scholarly journals Redox regulation of pyruvate kinase M2 by cysteine oxidation and S-nitrosation

2018 ◽  
Vol 475 (20) ◽  
pp. 3275-3291 ◽  
Author(s):  
Alice Rose Mitchell ◽  
Meng Yuan ◽  
Hugh P. Morgan ◽  
Iain W. McNae ◽  
Elizabeth A. Blackburn ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Pyruvate Kinase ◽  
Redox Regulation ◽  
In Vivo Studies ◽  
Cysteine Oxidation ◽  
Resistance To Oxidation ◽  
Biotin Switch ◽  
Tetrameric Form ◽  
Major Target

We show here that the M2 isoform of human pyruvate kinase (M2PYK) is susceptible to nitrosation and oxidation, and that these modifications regulate enzyme activity by preventing the formation of the active tetrameric form. The biotin-switch assay carried out on M1 and M2 isoforms showed that M2PYK is sensitive to nitrosation and that Cys326 is highly susceptible to redox modification. Structural and enzymatic studies have been carried out on point mutants for three cysteine residues (Cys424, Cys358, and Cys326) to characterise their potential roles in redox regulation. Nine cysteines are conserved between M2PYK and M1PYK. Cys424 is the only cysteine unique to M2PYK. C424S, C424A, and C424L showed a moderate effect on enzyme activity with 80, 100, and 140% activity, respectively, compared with M2PYK. C358 had been previously identified from in vivo studies to be the favoured target for oxidation. Our characterised mutant showed that this mutation stabilises tetrameric M2PYK, suggesting that the in vivo resistance to oxidation for the Cys358Ser mutation is due to stabilisation of the tetrameric form of the enzyme. In contrast, the Cys326Ser mutant exists predominantly in monomeric form. A biotin-switch assay using this mutant also showed a significant reduction in biotinylation of M2PYK, confirming that this is a major target for nitrosation and probably oxidation. Our results show that the sensitivity of M2PYK to oxidation and nitrosation is regulated by its monomer–tetramer equilibrium. In the monomer state, residues (in particular C326) are exposed to oxidative modifications that prevent reformation of the active tetrameric form.

scholarly journals Targeting Pyruvate Kinase M2 and Lactate Dehydrogenase A Is an Effective Combination Strategy for the Treatment of Pancreatic Cancer

Cancers ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 1372 ◽  
Author(s):  
Goran Hamid Mohammad ◽  
Vessela Vassileva ◽  
Pilar Acedo ◽  
Steven W. M. Olde Damink ◽  
Massimo Malago ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Cell Proliferation ◽  
Enzyme Activity ◽  
Lactate Dehydrogenase ◽  
Tumor Growth ◽  
Pyruvate Kinase ◽  
Glycolytic Enzymes ◽  
Pyruvate Kinase M2 ◽  
Tumor Tissues

Reprogrammed glucose metabolism is one of the hallmarks of cancer, and increased expression of key glycolytic enzymes, such as pyruvate kinase M2 (PKM2) and lactate dehydrogenase A (LDHA), has been associated with poor prognosis in various malignancies. Targeting these enzymes could attenuate aerobic glycolysis and inhibit tumor proliferation. We investigated whether the PKM2 activator, TEPP-46, and the LDHA inhibitor, FX-11, can be combined to inhibit in vitro and in vivo tumor growth in preclinical models of pancreatic cancer. We assessed PKM2 and LDHA expression, enzyme activity, and cell proliferation rate after treatment with TEPP-46, FX-11, or a combination of both. Efficacy was validated in vivo by evaluating tumor growth, PK and LDHA activity in plasma and tumors, and PKM2, LDHA, and Ki-67 expression in tumor tissues following treatment. Dual therapy synergistically inhibited pancreatic cancer cell proliferation and significantly delayed tumor growth in vivo without apparent toxicity. Treatment with TEPP-46 and FX-11 resulted in increased PK and reduced LDHA enzyme activity in plasma and tumor tissues and decreased PKM2 and LDHA expression in tumors, which was reflected by a decrease in tumor volume and proliferation. The targeting of glycolytic enzymes such as PKM2 and LDHA represents a promising therapeutic approach for the treatment of pancreatic cancer.

scholarly journals N-Butyldeoxygalactonojirimycin Induces Reversible Infertility in Male CD Rats

