RESUMO
Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine with roles in innate and adaptive human immune responses, as well as inflammation. MIF exerts its biological activity by binding to the cell surface receptor CD74 as well as intracellular signalling proteins. MIF also possesses keto-enol tautomerase activity. Inhibition of the tautomerase activity has been associated with loss of biological activity of MIF and a potential anticancer target. Isothiocyanates (ITCs) are a class of compounds present in cruciferous vegetables that inhibit the MIF tautomerase activity via covalent modification of the N-terminal proline. A range of substituted ITCs featuring benzyl, phenethyl and phenyl propyl isothiocyanates were designed, synthesised and tested to determine any structure activity relationship for inhibiting MIF. Crystal structures of covalent compounds 8 and 9 in complex with rhMIF revealed key hydrogen bonding and edge-to-face π stacking interactions. Compound 9 and 11 with sub micromolar activity were tested in the NCI60 cancer cell lines panel. Both compounds showed tissue-specific reduced growth in colon and renal cancer cell lines, while one of these showed potent, dose-dependent inhibition of growth against all seven colon cancer cell lines (GI50 < 2.5 µM) and all eight renal cancer cell lines (GI50 < 2.2 µM).
RESUMO
Variants in the gene encoding human cytochrome c (CYCS) cause mild autosomal dominant thrombocytopenia. Despite high sequence conservation between mouse and human cytochrome c, this phenotype is not recapitulated in mice for the sole mutant (G41S) that has been investigated. The effect of the G41S mutation on the in vitro activities of cytochrome c is also not conserved between human and mouse. Peroxidase activity is increased in both mouse and human G41S variants, whereas apoptosome activation is increased for human G41S cytochrome c but decreased for mouse G41S cytochrome c. These apoptotic activities of cytochrome c are regulated at least in part by conformational dynamics of the main chain. Here we use computational and in vitro approaches to understand why the impact of the G41S mutation differs between mouse and human cytochromes c. The G41S mutation increases the inherent entropy and main chain mobility of human but not mouse cytochrome c. Exclusively in human G41S cytochrome c this is accompanied by a decrease in occupancy of H-bonds between protein and heme during simulations. These data demonstrate that binding of cytochrome c to Apaf-1 to trigger apoptosome formation, but not the peroxidase activity of cytochrome c, is enhanced by increased mobility of the native protein conformation.
Assuntos
Citocromos c , Ativação Enzimática , Mutação , Conformação Proteica , Citocromos c/metabolismo , Citocromos c/genética , Citocromos c/química , Humanos , Animais , Camundongos , Especificidade da Espécie , Simulação de Dinâmica Molecular , Caspases/metabolismo , Caspases/genética , Caspases/químicaRESUMO
The problematic opportunistic pathogen Pseudomonas aeruginosa secretes a siderophore, pyoverdine. Pyoverdine scavenges iron needed by the bacteria for growth and for pathogenicity in a range of different infection models. PvdF, a hydroxyornithine transformylase enzyme, is essential for pyoverdine synthesis, catalysing synthesis of formylhydroxyornithine (fOHOrn) that forms part of the pyoverdine molecule and provides iron-chelating hydroxamate ligands. Using a mass spectrometry assay, we confirm that purified PvdF catalyses synthesis of fOHOrn from hydroxyornithine and formyltetrahydrofolate substrates. Site directed mutagenesis was carried out to investigate amino acid residues predicted to be required for enzymatic activity. Enzyme variants were assayed for activity in vitro and also in vivo, through measuring their ability to restore pyoverdine production to a pvdF mutant strain. Variants at two putative catalytic residues N168 and H170 greatly reduced enzymatic activity in vivo though did not abolish activity in vitro. Change of a third residue D229 abolished activity both in vivo and in vitro. A change predicted to block entry of N10-formyltetrahydrofolate (fTHF) to the active site also abolished activity both in vitro and in vivo. A co-purification assay showed that PvdF binds to an enzyme PvdA that catalyses synthesis of hydroxyornithine, with this interaction likely to increase the efficiency of fOHOrn synthesis. Our findings advance understanding of how P. aeruginosa synthesises pyoverdine, a key factor in host-pathogen interactions.
