Triuret (also known as carbonyldiurea, dicarbamylurea, or 2,4-diimidotricarbonic diamide) is a byproduct of purine degradation in living organisms. An abundant triuret precursor is uric acid, whose level is altered in multiple metabolic pathologies. Triuret can be generated via urate oxidation by peroxynitrite, the latter being produced by the reaction of nitric oxide radical with superoxide radical anion. From this standpoint, an excess production of superoxide radical anions could indirectly favor triuret formation; however very little is known about the potential in vivo roles of this metabolite. Triuret’s structure is suggestive of its ability to adopt various conformations and act as a flexible ligand for metal ions. In the current study, HPLC-MS/MS, energy-resolved mass spectrometry, selected ion monitoring, collision-induced dissociation, IRMPD spectroscopy, Fourier transform-ion cyclotron resonance mass spectrometry and computational methods were employed to characterize the structure of triuret and its metal complexes, to determine the triuret-alkali metal binding motif, and to evaluate triuret affinity toward alkali metal ions, as well as its affinity for Na+ and K+ relative to other organic ligands. The most favored binding motif was determined to be a bidentate chelation of triuret with the alkali metal cation involving two carbonyl oxygens. Using the complexation selectivity method, it was observed that in solution triuret has an increased affinity for potassium ions, compared to sodium and other alkali metal ions. We propose that triuret may act as a potential hypokalemic agent under pathophysiological conditions conducive to its excessive formation and thus contribute to electrolyte disorders. The collision- or photo-induced fragmentation channels of deprotonated and protonated triuret, as well as its alkali metal adducts, are likely to mimic the triuret degradation pathways in vivo.
Triuret; Uric acid degradation; Hypokalemia; Alkali metal adducts; Isomers; Electrospray ionization; Photodissociation; Collision-induced dissociation; IRMPD spectroscopy
Nanosecond pulsed electric field (nsPEF) is a novel modality for permeabilization of membranous structures and intracellular delivery of xenobiotics. We hypothesized that oxidative effects of nsPEF could be a separate primary mechanism responsible for bioeffects. ROS production in cultured cells and media exposed to 300-ns PEF (1–13 kV/cm) was assessed by oxidation of 2′,7′-dichlorodihydrofluoresein (H2DCF), dihidroethidium (DHE), or Amplex Red. When a suspension of H2DCF-loaded cells was subjected to nsPEF, the yield of fluorescent 2′,7′-dichlorofluorescein (DCF) increased proportionally to the pulse number and cell density. DCF emission increased with time after exposure in nsPEF-sensitive Jurkat cells, but remained stable in nsPEF-resistant U937 cells. In cell-free media, nsPEF facilitated the conversion of H2DCF into DCF. This effect was not related to heating and was reduced by catalase, but not by mannitol or superoxide dismutase. Formation of H2O2 in nsPEF-treated media was confirmed by increased oxidation of Amplex Red. ROS increase within individual cells exposed to nsPEF was visualized by oxidation of DHE. We conclude that nsPEF can generate both extracellular (electrochemical) and intracellular ROS, including H2O2 and possibly other species. Therefore, bioeffects of nsPEF are not limited to electropermeabilization; concurrent ROS formation may lead to cell stimulation and/or oxidative cell damage.