2019 ◽  
Vol 21 (1) ◽  
pp. 301
Author(s):  
Vijayalaxmi Gupta ◽  
Sheri A. Hild ◽  
Sudhakar R. Jakkaraj ◽  
Erick J. Carlson ◽  
Henry L. Wong ◽  
...  
Keyword(s):  
Major Effect ◽  
In Vivo Studies ◽  
Pharmacokinetic Studies ◽  
Male Mice ◽  
Lead Compounds ◽  
Contraceptive Effect ◽  
First Time ◽  
Major Target

This study shows for the first time that an iminosugar exerts anti-spermiogenic effect, inducing reversible infertility in a species that is not related to C57BL/6 male mice. In CD rats, N-butyldeoxygalactonojirimycin (NB-DGJ) caused reversible infertility at 150 mg/kg/day when administered daily as single oral dose. NB-DGJ inhibited CD rat-derived testicular β-glucosidase 2 (GBA2) activity at 10 µM but did not inhibit CD rat-derived testicular ceramide-specific glucosyltransferase (CGT) at doses up to 1000 µM. Pharmacokinetic studies revealed that sufficient plasma levels of NB-DGJ (50 µM) were achieved to inhibit the enzyme. Fertility was blocked after 35 days of treatment and reversed one week after termination of treatment. The rapid return of fertility indicates that the major effect of NB-DGJ may be epididymal rather than testicular. Collectively, our in vitro and in vivo studies in rats suggest that iminosugars should continue to be pursued as potential lead compounds for development of oral, non-hormonal male contraceptives. The study also adds evidence that GBA2, and not CGT, is the major target for the contraceptive effect of iminosugars.

scholarly journals Control in vivo of rat liver phosphofructokinase by glucagon and nutritional changes

1980 ◽  
Vol 186 (3) ◽  
pp. 953-957 ◽  
Author(s):  
A Nieto ◽  
J G Castaño
Keyword(s):  
Enzyme Activity ◽  
Pyruvate Kinase ◽  
High Protein Diet ◽  
High Carbohydrate Diet ◽  
Physiological Changes ◽  
Carbohydrate Diet ◽  
High Carbohydrate ◽  
Dietary Condition ◽  
Phosphofructokinase Activity

Glucagon (250 microgram/kg body wt.) intravenously injected into normal fed rats produces within 5 min a marked inactivation of liver phosphofructokinase, only observed when the enzyme activity is measured at subsaturating concentrations of fructose 6-phosphate. Since half-maximal inactivation is observed at a dose of glucagon of 0.32 microgram/body wt., a dose within the range of the physiological concentrations of the hormone, the inactivation of phosphofructokinase can occur in vivo in response to physiological changes in the concentration of glucagon. In gluconeogenic conditions (starved rats or high-protein-diet-fed rats), there is a marked inactivation of liver phosphofructokinase at subsaturating concentrations of fructose 6-phosphate similar to that found in normal fed rats after glucagon treatment. In these gluconeogenic conditions a 50% decrease in the Vmax. of the enzyme is also observed. No significant changes in phosphofructokinase activity either at subsaturating concentrations of fructose 6-phosphate or in the Vmax. of the enzyme are observed when rats are fed on a high-carbohydrate diet. In the last dietary condition, glucagon treatment produces similar effects to that described in the normal fed rats. Similar results have been obtained in the above condtions for pyruvate kinase L activity when measured at subsaturating concentrations of phosphoenolpyruvate.

scholarly journals Safety assessment of Lactobacillus reuteri IDCC 3701 based on phenotypic and genomic analysis

2021 ◽  
Vol 71 (1) ◽  
Author(s):  
Bo Som Lee ◽  
O-Hyun Ban ◽  
Won Yeong Bang ◽  
Seung A Chae ◽  
Sangki Oh ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Lactobacillus Reuteri ◽  
Lactate Production ◽  
Whole Genome Sequence ◽  
In Vivo Studies ◽  
Human Consumption ◽  
Human Breast Milk ◽  
Whole Genome ◽  
Phenotypic Analysis

