cytochrome-c-t and quinone

Cytochrome

Topics: Cryoelectron Microscopy; Cytochromes b; Cytochromes c; Electron Transport Complex III; Lipids; Oxidation-Reduction; Quinones; Rhodobacter sphaeroides; Styrene

A marine polycyclic quinone-type metabolite, halenaquinone (HQ), was found to inhibit the proliferation of Molt 4, K562, MDA-MB-231 and DLD-1 cancer cell lines, with IC50 of 0.48, 0.18, 8.0 and 6.76 μg/mL, respectively. It exhibited the most potent activity against leukemia Molt 4 cells. Accumulating evidence showed that HQ may act as a potent protein kinase inhibitor in cancer therapy. To fully understand the mechanism of HQ, we further explored the precise molecular targets in leukemia Molt 4 cells. We found that the use of HQ increased apoptosis by 26.23%-70.27% and caused disruption of mitochondrial membrane potential (MMP) by 17.15%-53.25% in a dose-dependent manner, as demonstrated by Annexin-V/PI and JC-1 staining assays, respectively. Moreover, our findings indicated that the pretreatment of Molt 4 cells with N-acetyl-l-cysteine (NAC), a reactive oxygen species (ROS) scavenger, diminished MMP disruption and apoptosis induced by HQ, suggesting that ROS overproduction plays a crucial rule in the cytotoxic activity of HQ. The results of a cell-free system assay indicated that HQ could act as an HDAC and topoisomerase catalytic inhibitor through the inhibition of pan-HDAC and topoisomerase IIα expression, respectively. On the protein level, the expression of the anti-apoptotic proteins p-Akt, NFκB, HDAC and Bcl-2, as well as hexokinase II was inhibited by the use of HQ. On the other hand, the expression of the pro-apoptotic protein Bax, PARP cleavage, caspase activation and cytochrome c release were increased after HQ treatment. Taken together, our results suggested that the antileukemic effect of HQ is ROS-mediated mitochondrial apoptosis combined with the inhibitory effect on HDAC and topoisomerase activities.

Topics: Animals; Antigens, Neoplasm; Apoptosis; bcl-2-Associated X Protein; Benzoquinones; Cell Line, Tumor; Cytochromes c; DNA Topoisomerases, Type II; DNA-Binding Proteins; Female; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; K562 Cells; Membrane Potential, Mitochondrial; Mice; Mice, Nude; Mitochondria; NF-kappa B; Oxidative Stress; Poly(ADP-ribose) Polymerases; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-bcl-2; Quinones; Reactive Oxygen Species

Biologically reactive intermediates are formed following metabolism of xenobiotics, and during normal oxidative metabolism. These reactive species are electrophilic in nature and are capable of forming stable adducts with target proteins. These covalent protein modifications can initiate processes that lead to acute tissue injury or chronic disease. Recent advancements in mass spectrometry techniques and data analysis has permitted a more detailed investigation of site-specific protein modifications by reactive electrophiles. Knowledge from such analyses will assist in providing a better understanding of how specific classes of electrophiles produce toxicity and disease progression via site-selective protein-specific covalent modification. Hydroquinone (HQ) is a known environmental toxicant, and its quinone-thioether metabolites, formed via the intermediate generation of 1,4-benzoquinone (1,4-BQ), elicit their toxic response via the covalent modification of target proteins and the generation of reactive oxygen species. We have utilized a model protein, cytochrome c, to guide us in identifying 1,4-BQ- and 1,4-BQ-thioether derived site-specific protein modifications. LC-MS/MS analyses reveals that these modifications occur selectively on lysine and glutamic acid residues of the target protein, and that these modifications occur within identifiable "electrophile binding motifs" within the protein. These motifs are found within lysine-rich regions of the protein and appear to be target sites of 1,4-BQ-thioether adduction. These residues also appear to dictate the nature of post-adduction chemistry and the final structure of the adduct. This model system will provide critical insight for in vivo adduct hunting following exposure to 1,4-BQ-thioethers, but the general approaches can also be extended to the identification of protein adducts derived from other classes of reactive electrophiles.