Assuntos
Proteínas de Bactérias/metabolismo , Hidroximetil e Formil Transferases/metabolismo , Oxigenases de Função Mista/metabolismo , Sideróforos/biossíntese , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Domínio Catalítico , Hidroximetil e Formil Transferases/genética , Hidroximetil e Formil Transferases/isolamento & purificação , Oxigenases de Função Mista/genética , Mutagênese Sítio-Dirigida , Oligopeptídeos/biossíntese , Mapas de Interação de Proteínas , Estabilidade Proteica , Pseudomonas aeruginosa/metabolismoRESUMO
Mutation of cytochrome c in humans causes mild autosomal dominant thrombocytopenia. The role of cytochrome c in platelet formation, and the molecular mechanism underlying the association of cytochrome c mutations with thrombocytopenia remains unknown, although a gain-of-function is most likely. Cytochrome c contributes to several cellular processes, with an exchange between conformational states proposed to regulate changes in function. Here, we use experimental and computational approaches to determine whether pathogenic variants share changes in structure and function, and to understand how these changes might occur. Three pathogenic variants (G41S, Y48H, A51V) cause an increase in apoptosome activation and peroxidase activity. Molecular dynamics simulations of these variants, and two non-naturally occurring variants (G41A, G41T), indicate that increased apoptosome activation correlates with the increased overall flexibility of cytochrome c, particularly movement of the Ω loops. Crystal structures of Y48H and G41T complement these studies which overall suggest that the binding of cytochrome c to apoptotic protease activating factor-1 (Apaf-1) may involve an 'induced fit' mechanism which is enhanced in the more conformationally mobile variants. In contrast, peroxidase activity did not significantly correlate with protein dynamics. Thus, the mechanism by which the variants increase peroxidase activity is not related to the conformational dynamics of the native hexacoordinate state of cytochrome c. Recent molecular dynamics data proposing conformational mobility of specific cytochrome c regions underpins changes in reduction potential and alkaline transition pK was not fully supported. These data highlight that conformational dynamics of cytochrome c drive some but not all of its properties and activities.
Assuntos
Apoptose/fisiologia , Citocromos c/química , Mutação de Sentido Incorreto , Mutação Puntual , Substituição de Aminoácidos , Apoptossomas , Cristalografia por Raios X , Citocromos c/genética , Citocromos c/isolamento & purificação , Citocromos c/metabolismo , Humanos , Ligação de Hidrogênio , Peróxido de Hidrogênio/metabolismo , Concentração de Íons de Hidrogênio , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Oxirredução , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Células U937RESUMO
Plasma lipoprotein (a) [Lp(a)] levels are largely determined by variation in the LPA gene, which codes for apo(a). Genome-wide association studies (GWASs) have identified nonsynonymous variants in LPA that associate with low Lp(a) levels, although their effect on apo(a) function is unknown. We investigated two such variants, R990Q and R1771C, which were present in four null Lp(a) individuals, for structural and functional effects. Sequence alignments showed the R990 and R1771 residues to be highly conserved and homologous to each other and to residues associated with plasminogen deficiency. Structural modeling showed both residues to make several polar contacts with neighboring residues that would be ablated on substitution. Recombinant expression of the WT and R1771C apo(a) in liver and kidney cells showed an abundance of an immature form for both apo(a) proteins. A mature form of apo(a) was only seen with the WT protein. Imaging of the recombinant apo(a) proteins in conjunction with markers of the secretory pathway indicated a poor transit of R1771C into the Golgi. Furthermore, the R1771C mutant displayed a glycosylation pattern consistent with ER, but not Golgi, glycosylation. We conclude that R1771 and the equivalent R990 residue facilitate correct folding of the apo(a) kringle structure and mutations at these positions prevent the proper folding required for full maturation and secretion. To our knowledge, this is the first example of nonsynonymous variants in LPA being causative of a null Lp(a) phenotype.