nanosecond pulses; electroporation; cell death; pulsed electric field; reactive oxygen species; membrane permeabilization
Optimal and efficient killing of ingested microbes by human neutrophils is mediated in large part by the action of hypochlorous acid produced by the myeloperoxidase-H2O2-chloride system in phagosomes. Myeloperoxidase gene transcription is limited to early myeloid precursors in the bone marrow, when myeloperoxidase is synthesized and stored in azurophilic granules for subsequent release from stimulated neutrophils. Promyeloperoxidase, the 90-kDa myeloperoxidase precursor synthesized in the endoplasmic reticulum (ER), contains a 125-amino acid pro-region whose function and fate during myeloperoxidase biosynthesis are unknown. Promyeloperoxidase has two fates during myeloperoxidase biosynthesis; the majority undergoes proteolytic processing to generate mature myeloperoxidase, while the remainder is constituvely secreted from the cells in bone marrow. We used a promyelocytic cell line that produces endogenous myelperoxidase as well as human embryonic kidney cells stably expressing normal and mutant forms of myeloperoxidase to examine proteolytic processing of promyeloperoxidase. We demonstrated that CMK-RVKR, an inhibitor of subtilisin-like proteinases, blocked cleavage of the pro-peptide of promyeloperoxidase in a post-ER compartment. Mutants with alanine substitution of basic residues in the predicted proteinase cleavage site failed to undergo maturation to normal myeloperoxidase subunits and were arrested at the promyeloperoxidase stage. Whereas specific mutants varied as to their stability, secreted promyeloperoxidase from the mutants retained the capacity to generate hypochlorous acid. Taken together, these studies demonstrate proconvertase-dependent cleavage of promyeloperoxidase as an essential step in normal proteolytic processing and granule targeting of myeloperoxidase. Furthermore, although mutations in the proteinase cleavage site reduced intracellular stability of the mutants, the integrity of the heme group was not compromised, as chlorinating activity was retained in the secreted promyeloperoxidase.
myeloperoxidase; proproteins; protein processing; biosynthesis
•Low H2O2 concentrations: fast but limited inactivation of MPO via Compound I.•High H2O2 concentrations: complete inactivation of MPO via Compound III.•H2O2 (⩾1000 fold molar excess) acts as a suicide substrate.•Inactivation: destruction of heme, oxidation and disruption of protein.
Human myeloperoxidase (MPO) uses hydrogen peroxide generated by the oxidative burst of neutrophils to produce an array of antimicrobial oxidants. During this process MPO is irreversibly inactivated. This study focused on the unknown role of hydrogen peroxide in this process. When treated with low concentrations of H2O2 in the absence of reducing substrates, there was a rapid loss of up to 35% of its peroxidase activity. Inactivation is proposed to occur via oxidation reactions of Compound I with the prosthetic group or amino acid residues. At higher concentrations hydrogen peroxide acts as a suicide substrate with a rate constant of inactivation of 3.9 × 10−3 s−1. Treatment of MPO with high H2O2 concentrations resulted in complete inactivation, Compound III formation, destruction of the heme groups, release of their iron, and detachment of the small polypeptide chain of MPO. Ten of the protein’s methionine residues were oxidized and the thermal stability of the protein decreased. Inactivation by high concentrations of H2O2 is proposed to occur via the generation of reactive oxidants when H2O2 reacts with Compound III. These mechanisms of inactivation may occur inside neutrophil phagosomes when reducing substrates for MPO become limiting and could be exploited when designing pharmacological inhibitors.
Myeloperoxidase; Neutrophils; Oxidative stress; Suicide inhibitor; Mechanism-based inhibition; Oxidative modification
Bisphosphonates (BPs) inhibit osteocyte and osteoblast apoptosis via opening of connexin (Cx) 43 hemichannels and activating the extracellular signal regulated kinases ERKs. Previously, we hypothesized that intracellular survival signaling is initiated by interaction of BPs with Cx43. However, using whole cell binding assays with [3H]-alendronate, herein we demonstrated the presence of saturable, specific and high affinity binding sites in the Cx43-expressing ROS 17/2.8 osteoblastic cells, authentic osteoblasts and MLO-Y4 cells expressing Cx43 or not, as well as in HeLa cells lacking Cx43 expression and ROS 17/2.8 cells pretreated with agents that disassemble Cx channels. In addition, both BPs and the PTP inhibitor Na3VO4 increased proliferation of cells expressing Cx43 or not. Furthermore, although BPs are internalized and inhibit intracellular enzymes in osteoclasts, whether the drugs penetrate non-resorptive bone cells is not known. To clarify this, we evaluated the osteoblastic uptake of AF-ALN, a fluorescently labeled analog of alendronate. AF-ALN was rapidly internalized in cells expressing Cx43 or not indicating that this process is not mediated via Cx43 hemichannels. Altogether, these findings suggest that although required for triggering intracellular survival signaling by BPs, Cx43 is dispensable for cellular BP binding, its uptake, as well as the proliferative effects of these agents.