Topics: Amino Acid Sequence; Animals; Benzoquinones; Binding Sites; Chromatography, Liquid; Cytochromes c; Molecular Sequence Data; Protein Processing, Post-Translational; Proteins; Substrate Specificity; Tandem Mass Spectrometry; Xenobiotics

Tetrahydroisoquinoline (TIQ) derivatives are putative neurotoxins that may contribute to the degeneration of dopaminergic neurons in Parkinson's disease. One TIQ, norsalsolinol (NorSAL), is present in dopamine-rich areas of human brain, including the substantia nigra. Here, we demonstrate that NorSAL reduces cell viability and induces apoptosis via cytochrome c release and caspase 3 activation in SH-SY5Y human neuroblastoma cells. Cytochrome c release, caspase 3 activation, and apoptosis induction were all inhibited by the antioxidant N-acetylcysteine. Thus, reactive oxygen species (ROS) contribute to apoptosis induced by NorSAL. Treatment with NorSAL also increased levels of oxidative damage to DNA, a stimulus for apoptosis, in SH-SY5Y. To clarify the mechanism of intracellular DNA damage, we examined the DNA damage caused by NorSAL using (32)P-5'-end-labeled isolated DNA fragments. NorSAL induced DNA damage in the presence of Cu(II). Catalase and bathocuproine, a Cu(I) chelator, inhibited this DNA damage, suggesting that ROS such as the Cu(I)-hydroperoxo complex derived from the reaction of H(2)O(2) with Cu(I), promote DNA damage by NorSAL. In summary, NorSAL-generated ROS induced oxidative DNA damage, which led to caspase-dependent apoptosis in neuronal cells.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Apoptosis; Autoradiography; Benzoquinones; Caspase 3; Cell Line, Tumor; Copper; Cytochromes c; Deoxyguanosine; DNA Damage; Dose-Response Relationship, Drug; Free Radical Scavengers; Humans; NAD; Neuroblastoma; Phenanthrolines; Phosphorus Isotopes; Salsoline Alkaloids; Tetrahydroisoquinolines; Tetrazolium Salts; Thiazoles; Time Factors; Tyrosine 3-Monooxygenase

The neurotoxin 6-hydroxydopamine (6-OHDA) has been widely used to generate an experimental model of Parkinson's disease. It has been reported that reactive oxygen species (ROS), such as the superoxide anion and hydrogen peroxide (H2O2), generated from 6-OHDA are involved in its cytotoxicity; however, the contribution and role of ROS in 6-OHDA-induced cell death have not been fully elucidated. In the present study using PC12 cells, we observed the generation of 50 microM H2O2 from a lethal concentration of 100 microM 6-OHDA within a few minutes, and compared the sole effect of H2O2 with 6-OHDA. Catalase, an H2O2-removing enzyme, completely abolished the cytotoxic effect of H2O2, while a significant but partial protective effect was observed against 6-OHDA. 6-OHDA induced peroxiredoxin oxidation, cytochrome c release, and caspase-3 activation. Catalase exhibited a strong inhibitory effect against the peroxiredoxin oxidation, and cytochrome c release induced by 6-OHDA; however, caspase-3 activation was not effectively inhibited by catalase. On the other hand, 6-OHDA-induced caspase-3 activation was inhibited in the presence of caspase-8, caspase-9, and calpain inhibitors. These results suggest that the H2O2 generated from 6-OHDA plays a pivotal role in 6-OHDA-induced peroxiredoxin oxidation, and cytochrome c release, while H2O2- and cytochrome c-independent caspase activation pathways are involved in 6-OHDA-induced neurotoxicity. These findings may contribute to explain the importance of generated H2O2 and secondary products as a second messenger of 6-OHDA-induced cell death signal linked to Parkinson's disease.