Abstract Purpose Lactobacillus reuteri is one of the most largely studied human-commensal bacteria and widely used as a form of probiotics. Safety of probiotics has become increasingly important for human consumption due to increasing health-concerns in food industry. In this study, the safety of L. reuteri IDCC 3701 isolated from human breast milk was thoroughly investigated. Methods Whole-genome sequence analysis was performed to identify antibiotic resistance and toxigenicity of L. reuteri IDCC 3701. Phenotypic analysis such as minimal inhibitory concentration, β-hemolysis, extracellular enzyme activity, and the production of biological amines and L/D-lactate, was investigated. Finally, acute oral toxicity test was performed to access L. reuteri IDCC 3701 safety. Results Although multiple resistances to gentamicin and kanamycin were observed in L. reuteri IDCC 3701, it was revealed that these resistances are intrinsic and not transferable through whole-genome analysis. In addition, various phenotypic analysis concerning hemolysis, enzyme activity, and D-lactate production did not show any negative results. Although L. reuteri IDCC 3701 harbors a histidine decarboxylase gene, no biogenic amines were detected. Finally, L. reuteri IDCC 3701 exhibited no evidence of acute toxicity according to an in vivo study. Conclusion Our findings demonstrate that L. reuteri IDCC 3701 is considered to be safe for human consumption as probiotics based on the in silico, in vitro and in vivo studies.

In vitro Thermal and Salt Stability of Pyruvate Kinase Are Increased by Proline Analogues and Trigonelline

1991 ◽  
Vol 18 (3) ◽  
pp. 279 ◽  
Author(s):  
a Shomer-Ilan ◽  
GP Jones ◽  
LG Paleg
Keyword(s):  
Zea Mays ◽  
Enzyme Activity ◽  
Pyruvate Kinase ◽  
Compatible Solutes ◽  
Rabbit Muscle ◽  
Protective Effects ◽  
Protective Properties ◽  
Nitrogenous Compounds

The nitrogenous compounds N-methyl-L-proline (MP), trans-4-hydroxy-N-methyl-L-proline (MHP) and trigonelline (T), which undergo stress-induced accumulation in some Australian plants, were analysed and compared with proline (P) and glycinebetaine (B) for possible protective roles. The activity of pyruvate kinase (PK), prepared from Zea mays leaves and rabbit muscle, was unaffected even in the presence of 750 mM of the proline analogues. Thus, MP and MHP, like P and B, have the properties to act in vivo as compatible osmotica. T was not as compatible, decreasing enzyme activity 20% at 0.5 M. Like P and B, however, MP, MHP and T all also exhibited protective properties. They increased, in vitro, the thermal stability of PK from both plant and animal sources, and they protected PK (Zea mays) from salt inhibition at two substrate levels. The effect of salt on PK (Zea mays) was substrate dependent; at low phosphoenolpyruvate (PEP) levels, salt inhibited the enzyme activity, while salt effects were less severe in the presence of higher substrate levels. In the presence of high NaCl concentrations, the protective effects of high substrate levels and the compatible solutes seem to be additive. The Km (PEP) value of the plant PK increased in the presence of salt but the effect was ameliorated by the compatible solute MHP.

Mechanisms and consequences of protein cysteine oxidation: the role of the initial short-lived intermediates

2020 ◽  
Vol 64 (1) ◽  
pp. 55-66
Author(s):  
Lucia Turell ◽  
Ari Zeida ◽  
Madia Trujillo
Keyword(s):  
Redox Regulation ◽  
Catalytic Mechanism ◽  
Cysteine Residue ◽  
Oxidation Reactions ◽  
Cysteine Oxidation ◽  
Deprotonated Form ◽  
Thiyl Radicals ◽  
Sulfenic Acids ◽  
Kinetics Of

Abstract Thiol groups in protein cysteine (Cys) residues can undergo one- and two-electron oxidation reactions leading to the formation of thiyl radicals or sulfenic acids, respectively. In this mini-review we summarize the mechanisms and kinetics of the formation of these species by biologically relevant oxidants. Most of the latter react with the deprotonated form of the thiol. Since the pKa of the thiols in protein cysteines are usually close to physiological pH, the thermodynamics and the kinetics of their oxidation in vivo are affected by the acidity of the thiol. Moreover, the protein microenvironment has pronounced effects on cysteine residue reactivity, which in the case of the oxidation mediated by hydroperoxides, is known to confer specificity to particular protein cysteines. Despite their elusive nature, both thiyl radicals and sulfenic acids are involved in the catalytic mechanism of several enzymes and in the redox regulation of protein function and/or signaling pathways. They are usually short-lived species that undergo further reactions that converge in the formation of different stable products, resulting in several post-translational modifications of the protein. Some of these can be reversed through the action of specific cellular reduction systems. Others damage the proteins irreversibly, and can make them more prone to aggregation or degradation.

