Redox coding
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SIGNIFICANCE: Hexokinases are key enzymes that are responsible for the first reaction of glycolysis, but they also moonlight other cellular processes, including mitochondrial redox signaling regulation. Modulation of hexokinase activity and spatiotemporal location by reactive oxygen and nitrogen species as well as other gasotransmitters serves as the basis for a unique, underexplored method of tight and flexible regulation of these fundamental enzymes. Recent Advances: Redox modifications of thiols serve as a molecular code that enables the precise and complex regulation of hexokinases. Redox regulation of hexokinases is also used by multiple parasites to cause widespread and severe diseases, including malaria, Chagas disease, and sleeping sickness. Redox-active molecules affect each other, and the moonlighting activity of hexokinases provides another feedback loop that affects the cellular redox status and is hijacked in malignantly transformed cells. CRITICAL ISSUES: Several compounds affect the redox status of hexokinases in vivo. These include the dehydroascorbic acid (oxidized form of vitamin C), pyrrolidinium porrolidine-1-carbodithioate (contraceptive), peroxynitrite (product of ethanol metabolism), alloxan (a glucose analog), and isobenzothiazolinone ebselen. However, very limited information is available regarding which amino acid residues in hexokinases are affected by redox signaling. Except in cases of monogenic diabetes, direct evidence is absent for disease phenotypes that are associated with variations within motifs that are susceptible to redox signaling. FUTURE DIRECTIONS: Further studies should address the propensity of hexokinases and their disease-associated variants to participate in redox regulation. Robust and straightforward proteomic methods are needed to understand the context and consequences of hexokinase-mediated redox regulation in health and disease.
- MeSH
- hexokinasa metabolismus MeSH
- lidé MeSH
- mitochondrie metabolismus MeSH
- oxidace-redukce MeSH
- signální transdukce MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
Stable isotope labeling by amino acids in cell culture (SILAC) and iodoacetyl tandem mass tag (iodoTMT) are well-implemented mass spectrometry-based approaches for quantification of proteins and for site-mapping of cysteine modification. We describe here a combination of SILAC and iodoTMT to assess ongoing changes in the global proteome and cysteine modification levels using liquid chromatography separation coupled with high-resolution mass spectrometry (LC-MS/MS).
Nucleosides and 2'-deoxyribonucleoside triphosphates (dNTPs) bearing phenothiazine (PT) attached to a nucleobase (cytosine or 7-deazaadenine) either directly or through an acetylene linker were prepared through Suzuki or Sonogashira cross-coupling and triphosphorylation, and were studied as building blocks for polymerase construction of modified DNA. The directly PT-substituted dNTPs were better substrates for polymerases than the alkyne-linked dNTPs but all of them were used in enzymatic synthesis of DNA using primer extension, nicking enzyme amplification, PCR or 3'-tail labelling by terminal deoxynucleotidyl transferase. The phenothiazine served as an oxidizable redox label (giving two analytically useful signals of oxidation on electrode) for nucleosides and DNA and was also used in orthogonal combination with previously developed benzofurazane or nitrophenyl labels for redox coding of DNA bases. Therefore, the title PT-linked dNTPs are useful additions to the portfolio of nucleotides for enzymatic synthesis of redox-labelled DNA for electrochemical analysis.
The thioredoxin system is a significant redox regulator in all organisms. Thioredoxins in bacteria are the major dithiol reductants in the cytosol (or an advanced equivalent to dithiotreitol of cells) thanks to the low redox potentials (Holmgren, 1985). In the genome of the studied model Streptomyces coelicolor A3(2) several genes were revealed which code proteins forming the thioredoxin system. It seems that this gram-positive soil bacteria have a very complex redox system, with a variety of reducing possibilities. In this work cloning, purification and characterization of further thioredoxins (TrxA2 and TrxA3) are described. Both proteins were overexpressed in E. coli cytoplasma as soluble active hexahistidine fusion proteins and isolated as homogenous substances.