Assuntos
Apoproteína(a)/genética , Lipoproteína(a)/genética , Plasminogênio/genética , Polimorfismo de Nucleotídeo Único/genética , Adulto , Idoso , Alelos , Linhagem Celular Tumoral , Estudos de Coortes , Humanos , Lipoproteína(a)/sangue , Masculino , Pessoa de Meia-Idade , Mutação , Plasminogênio/deficiênciaRESUMO
Thiol dioxygenases make up a class of ferrous iron-dependent enzymes that oxidize thiols to their corresponding sulfinates. X-ray diffraction structures of cysteine-bound cysteine dioxygenase show how cysteine is coordinated via its thiolate and amine to the iron and oriented correctly for O atom transfer. There are currently no structures with 3-mercaptopropionic acid or mercaptosuccinic acid bound to their respective enzymes, 3-mercaptopropionate dioxygenase or mercaptosuccinate dioxygenase. Sequence alignments and comparisons of known structures have led us to postulate key structural features that define substrate specificity. Here, we compare the rates and reactivities of variants of Rattus norvegicus cysteine dioxygenase and 3-mercaptopropionate dioxygenases from Pseudomonas aureginosa and Ralstonia eutropha (JMP134) and show how binary variants of three structural features correlate with substrate specificity and reactivity. They are (1) the presence or absence of a cis-peptide bond between residues Ser158 and Pro159, (2) an Arg or Gln at position 60, and (3) a Cys or Arg at position 164 (all RnCDO numbering). Different permutations of these features allow sulfination of l-cysteine, 3-mercaptopropionic acid, and ( R)-mercaptosuccinic acid to be promoted or impeded.
Assuntos
Ácido 3-Mercaptopropiônico/química , Cisteína Dioxigenase/química , Compostos de Sulfidrila/química , Sequência de Aminoácidos , Animais , Catálise , Cristalografia por Raios X , Cupriavidus necator/química , Cisteína/química , Ferro/química , Cinética , Simulação de Acoplamento Molecular , Oxirredução , Pseudomonas/química , Ratos , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
Cysteine dioxygenase is a non-heme mononuclear iron enzyme with unique structural features, namely an intramolecular thioether cross-link between cysteine 93 and tyrosine 157, and a disulfide bond between substrate L-cysteine and cysteine 164 in the entrance channel to the active site. We investigated how these posttranslational modifications affect catalysis through a kinetic, crystallographic and computational study. The enzyme kinetics of a C164S variant are identical to WT, indicating that disulfide formation at C164 does not significantly impair access to the active site at physiological pH. However, at high pH, the cysteine-tyrosine cross-link formation is enhanced in C164S. This supports the view that disulfide formation at position 164 can limit access to the active site. The C164S variant yielded crystal structures of unusual clarity in both resting state and with cysteine bound. Both show that the iron in the cysteine-bound complex is a mixture of penta- and hexa-coordinate with a water molecule taking up the final site (60 % occupancy), which is where dioxygen is believed to coordinate during turnover. The serine also displays stronger hydrogen bond interactions to a water bound to the amine of the substrate cysteine. However, the interactions between cysteine and iron appear unchanged. DFT calculations support this and show that WT and C164S have similar binding energies for the water molecule in the final site. This variant therefore provides evidence that WT also exists in an equilibrium between penta- and hexa-coordinate forms and the presence of the sixth ligand does not strongly affect dioxygen binding.
Assuntos
Cisteína Dioxigenase/química , Cisteína/química , Animais , Domínio Catalítico , Cisteína/metabolismo , Cisteína Dioxigenase/isolamento & purificação , Cisteína Dioxigenase/metabolismo , Modelos Moleculares , Teoria Quântica , Ratos , SoftwareRESUMO
Thiol dioxygenases catalyze the synthesis of sulfinic acids in a range of organisms from bacteria to mammals. A thiol dioxygenase from the bacterium Pseudomonas aeruginosa oxidizes both 3-mercaptopropionic acid and cysteine, with a â¼70 fold preference for 3-mercaptopropionic acid over all pHs. This substrate reactivity is widened compared to other thiol dioxygenases and was exploited in this investigation of the residues important for activity. A simple model incorporating two protonation events was used to fit profiles of the Michaelis-Menten parameters determined at different pH values for both substrates. The pKs determined using plots of k(cat)/Km differ at low pH, but not in a way easily attributable to protonation of the substrate alone and share a common value at higher pH. Plots of k(cat) versus pH are also quite different at low pH showing the monoprotonated ES complexes with 3-mercaptopropionic acid and cysteine have different pKs. At higher pH, k(cat) decreases sigmoidally with a similar pK regardless of substrate. Loss of reactivity at high pH is attributed to deprotonation of tyrosine 159 and its influence on dioxygen binding. A mechanism is proposed by which deprotonation of tyrosine 159 both blocks oxygen binding and concomitantly promotes cystine formation. Finally, the role of tyrosine 159 was further probed by production of a G95C variant that is able to form a cysteine-tyrosine crosslink homologous to that found in mammalian cysteine dioxygenases. Activity of this variant is severely impaired. Crystallography shows that when un-crosslinked, the cysteine thiol excludes tyrosine 159 from its native position, while kinetic analysis shows that the thioether bond impairs reactivity of the crosslinked form.