Topics: Animals; Antioxidants; Benzoquinones; Catalase; Cell Death; Cell Survival; Cytochromes c; Cytotoxins; Hydrogen Peroxide; Models, Biological; Oxidation-Reduction; Oxidopamine; PC12 Cells; Peroxidases; Peroxiredoxins; Rats; Signal Transduction

Electrophiles generated endogenously, or via the metabolic bioactivation of drugs and other environmental chemicals, are capable of binding to a variety of nucleophilic sites within proteins. Factors that determine site selective susceptibility to electrophile-mediated post-translational modifications, and the consequences of such alterations, remain largely unknown. To identify and characterize chemical-mediated protein adducts, electrophiles with known toxicity were utilized. Hydroquinone, and its mercapturic acid pathway metabolites, cause renal proximal tubular cell necrosis and nephrocarcinogenicity in rats. The adverse effects of HQ and its thioether metabolites are in part a consequence of their oxidation to the corresponding electrophilic 1,4-benzoquinones (BQ). We now report that BQ and 2-(N-acetylcystein-S-yl)benzoquinone (NAC-BQ) preferentially bind to solvent-exposed lysine-rich regions within cytochrome c. Furthermore, we have identified specific glutamic acid residues within cytochrome c as novel sites of NAC-BQ adduction. The microenvironment at the site of adduction governs both the initial specificity and the structure of the final adduct. The solvent accessibility and local pKa of the adducted and neighboring amino acids contribute to the selectivity of adduction. Postadduction chemistry subsequently alters the nature of the final adduct. Using molecular modeling, the impact of BQ and NAC-BQ adduction on cytochrome c was visualized, revealing the spatial rearrangement of critical residues necessary for protein-protein interactions. Consequently, BQ-adducted cytochrome c fails to initiate caspase-3 activation in native lysates and also inhibits Apaf-1 oligomerization into an apoptosome complex in a purely reconstituted system. In summary, a combination of mass spectroscopic, molecular modeling, and biochemical approaches confirms that electrophile-protein adducts produce structural alterations that influence biological function.

Topics: Acetylcysteine; Amino Acid Motifs; Amino Acid Sequence; Animals; Apoptosomes; Benzoquinones; Caspase 3; Caspase 9; Cell Line, Tumor; Chromatography, Liquid; Circular Dichroism; Cytochromes c; Horses; Humans; Hydrogen-Ion Concentration; Models, Molecular; Molecular Sequence Data; Molecular Structure; Protein Binding; Protein Conformation; Protein Structure, Secondary; Protein Structure, Tertiary; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Tandem Mass Spectrometry

The influence of a number of redox reagents on the charge state distribution in nanoelectrospray mass spectrometry was examined using cytochrome c and ubiquitin. The redox active species investigated were: 1,4-benzoquinone, quinhydrone, tetracyanoquinodimethane (TCNQ), hydroquinone, and ascorbic acid. The redox active species was mixed with the protein sample before injection into the nanoelectrospray emitter, and mass spectra were acquired using a triple quadrupole mass spectrometer. Under the same experimental conditions, the charge state distribution of cytochrome c was observed to shift from a weighted average charge state of 14.25 (in the absence of redox species) to 7.10 in the presence of 1,4-benzoquinone. When quinhydrone was mixed with cytochrome c, the charge state distribution of the protein also shifted to lower charge states (weighted average charge state = 9.43), indicative of less charge state reduction for quinhydrone than with 1,4-benzoquinone. Addition of the redox reagent had little effect on the conformation of cytochrome c, as indicated by far ultraviolet circular dichroism spectra. In contrast, the reagents TCNQ, hydroquinone, and ascorbic acid exhibited negligible effects on the observed charge state distribution of the protein. The differing results for these redox reagents can be rationalized in terms of the redox half reactions involving these species. The results observed with ubiquitin upon adding quinhydrone were analogous to those observed with cytochrome c.