scholarly journals Potential Modulation of Sirtuins by Oxidative Stress

2016 ◽  
Vol 2016 ◽  
pp. 1-12 ◽  
Author(s):  
Leonardo Santos ◽  
Carlos Escande ◽  
Ana Denicola
Keyword(s):  
Oxidative Stress ◽  
Posttranslational Modifications ◽  
Histone Deacetylases ◽  
Redox Regulation ◽  
Molecular Mechanisms ◽  
Compensatory Mechanism ◽  
Cysteine Oxidation ◽  
Catalytic Core

Sirtuins are a conserved family of NAD-dependent protein deacylases. Initially proposed as histone deacetylases, it is now known that they act on a variety of proteins including transcription factors and metabolic enzymes, having a key role in the regulation of cellular homeostasis. Seven isoforms are identified in mammals (SIRT1–7), all of them sharing a conserved catalytic core and showing differential subcellular localization and activities. Oxidative stress can affect the activity of sirtuins at different levels: expression, posttranslational modifications, protein-protein interactions, and NAD levels. Mild oxidative stress induces the expression of sirtuins as a compensatory mechanism, while harsh or prolonged oxidant conditions result in dysfunctional modified sirtuins more prone to degradation by the proteasome. Oxidative posttranslational modifications have been identifiedin vitroandin vivo, in particular cysteine oxidation and tyrosine nitration. In addition, oxidative stress can alter the interaction with other proteins, like SIRT1 with its protein inhibitor DBC1 resulting in a net increase of deacetylase activity. In the same way, manipulation of cellular NAD levels by pharmacological inhibition of other NAD-consuming enzymes results in activation of SIRT1 and protection against obesity-related pathologies. Nevertheless, further research is needed to establish the molecular mechanisms of redox regulation of sirtuins to further design adequate pharmacological interventions.

CarR, a MarR-family regulator from Corynebacterium glutamicum, modulated antibiotic and aromatic compound resistance

2019 ◽  
Vol 476 (21) ◽  
pp. 3141-3159 ◽  
Author(s):  
Meiru Si ◽  
Can Chen ◽  
Zengfan Wei ◽  
Zhijin Gong ◽  
GuiZhi Li ◽  
...  
Keyword(s):  
Stress Response ◽  
Ligand Binding ◽  
Corynebacterium Glutamicum ◽  
Conformational Changes ◽  
Cysteine Oxidation ◽  
Transcriptional Regulatory ◽  
Ligand Free ◽  
Redox Sensing

Abstract MarR (multiple antibiotic resistance regulator) proteins are a family of transcriptional regulators that is prevalent in Corynebacterium glutamicum. Understanding the physiological and biochemical function of MarR homologs in C. glutamicum has focused on cysteine oxidation-based redox-sensing and substrate metabolism-involving regulators. In this study, we characterized the stress-related ligand-binding functions of the C. glutamicum MarR-type regulator CarR (C. glutamicum antibiotic-responding regulator). We demonstrate that CarR negatively regulates the expression of the carR (ncgl2886)–uspA (ncgl2887) operon and the adjacent, oppositely oriented gene ncgl2885, encoding the hypothetical deacylase DecE. We also show that CarR directly activates transcription of the ncgl2882–ncgl2884 operon, encoding the peptidoglycan synthesis operon (PSO) located upstream of carR in the opposite orientation. The addition of stress-associated ligands such as penicillin and streptomycin induced carR, uspA, decE, and PSO expression in vivo, as well as attenuated binding of CarR to operator DNA in vitro. Importantly, stress response-induced up-regulation of carR, uspA, and PSO gene expression correlated with cell resistance to β-lactam antibiotics and aromatic compounds. Six highly conserved residues in CarR were found to strongly influence its ligand binding and transcriptional regulatory properties. Collectively, the results indicate that the ligand binding of CarR induces its dissociation from the carR–uspA promoter to derepress carR and uspA transcription. Ligand-free CarR also activates PSO expression, which in turn contributes to C. glutamicum stress resistance. The outcomes indicate that the stress response mechanism of CarR in C. glutamicum occurs via ligand-induced conformational changes to the protein, not via cysteine oxidation-based thiol modifications.

Sign in / Sign up

Export Citation Format

Share Document