Assuntos
Ácido 3-Mercaptopropiônico/química , Proteínas de Bactérias/química , Dioxigenases/química , Pseudomonas aeruginosa/enzimologia , Ácido 3-Mercaptopropiônico/isolamento & purificação , Proteínas de Bactérias/isolamento & purificação , Cristalografia por Raios X , Dioxigenases/isolamento & purificação , Concentração de Íons de Hidrogênio , Estrutura Secundária de Proteína , Especificidade por Substrato/fisiologiaRESUMO
Thiol dioxygenation is the initial oxidation step that commits a thiol to important catabolic or biosynthetic pathways. The reaction is catalyzed by a family of specific non-heme mononuclear iron proteins each of which is reported to react efficiently with only one substrate. This family of enzymes includes cysteine dioxygenase, cysteamine dioxygenase, mercaptosuccinate dioxygenase, and 3-mercaptopropionate dioxygenase. Using sequence alignment to infer cysteine dioxygenase activity, a cysteine dioxygenase homologue from Pseudomonas aeruginosa (p3MDO) has been identified. Mass spectrometry of P. aeruginosa under standard growth conditions showed that p3MDO is expressed in low levels, suggesting that this metabolic pathway is available to the organism. Purified recombinant p3MDO is able to oxidize both cysteine and 3-mercaptopropionic acid in vitro, with a marked preference for 3-mercaptopropionic acid. We therefore describe this enzyme as a 3-mercaptopropionate dioxygenase. Mössbauer spectroscopy suggests that substrate binding to the ferrous iron is through the thiol but indicates that each substrate could adopt different coordination geometries. Crystallographic comparison with mammalian cysteine dioxygenase shows that the overall active site geometry is conserved but suggests that the different substrate specificity can be related to replacement of an arginine by a glutamine in the active site.
Assuntos
Ácido 3-Mercaptopropiônico/química , Proteínas de Bactérias/química , Cisteína Dioxigenase/química , Pseudomonas aeruginosa/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Cisteína/química , Ferro/química , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Oxigênio/química , Consumo de Oxigênio , Peptídeos/química , Ligação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Espectrofotometria , Especificidade por Substrato , Compostos de SulfidrilaRESUMO
Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that has roles in the innate immune response, and also contributes to inflammatory disease. While the biological properties of MIF are closely linked to protein-protein interactions, MIF also has tautomerase activity. Inhibition of this activity interferes with the interaction of MIF with protein partners e.g. the CD74 receptor, and tautomerase inhibitors show promise in disease models including multiple sclerosis and colitis. Isothiocyanates inhibit MIF tautomerase activity via covalent modification of the N-terminal proline. We systematically explored variants of benzyl and phenethyl isothiocyanates, to define determinants of inhibition. In particular, substitution with hydroxyl, chloro, fluoro and trifluoro moieties at the para and meta positions were evaluated. In assays on treated cells and recombinant protein, the IC50 varied from 250 nM to >100 µM. X-ray crystal structures of selected complexes revealed that two binding modes are accessed by some compounds, perhaps owing to strain in short linkers between the isothiocyanate and aromatic ring. The variety of binding modes confirms the existence of two subsites for inhibitors and establishes a platform for the development of potent inhibitors of MIF that only need to target one of these subsites.
Assuntos
Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Oxirredutases Intramoleculares/antagonistas & inibidores , Oxirredutases Intramoleculares/metabolismo , Isotiocianatos/metabolismo , Isotiocianatos/farmacologia , Fatores Inibidores da Migração de Macrófagos/antagonistas & inibidores , Fatores Inibidores da Migração de Macrófagos/metabolismo , Desenho de Fármacos , Humanos , Oxirredutases Intramoleculares/química , Células Jurkat , Fatores Inibidores da Migração de Macrófagos/química , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação ProteicaRESUMO
Cysteine dioxygenase (CDO) is a non-heme monoiron enzyme with an unusual posttranslational modification in the proximity of the ferrous iron active site. This modification, a cysteine to tyrosine thioether bond, cross-links two ß-strands of the ß-barrel. We have investigated its role in catalysis through a combined crystallographic and kinetic approach. The C93G variant lacks the cross-link and shows little change in structure from that of the wild type, suggesting that the cross-link does not stabilize an otherwise unfavorable conformation. A pH-dependent kinetic study shows that both cross-linked and un-cross-linked CDO are active but the optimal pH decreases with the presence of the cross-link. This result reflects the effect of the thioether bond on the pKa of Y157 and this residue's role in catalysis. At higher pH values, kcat is also higher for the cross-linked form, extending the pH range of activity. We therefore propose that the cross-link also increases activity by controlling deleterious interactions involving the thiol/ate of C93.