A straightforward methodology of fluorine substitution by tritium/deuterium is reported. The described method is selective towards the F─C (sp3 ) group and leaves both the aromatic F─C (sp2 ) and F2 ─C (sp3 ) moieties unaffected. Alkylfluorides, readily synthesized from appropriate alcohols by treatment with diethylaminosulfur trifluoride (DAST) reagent in an overall yield up to 76%, undergoes activation with the boron-based Lewis acid B(C6 F5 )3 , and stoichiometric in situ reduction with a tritide/deuteride reagent-the [TMP2(3) H][2(3) HB(C6 F5 )3 ] system of frustrated Lewis pair. This methodology provides an isolated yield of up to 93% of regio-specifically labeled small organic compounds with superior 2 H-enrichment of over 95%. The specific activity of prepared 1-(2-[3 H]-ethyl)naphthalene was determined at 29.0 Ci/mmol. The site selectivity of the Lewis acid/ [TMP2(3) H][2(3) HB(C6 F5 )3 ] approach is orthogonal to currently used methods and allows for isotopic labeling of complementary positions in molecules. Reported labeling methodology proceeds well at ultra-mild reaction conditions (220 mbar of T2 ), allowing very low consumption of the radioactive source (4.2 Ci/156 GBq), and producing limited amount of radioactive waste.
3-Hydroxycyclopent-1-ene-1-carboxylic acid (HOCPCA (1)) is a potent ligand for high-affinity γ-hydroxybutyric acid binding sites in the central nervous system. Various approaches to the introduction of a hydrogen label onto the HOCPCA skeleton are reported. The outcomes of the feasible C─H activation of olefin carbon (C-2) by iridium catalyst are compared with the reduction of the carbonyl group (C-3) by freshly prepared borodeuterides. The most efficient iridium catalysts proved to be Kerr bulky phosphine N-heterocyclic species providing outstanding deuterium enrichment (up to 91%) in a short period of time. The highest deuterium enrichment (>99%) was achieved through the reduction of ketone precursor 2 by lithium trimethoxyborodeuteride. Hence, analogical conditions were used for the tritiation experiment. [3 H]-HOCPCA selectively labeled on the position C-3 was synthetized with radiochemical purity >99%, an isolated yield of 637 mCi and specific activity = 28.9 Ci/mmol.
A chromate-tolerant mutant chr1-663T bearing a stable one-gene mutation and its parental strain 6chr(+) were used to investigate the background of Cr(VI) tolerance in the fission yeast Schizosaccharomyces pombe. The mutant chr1-663T displayed a significantly decreased specific glutathione reductase (GR) activity coded by the pgr1 (+) gene compared with its parental strain. Transformants of the mutant chr1-663T with a nonintegrative pUR18N vector expressing the pgr1 (+) gene exhibited the same Cr(VI) sensitivity and specific GR activity as their parental strain, demonstrating the importance of the GR-NADPH system in Cr(VI) tolerance. Transformants, nevertheless, exhibited an increased intracellular peroxide concentration, a decreased Cr(VI)-reducing and HO*-producing ability, which suggested an unbalanced oxidoreduction state of cells and partial complementation of the GR function. No mutation was found in the sequences of the pgr1 (+) and the pap1 (+) (transcriptional regulatory gene of GR) genes of the Cr(VI)-tolerant mutant by sequence analysis.
20-Oxo-5β-[9,12,12-(2)H(3)]pregnan-3α-yl-l-glutamyl 1-ester 11 was synthesized as an internal standard for quantification of a neuroprotective NMDA receptor ligand, 20-oxo-5β-pregnan-3α-yl-l-glutamyl 1-ester 18 and its metabolites, in plasma and tissue. 11α-Hydroxy-progesterone (1) was reduced under basic conditions to yield the corresponding 5β-steroid. Protection of the 3- and 20-oxo groups and oxidation of the 11α-hydroxy group was then followed by a deuterium exchange, conducted under basic conditions using deuterated methanol. Next, the carbonyl moiety at C-11 was reduced and the 11α-hydroxyl group removed through utilization of the Barton-McCombie reaction. Subsequent deprotection of the 3- and 20-acetals and stereoselective reduction of the 3-oxo group gave the desired trideuterated pregnanolone (8). This was coupled with protected glutamic acid, which was then deprotected to yield [9,12,12-(2)H(3)]-pregnanolone glutamate (11) with >99% isotopic purity.
