2-Cys peroxiredoxin (2CP); TrxL2 was able to reduce 2CP with high efficiency. We achieved a complete in vitro reconstitution of the TrxL2/2CP redox cascade for oxidizing redox-regulated proteins and draining reducing power to hydrogen peroxide (H2O2). We further addressed the physiological relevance of this system by analyzing protein-oxidation dynamics. In Arabidopsis plants, a decreased level of 2CP led to the impairment of the reoxidation of redox-regulated proteins during light–dark transitions. A delayed response of protein reoxidation was concomitant with the prolonged accumulation of reducing power in TrxL2. These results suggest an in vivo function of the TrxL2/2CP redox cascade for driving oxidative thiol modulation in chloroplasts.

Distinct electron transfer from ferredoxin–thioredoxin reductase to multiple thioredoxin isoforms in chloroplasts

2017 ◽  
Vol 474 (8) ◽  
pp. 1347-1360 ◽  
Author(s):  
Keisuke Yoshida ◽  
Toru Hisabori
Keyword(s):  
Electron Transfer ◽  
Redox Regulation ◽  
Thioredoxin Reductase ◽  
Reducing Power ◽  
Redox Potentials ◽  
Regulation Network ◽  
Electron Transfers ◽  
Kinetics Of

Thiol-based redox regulation is considered to support light-responsive control of various chloroplast functions. The redox cascade via ferredoxin–thioredoxin reductase (FTR)/thioredoxin (Trx) has been recognized as a key to transmitting reducing power; however, Arabidopsis thaliana genome sequencing has revealed that as many as five Trx subtypes encoded by a total of 10 nuclear genes are targeted to chloroplasts. Because each Trx isoform seems to have a distinct target selectivity, the electron distribution from FTR to multiple Trxs is thought to be the critical branch point for determining the consequence of chloroplast redox regulation. In the present study, we aimed to comprehensively characterize the kinetics of electron transfer from FTR to 10 Trx isoforms. We prepared the recombinant FTR protein from Arabidopsis in the heterodimeric form containing the Fe–S cluster. By reconstituting the FTR/Trx system in vitro, we showed that FTR prepared here was enzymatically active and suitable for uncovering biochemical features of chloroplast redox regulation. A series of redox state determinations using the thiol-modifying reagent, 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonate, indicated that all chloroplast Trx isoforms are commonly reduced by FTR; however, significantly different efficiencies were evident. These differences were apparently correlated with the distinct midpoint redox potentials among Trxs. Even when the experiments were performed under conditions of hypothetical in vivo stoichiometry of FTR and Trxs, a similar trend in distinguishable electron transfers was observed. These data highlight an aspect of highly organized circuits in the chloroplast redox regulation network.

scholarly journals Determining the Rate-Limiting Step for Light-Responsive Redox Regulation in Chloroplasts

Antioxidants ◽  
2018 ◽  
Vol 7 (11) ◽  
pp. 153 ◽  
Author(s):  
Keisuke Yoshida ◽  
Toru Hisabori
Keyword(s):  
Redox Regulation ◽  
Reducing Power ◽  
Rubisco Activase ◽  
Power Transfer ◽  
Target Proteins ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting

Thiol-based redox regulation ensures light-responsive control of chloroplast functions. Light-derived signal is transferred in the form of reducing power from the photosynthetic electron transport chain to several redox-sensitive target proteins. Two types of protein, ferredoxin-thioredoxin reductase (FTR) and thioredoxin (Trx), are well recognized as the mediators of reducing power. However, it remains unclear which step in a series of redox-relay reactions is the critical bottleneck for determining the rate of target protein reduction. To address this, the redox behaviors of FTR, Trx, and target proteins were extensively characterized in vitro and in vivo. The FTR/Trx redox cascade was reconstituted in vitro using recombinant proteins from Arabidopsis. On the basis of this assay, we found that the FTR catalytic subunit and f-type Trx are rapidly reduced after the drive of reducing power transfer, irrespective of the presence or absence of their downstream target proteins. By contrast, three target proteins, fructose 1,6-bisphosphatase (FBPase), sedoheptulose 1,7-bisphosphatase (SBPase), and Rubisco activase (RCA) showed different reduction patterns; in particular, SBPase was reduced at a low rate. The in vivo study using Arabidopsis plants showed that the Trx family is commonly and rapidly reduced upon high light irradiation, whereas FBPase, SBPase, and RCA are differentially and slowly reduced. Both of these biochemical and physiological findings suggest that reducing power transfer from Trx to its target proteins is a rate-limiting step for chloroplast redox regulation, conferring distinct light-responsive redox behaviors on each of the targets.