Assuntos
Cisteína Dioxigenase/química , Cisteína/química , Tirosina/química , Substituição de Aminoácidos , Animais , Catálise , Cristalografia por Raios X , Cisteína/genética , Cisteína Dioxigenase/genética , Concentração de Íons de Hidrogênio , Mutação de Sentido Incorreto , Estrutura Secundária de Proteína , Ratos , Tirosina/genéticaRESUMO
The physiological function of urate is poorly understood. It may act as a danger signal, an antioxidant, or a substrate for heme peroxidases. Whether it reacts sufficiently rapidly with lactoperoxidase (LPO) to act as a physiological substrate remains unknown. LPO is a mammalian peroxidase that plays a key role in the innate immune defense by oxidizing thiocyanate to the bactericidal and fungicidal agent hypothiocyanite. We now demonstrate that urate is a good substrate for bovine LPO. Urate was oxidized by LPO to produce the electrophilic intermediates dehydrourate and 5-hydroxyisourate, which decayed to allantoin. In the presence of superoxide, high yields of hydroperoxides were formed by LPO and urate. Using stopped-flow spectroscopy, we determined rate constants for the reaction of urate with compound I (k1 = 1.1 × 10(7) M(-1) s(-1)) and compound II (k2 = 8.5 × 10(3) M(-1) s(-1)). During urate oxidation, LPO was diverted from its peroxidase cycle because hydrogen peroxide reacted with compound II to give compound III. At physiologically relevant concentrations, urate competed effectively with thiocyanate, the main substrate of LPO for oxidation, and inhibited production of hypothiocyanite. Similarly, hypothiocyanite-dependent killing of Pseudomonas aeruginosa was inhibited by urate. Allantoin was present in human saliva and associated with the concentration of LPO. When hydrogen peroxide was added to saliva, oxidation of urate was dependent on its concentration and peroxidase activity. Our findings establish urate as a likely physiological substrate for LPO that will influence host defense and give rise to reactive electrophilic metabolites.
Assuntos
Lactoperoxidase/metabolismo , Tiocianatos/metabolismo , Ácido Úrico/metabolismo , Animais , Antibacterianos/metabolismo , Ligação Competitiva , Bovinos , Humanos , Imunidade Inata , Cinética , Lactoperoxidase/imunologia , Modelos Biológicos , Oxirredução , Pseudomonas aeruginosa/imunologia , Saliva/imunologia , Saliva/metabolismo , Especificidade por SubstratoRESUMO
A fast and easy method for enzyme activity assays using the chromogenic Ellman reagent, 5,5'-dithiobis(2-nitrobenzoic acid), was developed. The method was used to measure the activity of the nonheme mono-iron enzyme cysteine dioxygenase. Quantifying the depletion of the substrate, cysteine, allowed standard kinetic parameters to be determined for the enzyme from Rattus norvegicus. The assay was also used to quickly test the effects of ionic strength, pH, enzyme storage conditions, and potential inhibitors and activators. This assay facilitates a higher throughput than available HPLC-based assays, as it enjoys the advantages of fewer sample handling steps, implementation in a 96-well format, and speed. In addition, the relative specificity of Ellman's reagent, coupled with its reaction with a wide range of thiols, means that this assay is applicable to many enzymes. Finally, the use of readily available reagents and instrumentation means that this assay can be used by practically any research group to compare results with those of other groups.