bisphosphonates; Cx43 hemichannels; AF-ALN uptake; osteoblast survival
Cultured lung endothelial cells (LEC) respond to VEGF or arachidonic acid with increases in cell proliferation, the formation of tube-like structures, and the activation of Akt and ERK1/2 mediated growth pathways. LECs express a VEGF inducible Cyp2c44 epoxygenase and its 11,12- and 14,15-EET metabolites increase cell proliferation, tubulogenic activity, and the phosphorylation states of the ERK1/2 and Akt kinases. Ketoconazole, an epoxygenase inhibitor, blocks the cellular responses to VEGF. LECs expressing a Cyp2c44 epoxygenase small interference RNA show reductions in Cyp2c44 mRNA levels, and in their VEGF-stimulated proliferative and tubulogenic capacities; effects that are associated with decreases in VEGF-induced phosphorylation of the ERK1/2 and Akt kinases. We conclude that the Cyp2c44 arachidonic acid epoxygenase is a component of the signaling pathways associated with VEGF-stimulated angiogenesis, and suggest a role for EETs in the growth factor-induced changes in the activation states of the ERK1/2 and Akt kinase pathways.
Arachidonic acid epoxygenase; EETs; Angiogenesis; Mitogen activated kinases; Cytochrome P450; EETs and angiogenesis; Vascular endothelial growth factor; Cell signaling
Non-heme manganese catalases are widely distributed over microbial life and represent an environmentally important alternative to heme-containing catalases in antioxidant defense. Manganese catalases contain a binuclear manganese complex as their catalytic active site rather than a heme, and cycle between Mn2(II,II) and Mn2(III,III) states during turnover. X-ray crystallography has revealed the key structural elements of the binuclear manganese active site complex that can serve as the starting point for computational studies on the protein. Four manganese catalase enzymes have been isolated and characterized, and the enzyme appears to have a broad phylogenetic distribution including both bacteria and archae. More than 100 manganese catalase genes have been annotated in genomic databases, although the assignment of many of these putative manganese catalases needs to be experimentally verified. Iron limitation, exposure to low levels of peroxide stress, thermostability and cyanide resistance may provide the biological and environmental context for the occurrence of manganese catalases.
catalase; manganese; peroxide; antioxidant; oxidative stress; reactive oxygen species
Hydrogen peroxide (H2O2) is continuously formed by the autoxidation of redox enzymes in aerobic cells, and it also enters from the environment, where it can be generated both by chemical processes and by the deliberate actions of competing organisms. Because H2O2 is acutely toxic, bacteria elaborate scavenging enzymes to keep its intracellular concentration at nanomolar levels. Mutants that lack such enzymes grow poorly, suffer from high rates of mutagenesis, or even die. In order to understand how bacteria cope with oxidative stress, it is important to identify the key enzymes involved in H2O2 degradation. Catalases and NADH peroxidase (Ahp) are primary scavengers in many bacteria, and their activities and physiological impacts have been unambiguously demonstrated through phenotypic analysis and through direct measurements of H2O2 clearance in vivo. Yet a wide variety of additional enzymes have been proposed to serve similar roles: thiol peroxidase, bacterioferritin comigratory protein, glutathione peroxidase, cytochrome c peroxidase, and rubrerythrins. Each of these enzymes can degrade H2O2 in vitro, but their contributions in vivo remain unclear. In this review we examine the genetic, genomic, regulatory, and biochemical evidence that each of these is a bona fide scavenger of H2O2 in the cell. We also consider possible reasons that bacteria might require multiple enzymes to catalyze this process, including differences in substrate specificity, compartmentalization, cofactor requirements, kinetic optima, and enzyme stability. It is hoped that the resolution of these issues will lead to an understanding of stress resistance that is more accurate and perceptive.