scholarly journals Staphylococcus aureus NrdH Redoxin Is a Reductant of the Class Ib Ribonucleotide Reductase

2010 ◽  
Vol 192 (19) ◽  
pp. 4963-4972 ◽  
Author(s):  
Inbal Rabinovitch ◽  
Michaela Yanku ◽  
Adva Yeheskel ◽  
Gerald Cohen ◽  
Ilya Borovok ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Ribonucleotide Reductase ◽  
Thioredoxin Reductase ◽  
Reducing Power ◽  
Anaerobic Growth ◽  
Structural Motif ◽  
Thiol Redox ◽  
Class Ib

ABSTRACT Staphylococci contain a class Ib NrdEF ribonucleotide reductase (RNR) that is responsible, under aerobic conditions, for the synthesis of deoxyribonucleotide precursors for DNA synthesis and repair. The genes encoding that RNR are contained in an operon consisting of three genes, nrdIEF, whereas many other class Ib RNR operons contain a fourth gene, nrdH, that determines a thiol redoxin protein, NrdH. We identified a 77-amino-acid open reading frame in Staphylococcus aureus that resembles NrdH proteins. However, S. aureus NrdH differs significantly from the canonical NrdH both in its redox-active site, C-P-P-C instead of C-M/V-Q-C, and in the absence of the C-terminal [WF]SGFRP[DE] structural motif. We show that S. aureus NrdH is a thiol redox protein. It is not essential for aerobic or anaerobic growth and appears to have a marginal role in protection against oxidative stress. In vitro, S. aureus NrdH was found to be an efficient reductant of disulfide bonds in low-molecular-weight substrates and proteins using dithiothreitol as the source of reducing power and an effective reductant for the homologous class Ib RNR employing thioredoxin reductase and NADPH as the source of the reducing power. Its ability to reduce NrdEF is comparable to that of thioredoxin-thioredoxin reductase. Hence, S. aureus contains two alternative thiol redox proteins, NrdH and thioredoxin, with both proteins being able to function in vitro with thioredoxin reductase as the immediate hydrogen donors for the class Ib RNR. It remains to be clarified under which in vivo physiological conditions the two systems are used.

scholarly journals Mammalian thioredoxin reductase 1: roles in redox homoeostasis and characterization of cellular targets

2010 ◽  
Vol 430 (2) ◽  
pp. 285-293 ◽  
Author(s):  
Anton A. Turanov ◽  
Sebastian Kehr ◽  
Stefano M. Marino ◽  
Min-Hyuk Yoo ◽  
Bradley A. Carlson ◽  
...  
Keyword(s):  
Redox Regulation ◽  
Thioredoxin Reductase ◽  
In Vitro Assays ◽  
Mouse Serum ◽  
Main Function ◽  
Major Pathway ◽  
Thioredoxin System