Assuntos
Compostos Cromogênicos/química , Cisteína Dioxigenase/metabolismo , Ácido Ditionitrobenzoico/química , Ensaios Enzimáticos/métodos , Animais , Cisteína/metabolismo , Cisteína Dioxigenase/antagonistas & inibidores , Ativadores de Enzimas/farmacologia , Inibidores Enzimáticos/farmacologia , Ratos , Especificidade por SubstratoRESUMO
Photosystem II (PS II) is a macromolecular complex responsible for light-driven oxidation of water and reduction of plastoquinone as part of the photosynthetic electron transport chain found in thylakoid membranes. Each PS II complex is composed of at least 20 protein subunits and over 80 cofactors. The biogenesis of PS II requires further hydrophilic and membrane-spanning proteins which are not part of the active holoenzyme. Many of these biogenesis proteins make transient interactions with specific PS II assembly intermediates: sometimes these are essential for biogenesis while in other examples they are required for optimizing assembly of the mature complex. In this review the function and structure of the Psb27, Psb28 and Ycf48 hydrophilic assembly factors is discussed by combining structural, biochemical and physiological information. Each of these assembly factors has homologues in all oxygenic photosynthetic organisms. We provide a simple overview for the roles of these protein factors in cyanobacterial PS II assembly emphasizing their participation in both photosystem biogenesis and recovery from photodamage.
Assuntos
Modelos Moleculares , Complexo de Proteína do Fotossistema II/metabolismo , Synechocystis/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Luz , Complexo de Proteína do Fotossistema II/química , Synechocystis/efeitos da radiaçãoRESUMO
The peroxidase activity of cytochrome c may play a key role in the release of cytochrome c from the mitochondrial intermembrane space in the intrinsic apoptosis pathway. Induction of the peroxidase activity of cytochrome c is ascribed to partial unfolding and loss of axial co-ordination between the haem Fe and Met80, and is thought to be triggered by interaction of cytochrome c with cardiolipin (diphosphatidylglycerol) in vivo. However, the reaction mechanism for the peroxidase activity of either native or cardiolipin-bound cytochrome c is uncertain. In the present study we analyse the peroxidase activity of human and mouse cytochrome c residue 41 variants and demonstrate that stimulation of peroxidase activity can occur without prior loss of Fe-Met80 co-ordination or partial unfolding. The effects of cardiolipin and mutation of residue 41 are not additive, suggesting that cardiolipin stimulates peroxidase activity by the same mechanism as residue 41 mutation. Consistent with this, mutation of residue 41 did not enhance apoptotic release of cytochrome c from mitochondria. We propose that mutation of residue 41, and interaction with cardiolipin, increase peroxidase activity by altering the 40-57 Ω loop and its hydrogen bond network with the propionate of haem ring A. These changes enhance access of hydrogen peroxide and substrate to the haem.
Assuntos
Citocromos c/metabolismo , Mutação/genética , Peroxidase/genética , Peroxidase/metabolismo , Animais , Ativação Enzimática/genética , Variação Genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias Hepáticas/metabolismo , Peroxidase/química , Desdobramento de ProteínaRESUMO
Describing the organization of substrates and substrate analogues in the active site of cysteine dioxygenase identifies potential intermediates in this critical yet poorly understood reaction, the oxidation of cysteine to cysteine sulfinic acid. The fortuitous formation of persulfides under crystallization conditions has allowed their binding in the active site of cysteine dioxygenase to be studied. The crystal structures of cysteine persulfide and 3-mercaptopropionic acid persulfide bound to iron(II) in the active site show that binding of the persulfide occurs via the distal sulfide and, in the case of the cysteine persulfide, the amine also binds. Persulfide was detected by mass spectrometry in both the crystal and the drop, suggesting its origin is chemical rather than enzymatic. A mechanism involving the formation of the relevant disulfide from sulfide produced by hydrolysis of dithionite is proposed. In comparison, persulfenate {observed bound to cysteine dioxygenase [Simmons, C. R., et al. (2008) Biochemistry 47, 11390]} is shown through mass spectrometry to occur only in the crystal and not in the surrounding drop, suggesting that in the crystalline state the persulfenate does not lie on the reaction pathway. Stabilization of both the persulfenate and the persulfides does, however, suggest the position in which dioxygen binds during catalysis.