Thiol peroxidase; catalase; alkyl hydroperoxide reducatse; bacterioferritin comigratory protein; cytochrome c peroxidase; rubrerythrin
The appearance of a high molecular weight gelatinolytic enzyme (230 kDa) correlated with cartilage collagen loss in chick embryonic tibias cultured with lipopolysaccharide. This 230 kDa enzyme was purified and its activity was measured on synthetic and natural substrates. The enzyme was activated by aminophenylmercuric acetate and inhibited by ethylenediaminetetraacetic acid, phenanthroline, marimastat or tissue inhibitors of metalloproteinases. Amino acid sequences of peptides derived from the purified enzyme showed identity with avian MMP-9. Digestion of the intact enzyme with chondroitinase decreased the size of the molecule to 80 kDa on SDS-PAGE. When chick embryonic tibia cultures were radiolabeled with 35S-sulfate, the radiolabel co-purified with the 230 kDa gelatinase. Chondroitinase treated 230 kDa gelatinase also reacted with specific anti-chondroitin sulfate antibodiesand FACE analysis revealed a predominance of chondroitin-4-sulfate. These results demonstrate this avian matrix metalloproteinase contained glycosaminoglycan chains. To our knowledge, this is the first report of a matrix metalloproteinase in a proteoglycan form.
Avian; embryonic bone formation; extracellular matrix; gelatinase; matrix metalloproteinase; proteoglycan
Ceramidases play a critical role in generating sphingosine-1-phosphate by hydrolyzing ceramide into sphingosine, a substrate for sphingosine kinase. In order to elucidate its transcriptional regulation, we identify here a putative promoter region in the 5’-UTR of the human neutral CDase (nCDase) gene. Using human genomic DNA, we cloned a 3000 base pair region upstream of the translational start site of the nCDase gene. Luciferase reporter analyses demonstrated that this 3000 bp region had promoter activity, with the strongest induction occurring within the first 200 bp. Computational analysis revealed the 200 bp essential promoter region contained several well-characterized promoter elements, lacked a conical TATA box, but did contain a reverse oriented CCAAT box, a feature common to housekeeping genes. Electrophoretic mobility shift assays demonstrated that the identified candidate transcriptional response elements (TRE) bind their respective transcription factors, including NF-Y, AP-2, Oct-1, and GATA. Mutagenic analyses of the TRE revealed that these sites regulated promoter activity and mutating an individual site decreased promoter reporter activity by up to 50%. Together, our findings suggest that regulation of nCDase expression involves coordinated TATA-less transcriptional activity.
ceramidase; ceramide; sphingosine; promoter; transcriptional regulation; AP-1
Many forms of cellular stress cause an elevation of endogenous ceramide levels leading to growth arrest or apoptosis. Ceramidases (CDase) play a critical role in regulating apoptosis by hydrolyzing ceramide into sphingosine, a precursor for promitogenic sphingosine-1-phosphate. Growth factor induction of neutral CDase (nCDase) has been shown to have a cytoprotective effect against cytokine-induced increases in ceramide levels. To further define the physiological regulation of nCDase, we identified a 200 bp promoter region and demonstrated that serum activated this proximal promoter, which correlated with a serum-induced increase in human nCDase mRNA expression. Computational analysis revealed a putative cis-element for AP-1, a transcription factor activated by serum. Electrophoretic mobility shift assays demonstrated that the identified transcriptional response element binds to AP-1 transcription factors. RNA interference-mediated knockdown of the AP-1 subunit, c-Jun, inhibited the activity of the human nCDase proximal promoter, whereas, c-Jun overexpression increased promoter activity, which directly correlated with human nCDase mRNA transcription, decreased ceramide mass, and protection against caspase 3/7-dependent apoptosis. Taken together, our findings suggest that c-Jun/AP-1 signaling may, in part, regulate serum-induced human nCDase gene transcription.