The classical Trx (thioredoxin) system, composed of TR (Trx reductase), Trx and NADPH, defines a major pathway of cellular thiol-based redox regulation. Three TRs have been identified in mammals: (i) cytosolic TR1, (ii) mitochondrial TR3 and (iii) testes-specific TGR (Trx-glutathione reductase). All three are selenocysteine-containing enzymes with broad substrate specificity in in vitro assays, but which protein substrates are targeted by TRs in vivo is not well understood. In the present study, we used a mechanism-based approach to characterize the molecular targets of TR1. Cytosolic Trx1 was the major target identified in rat and mouse liver, as well as in rat brain and mouse serum. The results suggest that the main function of TR1 is to reduce Trx1. We also found that TR1-based affinity resins provide a convenient tool for specific isolation of Trxs from a variety of biological samples. To better assess the role of TRs in redox homoeostasis, we comparatively analysed TR1- and TR3-knockdown cells. Although cells deficient in TR1 were particularly sensitive to diamide, TR3-knockdown cells were more sensitive to hydrogen peroxide. To further examine the TR1–Trx1 redox pair, we used mice with a liver-specific knockout of selenocysteine tRNA. In this model, selenocysteine insertion into TR1 was blocked, but the truncated form of this protein was not detected. Instead, TR1 and TR3 levels were decreased in the knockout samples. Diminished hepatic TR1 function was associated with elevated Trx1 levels, but this protein was mostly in the oxidized state. Overall, this study provides evidence for the key role of the TR1–Trx1 pair in redox homoeostasis.

Different Forms of Fructose 1,6-Bisphosphatase in Chlorella

1993 ◽  
Vol 48 (1-2) ◽  
pp. 22-27 ◽  
Author(s):  
N. Grotjohann ◽  
G. Schneider ◽  
W. Kowallik
Keyword(s):  
Deae Cellulose ◽  
Reducing Power ◽  
Exchange Chromatography ◽  
Substrate Affinity ◽  
Kinetic Properties ◽  
Cell Extracts ◽  
Fructose 1,6 Bisphosphatase ◽  
Chloroplast Stroma

In crude extracts of Chlorella kessleri two forms of fructosebisphosphatase can be separated by ion exchange chromatography or by acid precipitation. FBPase I is eluted from DEAE cellulose at 200mᴍ KC1 and precipitated at pH 4.5, FBPase II is correspondingly eluted at 310 mᴍ KC1 and soluble at pH 4.5. Both enzymes differ in substrate affinity and degree of cooperativity. Based on literature data, FBPase I is assumed to be of cytosolic and FBPase II of chloroplastic origin. The mole mass of FBPase I is identical (51-65 kDa) at pH 6.5 and pH 8.5. T hat of FBPase II is however, about four times larger at pH 6.5 (257 kDa) than at pH 8.5 (67 kDa). Other pH values have only been tried with crude cell extracts in which still larger mole masses of FBPase resulted at more acidic pH (1349 kD a at pH 6.0). The lower mole mass form of FBPase II shows three times higher catalytic activity. Reducing agents, such as DTT, also increase the activity of FBPase II in vitro. In vivo, alkalization and production of reducing power occurs in the chloroplast stroma during illumination. If the above alterations exist in vivo, they would be a means to activate FBPase in the light. Oligomerization of FBPase II to aggregates with altered catalytic activities and kinetic properties is discussed as result of the action of specific wavelengths and to be responsible for differences in carbohydrate metabolism of Chlorella exposed to red or blue light.

scholarly journals Oxidative regulation of chloroplast enzymes by thioredoxin and thioredoxin-like proteins in Arabidopsis thaliana

2021 ◽  
Vol 118 (51) ◽  
pp. e2114952118
Author(s):  
Yuichi Yokochi ◽  
Yuka Fukushi ◽  
Ken-ichi Wakabayashi ◽  
Keisuke Yoshida ◽  
Toru Hisabori
Keyword(s):  
Redox Regulation ◽  
Reducing Power ◽  
Photosynthetic Electron Transport ◽  
Rubisco Activase ◽  
Thermal Dissipation ◽  
Fructose 1,6 Bisphosphatase ◽  
Related Proteins ◽  
Oxidative Regulation

Thioredoxin (Trx) is a protein that mediates the reducing power transfer from the photosynthetic electron transport system to target enzymes in chloroplasts and regulates their activities. Redox regulation governed by Trx is a system that is central to the adaptation of various chloroplast functions to the ever-changing light environment. However, the factors involved in the opposite reaction (i.e., the oxidation of various enzymes) have yet to be revealed. Recently, it has been suggested that Trx and Trx-like proteins could oxidize Trx-targeted proteins in vitro. To elucidate the in vivo function of these proteins as oxidation factors, we generated mutant plant lines deficient in Trx or Trx-like proteins and studied how the proteins are involved in oxidative regulation in chloroplasts. We found that f-type Trx and two types of Trx-like proteins, Trx-like 2 and atypical Cys His-rich Trx (ACHT), seemed to serve as oxidation factors for Trx-targeted proteins, such as fructose-1,6-bisphosphatase, Rubisco activase, and the γ-subunit of ATP synthase. In addition, ACHT was found to be involved in regulating nonphotochemical quenching, which is the mechanism underlying the thermal dissipation of excess light energy. Overall, these results indicate that Trx and Trx-like proteins regulate chloroplast functions in concert by controlling the redox state of various photosynthesis-related proteins in vivo.