- MeSH
- chromatografie na tenké vrstvě MeSH
- deuterium chemie MeSH
- glutamáty chemie MeSH
- hydroxyprogesterony chemie MeSH
- izotopové značení metody MeSH
- magnetická rezonanční spektroskopie MeSH
- molekulární struktura MeSH
- oxidace-redukce MeSH
- pregnanolon analogy a deriváty chemická syntéza chemie MeSH
- receptory N-methyl-D-aspartátu antagonisté a inhibitory MeSH
- rozpouštědla chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Several diseases (atherosclerosis, diabetes mellitus, chronic renal failure) are associated with oxidative and carbonyl stress, microinflammation and eventually autoimmune reaction. Both oxidative and carbonyl stress cause damage to important biological structures-proteins, carbohydrates, lipids and nucleic acids and may enhance inflammatory response. New compounds and modified structures are formed, among them advanced oxidation protein products (AOPP), advanced glycation end products (AGEs-e.g. pentosidine, carboxymethyllysine) and advanced lipoperoxidation end products (ALEs). Accumulation of glycoxidation products, upregulation of protective mechanisms like glyoxalase I as well as enhanced transcription of genes coding for cytokines, growth factors and adhesive molecules via AGE-RAGE (receptor for AGEs) interaction and subsequent increase of classical acute phase reactants (e.g. CRP-C-reactive protein or orosomucoid) can be observed in a variety of chronic diseases. Additionally, several RAGE gene polymorphisms have shown association with some pathological states-diabetic complications, vascular damage, inflammatory response or antioxidant status. Recent advances in understanding the pathogenesis of chronic diseases provide new possibilities for diagnostics and monitoring of severely ill patients, however, further studies are still required to establish efficient therapeutical strategies.
- MeSH
- chronická nemoc MeSH
- chronické selhání ledvin komplikace metabolismus MeSH
- diabetes mellitus genetika metabolismus MeSH
- financování organizované MeSH
- klinická chemie metody MeSH
- laktoylglutathionlyasa genetika metabolismus MeSH
- lidé MeSH
- mitogenem aktivované proteinkinasy genetika metabolismus MeSH
- nukleotidy metabolismus MeSH
- oxidace-redukce MeSH
- oxidační stres MeSH
- polymorfismus genetický MeSH
- produkty pokročilé glykace analýza metabolismus MeSH
- receptory cytoplazmatické a nukleární metabolismus MeSH
- zánět etiologie MeSH
- Check Tag
- lidé MeSH
SIGNIFICANCE: Redox modifications of thiols serve as a molecular code enabling precise and complex regulation of protein tyrosine phosphatases (PTPs) and other proteins. Particular gasotransmitters and even the redox modifications themselves affect each other, of which a typical example is S-nitrosylation-mediated protection against the further oxidation of protein thiols. RECENT ADVANCES: For a long time, PTPs were considered constitutively active housekeeping enzymes. This view has changed substantially over the last two decades, and the PTP family is now recognized as a group of tightly and flexibly regulated fundamental enzymes. In addition to the conventional ways in which they are regulated, including noncovalent interactions, phosphorylation, and oxidation, the evidence that has accumulated during the past two decades suggests that many of these enzymes are also modulated by gasotransmitters, namely by nitric oxide (NO) and hydrogen sulfide (H2S). CRITICAL ISSUES: The specificity and selectivity of the methods used to detect nitrosylation and sulfhydration remains to be corroborated, because several researchers raised the issue of false-positive results, particularly when using the most widespread biotin switch method. Further development of robust and straightforward proteomic methods is needed to further improve our knowledge of the full extent of the gasotransmitters-mediated changes in PTP activity, selectivity, and specificity. FURTHER DIRECTIONS: Results of the hitherto performed studies on gasotransmitter-mediated PTP signaling await translation into clinical medicine and pharmacotherapeutics. In addition to directly affecting the activity of particular PTPs, the use of reversible S-nitrosylation as a protective mechanism against oxidative stress should be of high interest.
- MeSH
- aktivace enzymů MeSH
- lidé MeSH
- reaktivní formy dusíku metabolismus MeSH
- sulfan metabolismus MeSH
- tyrosinfosfatasy metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