ceramidase; ceramide; sphingosine; promoter; transcriptional regulation; AP-1
Ala/Asp substitutions at Ser23/24 have been employed to investigate the functional impact of cardiac troponin I (cTnI) phosphorylation by protein kinase A (PKA). Some limitations of previous studies include the use of heterologous proteins and confounding effects arising from phosphorylation of cardiac myosin binding protein-C. Our goal was to probe the effects of cTnI phosphorylation using a homologous assay, so that altered function could be solely attributed to changes in cTnI. We reconstituted detergent-skinned rat cardiac papillary fibers with homologous rat cardiac troponin subunits to study the impact of Ala and Asp substitutions at Ser23/24 of rat cTnI (RcTnI S23A/24A and RcTnI S23D/24D). Both RcTnI S23A/24A and RcTnI S23D/24D showed a ~36% decrease in Ca2+-activated maximal tension. Both RcTnI S23A/24A and RcTnI S23D/24D showed a ~18% decrease in ATPase activity. Muscle fiber stiffness measurements suggested that the decrease in thin filament activation observed in RcTnI S23A/24A and RcTnI S23D/24D was due to a decrease in the number of strongly-bound crossbridges. Another major finding was that Ala and Asp substitutions in cTnI did not affect crossbridge detachment kinetics.
Cardiac troponin I; phosphorylation; substitutions; reconstitution; homologous proteins
Experimental studies in hemeproteins and model Tyr/Cys-containing peptides exposed to oxidizing and nitrating species suggest that intramolecular electron transfer (IET) between tyrosyl radicals (Tyr-O●) and Cys residues controls oxidative modification yields. The molecular basis of this IET process is not sufficiently understood with structural atomic detail. Herein, we analyzed using molecular dynamics and quantum mechanics-based computational calculations, mechanistic possibilities for the radical transfer reaction in Tyr/Cys-containing peptides in solution and correlated them with existing experimental data. Our results support that Tyr-O● to Cys radical transfer is mediated by an acid/base equilibrium that involves deprotonation of Cys to form the thiolate, followed by a likely rate-limiting transfer process to yield cysteinyl radical and a Tyr phenolate; proton uptake by Tyr completes the reaction. Both, the pKa values of the Tyr phenol and Cys thiol groups and the energetic and kinetics of the reversible IET are revealed as key physico-chemical factors. The proposed mechanism constitutes a case of sequential, acid/base equilibrium-dependent and solvent-mediated, proton-coupled electron transfer and explains the dependency of oxidative yields in Tyr/Cys peptides as a function of the number of alanine spacers. These findings contribute to explain oxidative modifications in proteins that contain sequence and/or spatially close Tyr-Cys residues.
Electron Transfer; Tyrosyl Radical; Oxidation; Nitration; Computer Simulation
The movement of a conserved protein loop (the WPD-loop) is important in catalysis by protein tyrosine phosphatases (PTPs). Using kinetics, isotope effects, and X-ray crystallography, the different effects arising from mutation of the conserved tryptophan in the WPD-loop were compared in two PTPs, the human PTP1B, and the bacterial YopH from Yersinia. Mutation of the conserved tryptophan in the WPD-loop to phenylalanine has a negligible effect on kcat in PTP1B and full loop movement is maintained. In contrast, the corresponding mutation in YopH reduces kcat by two orders of magnitude and the WPD loop locks in an intermediate position, disabling general acid catalysis. During loop movement the indole moiety of the WPD-loop tryptophan moves in opposite directions in the two enzymes. Comparisons of mammalian and bacterial PTPs reveal differences in the residues forming the hydrophobic pocket surrounding the conserved tryptophan. Thus, although WPD-loop movement is a conserved feature in PTPs, differences exist in the molecular details, and in the tolerance to mutation, in PTP1B compared to YopH. Despite high structural similarity of the active sites in both WPD-loop open and closed conformations, differences are identified in the molecular details associated with loop movement in PTPs from different organisms.