Antioxidant Activity, Phenolic Content and Hematoprotective Effects of Cleome arabica Polyphenolic Leaf Extract

2019 ◽  
Author(s):  
C. Tigrine ◽  
A. Kameli
Keyword(s):  
Antioxidant Activity ◽  
Biological Effects ◽  
Reducing Power ◽  
Hematological Toxicity ◽  
Protective Effects ◽  
Ferrous Ions ◽  
Polyphenolic Extract ◽  
Cleome Arabica

In this study a polyphenolic extract from Cleome arabica leaves (CALE) was investigated for its antioxidant activity in vitro using DPPH•, metal chelating and reducing power methods and for its protective effects against AraC-induced hematological toxicity in vivo using Balb C mice. Results indicated that CALE exhibited a strong and dose-dependent scavenging activity against the DPPH• free radical (IC50 = 4.88 μg/ml) and a high reducing power activity (EC50 = 4.85 μg/ml). Furthermore, it showed a good chelating effects against ferrous ions (IC50 = 377.75 μg/ml). The analysis of blood showed that subcutaneous injection of AraC (50 mg/kg) to mice during three consecutive days caused a significant myelosupression (P < 0.05). The combination of CALE and AraC protected blood cells from a veritable toxicity. Where, the number of the red cells, the amount of hemoglobin and the percentage of the hematocrite were significantly high. On the other hand, AraC cause an elevation of body temperature (39 °C) in mice. However, the temperature of the group treated with CALE and AraC remained normal and did not exceed 37.5 °C. The observed biological effects of CALE, in vitro as well as in vivo, could be due to the high polyphenol and flavonoid contents. In addition, the antioxidant activity of CALE suggested to be responsible for its hematoprotective effect.

scholarly journals Hepatoprotective studies on methanolic extract fractions of Lindernia ciliata and development of qualitative analytical profile for the bioactive extract

2019 ◽  
Vol 5 (1) ◽  
Author(s):  
Praneetha Pallerla ◽  
Narsimha Reddy Yellu ◽  
Ravi Kumar Bobbala
Keyword(s):  
Methanolic Extract ◽  
Antioxidant Properties ◽  
Radical Scavenging ◽  
Liver Homogenate ◽  
Reducing Power ◽  
Hepatoprotective Activity ◽  
Total Phenolic ◽  
Effective Fraction

Abstract Background The objective of the study is to evaluate the hepatoprotective activity of methanolic extract fractions of Lindernia ciliata (LC) and development of qualitative analytical profile of the bioactive fraction using HPLC fingerprinting analysis. All the fractions of methanolic extract of Lindernia ciliata (LCME) are assessed for their total phenolic, flavonoid contents and in vitro antioxidant properties by using DPPH, superoxide, nitric oxide, hydroxyl radical scavenging activities and reducing power assay. Acute toxicity study was conducted for all the fractions and the two test doses 50 and 100 mg/kg were selected for the hepatoprotective study. Liver damage was induced in different groups of rats by administering 3 g/kg.b.w.p.o. paracetamol and the effect of fractions were tested for hepatoprotective potential by evaluating serum biochemical parameters and histology of liver of rats. The effective fraction was evaluated for its antihepatotoxic activity against D-Galactosamine (400 mg/kg b.w. i.p.) and in vivo antioxidant parameters viz., Glutathione (GSH), Melondialdehyde (MDA) and Catalase (CAT) levels are estimated using liver homogenate. Results Among all the fractions, butanone fraction of LCME, (BNF-LCME) has shown better hepatoprotective activity and hence it is selected to evaluate the antihepatotoxicity against D-GaIN. The activity of BNF-LCME is well supported in in vitro and in vivo antioxidant studies and may be attributed to flavonoidal, phenolic compounds present in the fraction. Hence, BNF-LCME was subjected to the development of qualitative analytical profile using HPLC finger printing analysis. Conclusions All the fractions of LCME exhibited significant hepatoprotective activity and BNF-LCME (50 mg/kg) was identified as the most effective fraction.