Topics: Benzoquinones; Cytochromes c; Hydroquinones; Indicators and Reagents; Nanotechnology; Oxidation-Reduction; Proteins; Spectrometry, Mass, Electrospray Ionization; Ubiquitin

Cytochrome c (cyt c) was reduced by a tyrosine-containing peptide, tyrosyltyrosylphenylalanine (TyrTyrPhe), at pH 6.0-8.0, while tyrosinol or tyrosyltyrosine (TyrTyr) could not reduce cyt c effectively under the same condition. Cyt c was reduced at high peptide concentration, whereas the reaction did not occur effectively at low concentration. The reaction rate varied with time owing to a decrease in the TyrTyrPhe concentration and the production of tyrosine derivatives during the reaction. The initial rate constants were 2.4 x 10(-4) and 8.1 x 10(-4) s(-1) at pH 7.0 and 8.0, respectively, for the reaction with 1.0 mM TyrTyrPhe in 10 mM phosphate buffer at 15 degrees C. The reciprocal initial rate constant (1/k(int)) increased linearly against the reciprocal peptide concentration and against the linear proton concentration, whereas logk(int) decreased linearly against the root of the ionic strength. These results show that deprotonated (TyrTyrPhe)(-), presumably deprotonated at a tyrosine site, reduces cyt c by formation of an electrostatic complex. No significant difference in the reaction rate was observed between the reaction under nitrogen and oxygen atmospheres. From the matrix-assisted laser desorption ionization time-of-flight mass spectra of the reaction products, formation of a quinone and other tyrosine derivatives of the peptide was supported. These products should have been produced from a tyrosyl radical. We interpret the results that a cyt c(ox)/(TyrTyrPhe)(-)right harpoon over left harpooncyt c(red)/(TyrTyrPhe)(*) equilibrium is formed, which is usually shifted to the left. This equilibrium may shift to the right by reaction of the produced tyrosyl radical with the tyrosine sites of unreacted TyrTyrPhe peptides.

Topics: Animals; Benzoquinones; Cytochromes c; Horses; Kinetics; Oligopeptides; Oxidation-Reduction; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Identification of multiple and novel posttranslational modifications remains a major challenge in proteomics. The present approach uses comparative analysis by matrix-assisted laser/desorption ionization (MALDI) MS of proteolytic digests from control and treated proteins to target differences due to modifications, without initial assumption as to type or residue localization. Differences between modified and unmodified digest MS spectra highlight peptides of interest for subsequent tandem mass spectrometry (MS/MS) analysis. Targeted HPLC-electrospray ionization (ESI)-MS/MS is then used to fragment peptides, and manual de novo sequencing is used to determine the amino acid sequence and type of modification. This strategy for identifying posttranslational modifications in an unbiased manner is particularly useful for finding modifications produced by exogenous chemicals. Successful characterization of chemically induced posttranslational modifications and novel chemical adducts is given as an example of the use of this strategy. Histone H4 from butyrate-treated LLC-PK1 cells is separated on a gel into bands representing different overall charge state. Bands are analyzed by comparative MALDI-MS and LC-MS/MS to identify the sites of methylation and acetylation. Previous attempts to identify chemically adducted proteins in vivo have been unsuccessful in part due to a lack of understanding of the final adduct form. Cytochrome c is adducted in vitro with benzoquinone, an electrophilic metabolite of benzene capable of interacting with nucleophilic sites within proteins. De novo sequencing identifies a novel cyclized diquinone adduct species as the major reaction product, targeting Lys and His residues at two specific locations on the protein surface. This unpredicted reaction product is characterized using our unbiased methods for detection and demonstrates the important influence of protein structure on chemical adduction.

Topics: Acetylation; Animals; Benzoquinones; Butyrates; Cell Line; Chromatography, High Pressure Liquid; Cytochromes c; Histones; Horses; Methylation; Models, Molecular; Protein Processing, Post-Translational; Spectrometry, Mass, Electrospray Ionization; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Swine, Miniature