phosphatase; protein-tyrosine phosphatase
Tyrosine autophosphorylation within the cytoplasmic tail of EGF-receptor is a key event, which in turn recruits several factors including Shc, Grb2 and Rin1 that are essential activities for receptor-mediated endocytosis and signaling. In this study, we demonstrated that treatment with AG1478, an EGF-receptor kinase inhibitor, blocked the formation of Rab5-positive endosomes as well as the activation of Rab5 upon addition of EGF. We also found that EGF-receptor catalytically inactive mutant failed to activate Rab5 upon EGF stimulation. Additionally, endosomal co-localization of Rab5 and EGF-receptor was inhibited by AG1478. Interestingly, AG1478 inhibitor did not block the formation of enlarged Rab5-positive endosomes in cells expressing Rab5 GTP hydrolysis defective mutant (Rab5: Q79L). AG1478 inhibitor also blocked the in vitro endosome fusion in a concentration-dependent manner, and more importantly, Rab5: Q79L mutant rescued it. Furthermore, addition of Rin1, a Rab5 guanine nucleotide exchange factor, partially restored enodosome fusion in the presence of AG1478 inhibitor. Consistent with these observations, we also observed that Rin1 was unable to localize to membranes upon EGF-stimulation in the presence of AG1478 inhibitor. These results constitute first evidence that the enzymatic activity of a tyrosine kinase receptor is required endosome fusion via the activation of Rab5.
receptor tyrosine kinase; small GTPase; endosome fusion; receptor-mediated encoytosis; kinase inhibitors
To characterize the function of the sodium/inositol symporter SMIT2 in skeletal muscle, human SMIT2 cDNA was transfected into L6 myoblasts using pcDNA3.1 expression vector. Compared with the pcDNA3.1 vector only transfection, this overexpression increased the uptake of [3H]D-chiro-inositol (DCI) by 159-fold. [3H]myo-Inositol uptake increased by 37-fold. In contrast, [14C]D-glucose, [14C]2-deoxy-D-glucose, or [14C]3-O-methyl-D-glucose uptake remained unchanged in the presence of either 0, 5.5, or 25 mM unlabeled glucose. The Km of DCI and myo-inositol for DCI uptake was 111.0 and 158.0 μM, respectively, whereas glucose competed for DCI uptake with a Ki of 6.1 mM. Insulin treatment of non-transfected L6 cells (2 μM for 24 hours) increased [3H]DCI specific uptake 18-fold. DCI transport is up regulated by insulin and competitively inhibited by millimolar levels of glucose. Therefore, expression and/or function of SMIT2, a high affinity transporter specific for DCI and myo-inositol, may be reduced in diabetes mellitus, insulin resistance and polycystic ovary syndrome causing the abnormal DCI metabolism observed in these conditions.
SMIT2; D-chiro-inositol; myo-inositol; insulin resistance; diabetes; PCOS; DCI; glucose; pinitol
Gap junction channels provide a conduit for communication between neighboring cells. The function of gap junction channels is regulated by posttranslational modifications of connexins, the proteins that comprise these channels. Ubiquitination of connexins has increasingly been viewed as one mechanism by which cells regulate the level of connexins present in cells, as well as the corresponding intercellular communication. Here we review the current knowledge of connexin ubiquitination and the effects this may have on gap junctional communication.
connexin; ubiquitination; degradation; proteasome; lysosome
Gap Junctions (GJ) and hemichannels (HC) formed from the protein subunits called connexins are transmembrane conduits for the exchange of small molecules and ions. Connexins and another group of HC-forming proteins, pannexins comprise the two families of transmembrane proteins ubiquitously distributed in vertebrates. Most cell types express more than one connexin or pannexin. While connexin expression and channel activity may vary as a function of physiological and pathological states of the cell and tissue, only a few studies suggest the involvement of pannexin HC in acquired pathological conditions. Importantly, genetic mutations in connexin appear to interfere with GJ and HC function which results in several diseases. Thus connexins could serve as potential drug target for therapeutic intervention. Growing evidence suggests that diseases resulting from HC dysfunction might open a new direction for development of specific HC reagents. This review provides a comprehensive overview of the current studies of GJ and HC formed by connexins and pannexins in various tissue and organ systems including heart, central nervous system, kidney, mammary glands, ovary, testis, lens, retina, inner ear, bone, cartilage, lung and liver. In addition, present knowledge of the role of GJ and HC in cell cycle progression, carcinogenesis and stem cell development is also discussed.