Assuntos
Cisteína Dioxigenase/metabolismo , Modelos Moleculares , Sulfetos/metabolismo , Ácido 3-Mercaptopropiônico/química , Ácido 3-Mercaptopropiônico/metabolismo , Animais , Biocatálise , Domínio Catalítico , Cisteína/análogos & derivados , Cisteína/química , Cisteína/metabolismo , Cisteína Dioxigenase/química , Cisteína Dioxigenase/genética , Dissulfetos/química , Dissulfetos/metabolismo , Ligantes , Conformação Molecular , Oxirredução , Ligação Proteica , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Solubilidade , Espectrometria de Massas por Ionização por Electrospray , Sulfetos/química , Difração de Raios XRESUMO
GLI pathogenesis-related 1 (GLIPR1) was previously identified as an epigenetically regulated tumor suppressor in prostate cancer and, conversely, an oncoprotein in glioma. More recently, GLIPR1 was shown to be differentially expressed in other cancers including ovarian, acute myeloid leukemia, and Wilms' tumor. Here we investigated GLIPR1 expression in metastatic melanoma cell lines and tissue. GLIPR1 was variably expressed in metastatic melanoma cells, and transcript levels correlated with degree of GLIPR1 promoter methylation in vitro. Elevated GLIPR1 levels were correlated with increased invasive potential, and siRNA-mediated knockdown of GLIPR1 expression resulted in reduced cell migration and proliferation in vitro. Immunohistochemical studies of melanoma tissue microarrays showed moderate to high staining for GLIPR1 in 50% of specimens analyzed. GLIPR1 staining was observed in normal skin in merocrine sweat glands, sebaceous glands, and hair follicles within the dermis.
RESUMO
Psb27 associates with the CP43 subunit of photosystem II during biogenesis of the photosystem. Several models have been proposed for the interaction between Psb27 and CP43. The utility of predictions and hypotheses arising from these models depends on the accuracy of the Psb27 structure used in the model. Two of the Psb27 structures used to model the Psb27-CP43 interaction place residue E98 on the surface of Psb27 and D14 in a position to form hydrogen bonds that stabilise the fold of the protein; however, a third structure questions the surface exposure of E98 and does not identify significant interactions of D14. Here we present evidence that D14 contributes to the thermal stability of Psb27 and that E98 is located on the surface. A D14A mutation was shown to reduce the apparent midpoint of unfolding of Psb27 by 16 °C. Four highly conserved surface residues and E98 were subject to charge-reversal mutations (R54E, R94E, E98R, E103R, R108E). The stabilities of the charge-reversal variants and the unmodified control were similar, suggesting E98 is a surface residue. Placing E98 in the correct, surface position will support more reliable models of the interaction of Psb27 with CP43.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Mutação , Complexo de Proteína do Fotossistema II/metabolismo , Synechocystis , Proteínas de Bactérias/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ligação Proteica/genética , Conformação Proteica , Dobramento de Proteína , Estabilidade Proteica , Temperatura , TermodinâmicaRESUMO
Candida glabrata has emerged as an important fungal pathogen with intrinsic resistance to azole drugs. The limited efficacy of and resistance to existing antifungals is driving the need to identify new drug targets. The enzyme 6,7-dimethyl-8-(D-ribityl)lumazine synthase is part of the riboflavin-biosynthesis pathway essential to fungi and bacteria and is a potential drug target for the development of broad-spectrum antifungal drugs. The X-ray crystal structure of recombinant lumazine synthase from C. glabrata was obtained at 2.24â Å resolution and revealed a dimer of homopentamers, with one in five subunits containing a product molecule from the catalytic reaction.
Assuntos
Candida glabrata/metabolismo , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Candida glabrata/patogenicidade , Catálise , Domínio Catalítico , Cristalografia por Raios X , Modelos Moleculares , Complexos Multienzimáticos/genética , Conformação Proteica , Multimerização Proteica , Pteridinas/química , Pteridinas/metabolismoRESUMO
Cytochrome c is a highly conserved protein, with 20 residues identical in all eukaryotic cytochromes c. Gly-41 is one of these invariant residues, and is the position of the only reported naturally occurring mutation in cytochrome c (human G41S). The basis, if any, for the conservation of Gly-41 is unknown. The mutation of Gly-41 to Ser enhances the apoptotic activity of cytochrome c without altering its role in mitochondrial electron transport. Here we have studied additional residue 41 variants and determined their effects on cytochrome c functions and conformation. A G41T mutation decreased the ability of cytochrome c to induce caspase activation and decreased the redox potential, whereas a G41A mutation had no impact on caspase induction but the redox potential increased. All residue 41 variants decreased the pK (a) of a structural transition of oxidized cytochrome c to the alkaline conformation, and this correlated with a destabilization of the interaction of Met-80 with the heme iron(III) at physiological pH. In reduced cytochrome c the G41T and G41S mutations had distinct effects on a network of hydrogen bonds involving Met-80, and in G41T the conformational mobility of two Ω-loops was altered. These results suggest the impact of residue 41 on the conformation of cytochrome c influences its ability to act in both of its physiological roles, electron transport and caspase activation.