scholarly journals Sulfasalazine modifies metabolic profiles and enhances cisplatin chemosensitivity on cholangiocarcinoma cells in in vitro and in vivo models

2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Malinee Thanee ◽  
Sureerat Padthaisong ◽  
Manida Suksawat ◽  
Hasaya Dokduang ◽  
Jutarop Phetcharaburanin ◽  
...  
Keyword(s):  
Redox Regulation ◽  
Treated Group ◽  
Control Group ◽  
High Recurrence Rate ◽  
Nucleic Acid Metabolism ◽  
In Vivo Models ◽  
Tryptophan Degradation ◽  
Xenograft Mice

Abstract Background Sulfasalazine (SSZ) is widely known as an xCT inhibitor suppressing CD44v9-expressed cancer stem-like cells (CSCs) being related to redox regulation. Cholangiocarcinoma (CCA) has a high recurrence rate and no effective chemotherapy. A recent report revealed high levels of CD44v9-positive cells in CCA patients. Therefore, a combination of drugs could prove a suitable strategy for CCA treatment via individual metabolic profiling. Methods We examined the effect of xCT-targeted CD44v9-CSCs using sulfasalazine combined with cisplatin (CIS) or gemcitabine in CCA in vitro and in vivo models and did NMR-based metabolomics analysis of xenograft mice tumor tissues. Results Our findings suggest that combined SSZ and CIS leads to a higher inhibition of cell proliferation and induction of cell death than CIS alone in both in vitro and in vivo models. Xenograft mice showed that the CD44v9-CSC marker and CK-19-CCA proliferative marker were reduced in the combination treatment. Interestingly, different metabolic signatures and significant metabolites were observed in the drug-treated group compared with the control group that revealed the cancer suppression mechanisms. Conclusions SSZ could improve CCA therapy by sensitization to CIS through killing CD44v9-positive cells and modifying the metabolic pathways, in particular tryptophan degradation (i.e., kynurenine pathway, serotonin pathway) and nucleic acid metabolism.

scholarly journals A novel dephosphorylation targeting chimera selectively promoting tau removal in tauopathies

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Jie Zheng ◽  
Na Tian ◽  
Fei Liu ◽  
Yidian Zhang ◽  
Jingfen Su ◽  
...  
Keyword(s):  
Neurodegenerative Disorders ◽  
Protein Phosphatase ◽  
High Efficiency ◽  
Microtubule Assembly ◽  
Hyperphosphorylated Tau ◽  
Phosphatase 2A ◽  
Dependent Learning ◽  
The Brain

AbstractIntraneuronal accumulation of hyperphosphorylated tau is a hallmark pathology shown in over twenty neurodegenerative disorders, collectively termed as tauopathies, including the most common Alzheimer’s disease (AD). Therefore, selectively removing or reducing hyperphosphorylated tau is promising for therapies of AD and other tauopathies. Here, we designed and synthesized a novel DEPhosphorylation TArgeting Chimera (DEPTAC) to specifically facilitate the binding of tau to Bα-subunit-containing protein phosphatase 2A (PP2A-Bα), the most active tau phosphatase in the brain. The DEPTAC exhibited high efficiency in dephosphorylating tau at multiple AD-associated sites and preventing tau accumulation both in vitro and in vivo. Further studies revealed that DEPTAC significantly improved microtubule assembly, neurite plasticity, and hippocampus-dependent learning and memory in transgenic mice with inducible overexpression of truncated and neurotoxic human tau N368. Our data provide a strategy for selective removal of the hyperphosphorylated tau, which sheds new light for the targeted therapy of AD and related-tauopathies.

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