To assess whether ascorbic acid decreases the cytotoxicity of oxidized human low density lipoprotein (oxLDL) in cells involved in atherosclerosis, its interaction with oxLDL was studied in murine RAW264.7 macrophages. Macrophages took up ascorbate to millimolar intracellular concentrations and retained it with little loss over 18 h in culture. Culture of the macrophages with oxLDL enhanced ascorbate uptake. This was associated with increased expression of the ascorbate transporter (SVCT2), which was prevented by ascorbate and by inhibiting the NF-κB pathway. Culture of RAW264.7 macrophages with oxLDL increased intracellular dihydrofluorescein oxidation and lipid peroxidation, both of which were decreased by intracellular ascorbate. Ascorbate also protected the cells against oxLDL-induced cytotoxicity and apoptosis, but it did not affect macrophage accumulation of lipid from oxLDL or oxLDL-induced increases in macrophage cytokine secretion. These results suggest that ascorbate protects macrophages against oxLDL-induced oxidant stress and subsequent apoptotic death without impairing their function.
Ascorbate transport; oxidant stress; RAW264.7 macrophages; oxidized LDL; SVCT2; dihydrofluorescein; apoptosis; malondialdehyde; atherosclerosis; NF-κB
Human manganese superoxide dismutase (Sod2p) has been expressed in yeast and the protein purified from isolated yeast mitochondria, yielding both the metallated protein and the less stable apoprotein in a single chromatographic step. At 30 °C growth temperature, more than half of the purified enzyme is apoprotein that can be fully activated following reconstitution, while the remainder contains a mixture of manganese and iron. In contrast, only fully metallated enzyme was isolated from a similarly constructed yeast strain expressing the homologous yeast manganese superoxide dismutase. Both the manganese content and superoxide dismutase activity of the recombinant human enzyme increased with increasing growth temperatures. The dependence of in vivo metallation state on growth temperature resembles the in vitro thermal activation behavior of human manganese superoxide dismutase observed in previous studies. Partially metallated human superoxide dismutase is fully active in protecting yeast against superoxide stress produced by addition of paraquat to the growth medium. However, a splice variant of human manganese superoxide dismutase (isoform B) is expressed as insoluble protein in both Escherichia coli and yeast mitochondria and did not protect yeast against superoxide stress.
Manganese; Superoxide dismutase; Thermal activation; Metallation; Splice variant; Mitochondria; Isoform
The response and functions of proteasome regulators Pa28αβ (or 11S), Pa28γ, and Pa200 in oxidative-stress adaptation (also called hormesis) was studied in murine embryonic fibroblasts (MEF), using a well-characterized model of cellular adaptation to low concentrations (1.0 to 10.0μM) of hydrogen peroxide (H2O2), which alter gene expression profiles, increasing resistance to higher levels of oxidative-stress. Pa28αβ bound to 20S proteasomes immediately upon H2O2-treatment, whereas 26S proteasomes were disassembled at the same time. Over the next 24 hours, the levels of Pa28αβ, Pa28γ, and Pa200 proteasome regulators increased during H2O2-adaptation, whereas the 19S regulator was unchanged. Purified Pa28αβ, and to a lesser extent Pa28γ, significantly increased the ability of purified 20S proteasome to selectively degrade oxidized proteins; Pa28αβ also increased the capacity of purified immunoproteasome to selectively degrade oxidized proteins but Pa28γ did not. Pa200 regulator actually decreased 20S proteasome and immunoproteasome’s ability to degrade oxidized proteins but Pa200 and poly-ADP ribose polymerase may cooperate in enabling initiation of DNA repair. Our results indicate that cytoplasmic Pa28αβ and nuclear Pa28γ may both be important regulators of proteasome’s ability to degrade oxidatively-damaged proteins, and induced-expression of both 20S proteasome and immunoproteasome, and their Pa28αβ and Pa28γ regulators are important for oxidative-stress adaptation.
Oxidative stress adaptation (hormesis); Ubiquitin-Proteasome System; Protein Degradation; Proteasome regulators; Pa28 (11S) regulator; Immunoproteasome
► 89 genes encoding flavoproteins were identified in the human genome. ► Two thirds of human flavoproteins are linked to human diseases. ► Flavoenzymes are essential for the biosynthesis of other coenzymes and hormones. ► Flavoenzymes play a critical role in folate and cobalamin metabolism.
Vitamin B2 (riboflavin) is an essential dietary compound used for the enzymatic biosynthesis of FMN and FAD. The human genome contains 90 genes encoding for flavin-dependent proteins, six for riboflavin uptake and transformation into the active coenzymes FMN and FAD as well as two for the reduction to the dihydroflavin form. Flavoproteins utilize either FMN (16%) or FAD (84%) while five human flavoenzymes have a requirement for both FMN and FAD. The majority of flavin-dependent enzymes catalyze oxidation–reduction processes in primary metabolic pathways such as the citric acid cycle, β-oxidation and degradation of amino acids. Ten flavoproteins occur as isozymes and assume special functions in the human organism. Two thirds of flavin-dependent proteins are associated with disorders caused by allelic variants affecting protein function. Flavin-dependent proteins also play an important role in the biosynthesis of other essential cofactors and hormones such as coenzyme A, coenzyme Q, heme, pyridoxal 5′-phosphate, steroids and thyroxine. Moreover, they are important for the regulation of folate metabolites by using tetrahydrofolate as cosubstrate in choline degradation, reduction of N-5.10-methylenetetrahydrofolate to N-5-methyltetrahydrofolate and maintenance of the catalytically competent form of methionine synthase. These flavoenzymes are discussed in detail to highlight their role in health and disease.
Coenzyme A; Coenzyme Q; Folate; Heme; Pyridoxal 5′-phosphate; Steroids; Thyroxine; Vitamins
The tryptophan synthase α2β2 bi-enzyme complex catalyzes the last two steps in the synthesis of L-tryptophan (L-Trp). The α-subunit catalyzes cleavage of 3-indole-D-glycerol 3’-phosphate (IGP) to give indole and D-glyceraldehyde 3’-phosphate (G3P). Indole is then transferred (channeled) via an interconnecting 25 Å-long tunnel, from the α-subunit to the (β-subunit where it reacts with L-Ser in a pyridoxal 5’-phosphate-dependent reaction to give L-Trp and a water molecule. The efficient utilization of IGP and L-Ser by tryptophan synthase to synthesize L-Trp utilizes a system of allosteric interactions that (1) function to switch the α-site on and off at different stages of the β-subunit catalytic cycle, and (2) prevent the escape of the channeled intermediate, indole, from the confines of the α- and β-catalytic sites and the interconnecting tunnel. This review discusses in detail the chemical origins of the allosteric interactions responsible both for switching the α-site on and off, and for triggering the conformational changes between open and closed states which prevent the escape of indole from the bienzyme complex.
Epidemiologic data suggest that the incidence and severity of many types of cancer inversely correlates with indices of vitamin D status. The vitamin D receptor (VDR) is highly expressed in epithelial cells at risk for carcinogenesis including those resident in skin, breast, prostate and colon, providing a direct molecular link by which vitamin D status impacts on carcinogenesis. Consistent with this concept, activation of VDR by its ligand 1,25-dihydroxyvitamin D (1,25D) triggers comprehensive genomic changes in epithelial cells that contribute to maintenance of the differentiated phenotype, resistance to cellular stresses and protection of the genome. Many epithelial cells also express the vitamin D metabolizing enzyme CYP27B1 which enables autocrine generation of 1,25D from the circulating vitamin D metabolite 25-hydroxyvitamin D (25D), critically linking overall vitamin D status with cellular anti-tumor actions. Furthermore, pre-clinical studies in animal models has demonstrated that dietary supplementation with vitamin D or chronic treatment with VDR agonists decreases tumor development in skin, colon, prostate and breast. Conversely, deletion of the VDR gene in mice alters the balance between proliferation and apoptosis, increases oxidative DNA damage, and enhances susceptibility to carcinogenesis in these tissues. Because VDR expression is retained in many human tumors, vitamin D status may be an important modulator of cancer progression in persons living with cancer. Collectively, these observations have reinforced the need to further define the molecular actions of the VDR and the human requirement for vitamin D in relation to cancer development and progression.
vitamin D receptor; cancer; vitamin D; prevention; diet