RESUMO
Variants in the 5' UTR of ANKRD26 are a common cause of inherited thrombocytopenia (ANKRD26-RT), and are associated with sustained ANKRD26 expression, which inhibits megakaryocyte maturation and proplatelet formation. ANKRD26 expression is controlled by the binding of a RUNX1/FLI1 complex to the 5' UTR. To date, all reported ANKRD26-RD associated variants have been within the RUNX1 binding site and a 22 base pair flanking region. Here, we report a novel variant in the 5' UTR of ANKRD26, c.-107C>T. This variant is in the FLI1 binding site, and is predicted to disrupt FLI1 binding due to loss of a hydrogen bond with FLI1. Differentiated PBMCs from affected family members showed impaired megakaryocyte maturation and proplatelet formation and sustained expression of ANKRD26, and platelets from affected family members had higher ANKRD26 expression than control platelets. The variant increased activity of the ANKRD26 promotor in a reporter assay. We also provide evidence that the previously reported c.-140C>G ANKRD26 5' UTR variant is benign and not associated with thrombocytopenia. Identification of the c.-107C>T variant extends the range of the regulatory region in the 5' UTR of ANKRD26 that is associated with ANKRD26-RT.
Assuntos
Regiões 5' não Traduzidas , Proteína Proto-Oncogênica c-fli-1 , Trombocitopenia , Humanos , Trombocitopenia/genética , Trombocitopenia/patologia , Regiões 5' não Traduzidas/genética , Sítios de Ligação , Proteína Proto-Oncogênica c-fli-1/genética , Proteína Proto-Oncogênica c-fli-1/metabolismo , Masculino , Feminino , Linhagem , Megacariócitos/metabolismo , Megacariócitos/patologia , Plaquetas/metabolismo , Plaquetas/patologia , Subunidade alfa 2 de Fator de Ligação ao Core/genética , Ligação Proteica , Predisposição Genética para Doença , Peptídeos e Proteínas de Sinalização IntercelularRESUMO
Five pathogenic variants in the gene encoding cytochrome c (CYCS) associated with mild autosomal dominant thrombocytopenia have been reported. Previous studies of peripheral blood CD34+ or CD45+ cells from subjects with the G42S CYCS variant showed an acceleration in megakaryopoiesis compared to wild-type (WT) cells. To determine whether this result reflects a common feature of the CYCS variants, the c.145T>C mutation (Y49H variant) was introduced into the endogenous CYCS locus in K-562 cells, which undergo megakaryocytic maturation in response to treatment with a phorbol ester. The c.145T>C (Y49H) variant enhanced the megakaryocyte maturation of the K-562 cells, and this effect was seen when the cells were cultured at both 18 % and 5 % oxygen. Thus, alteration of megakaryopoiesis is common to both the G42S and Y49H CYCS variants and may contribute to the low platelet phenotype. The Y49H CYCS variant has previously been reported to impair mitochondrial respiratory chain function in vitro, however using extracellular flux analysis the c.145T>C (Y49H) variant does not alter mitochondrial bioenergetics of the K-562 cells, consistent with the lack of a phenotype characteristic of mitochondrial diseases in CYCS variant families. The Y49H variant has also been reported to enhance the ability of cytochrome c to trigger caspase activation in the intrinsic apoptosis pathway. However, as seen in peripheral blood cells from G42S CYCS variant carriers, the presence of Y49H cytochrome c in K-562 cells did not significantly change their response to an apoptotic stimulus.
Assuntos
Citocromos c , Megacariócitos , Mitocôndrias , Humanos , Citocromos c/metabolismo , Citocromos c/genética , Megacariócitos/metabolismo , Megacariócitos/citologia , Mitocôndrias/metabolismo , Mitocôndrias/genética , Células K562 , Trombocitopenia/genética , Trombocitopenia/metabolismo , Trombocitopenia/patologia , Apoptose/genética , Trombopoese/genética , MutaçãoRESUMO
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
A key signalling pathway required for clearance of viruses from host cells relies on the receptor protein, retinoic acid-inducible gene I (RIG-I). The activity of RIG-I is tightly controlled, and once bound to viral dsRNA, addition of lysine 63-linked ubiquitin chains activates signalling. Meanwhile, the addition of lysine 48-linked ubiquitin chains to RIG-I is required to terminate signalling when the infection has been resolved. Really interesting new gene (RING) finger protein 125 (RNF125) is the E3 ligase responsible for addition of the ubiquitin chains that terminate signalling, with disruption of its function associated with Tenorio syndrome. Here we describe a novel RNF125 gene variant in an individual with clinical symptoms including intellectual disability, macrocephaly and congenital heart disease, consistent with Tenorio syndrome. The newly identified Tenorio syndrome-associated variant [(NM_017831.4):c.670G>C p.Glu224Gln] is the first to be found in the ubiquitin interaction motif (UIM) of RNF125. While the E3 ligase activity of this RNF125 variant is retained, it has an impaired ability to interact with lysine 63-linked ubiquitin chains. The function of the UIM in RNF125 is uncertain; however, this study suggests that the UIM binds lysine 63-linked ubiquitin chains, and that this interaction is required for the normal function of RNF125.
Assuntos
Lisina , Ubiquitina-Proteína Ligases , Humanos , Lisina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ligação Proteica , Ubiquitina/genética , Ubiquitina/metabolismo , Proteínas de TransporteRESUMO
Hydrogen peroxide (H2O2) is a ubiquitous oxidant produced in a regulated manner by various enzymes in mammalian cells. H2O2 reversibly oxidizes thiol groups of cysteine residues to mediate intracellular signaling. While examples of H2O2-dependent signaling have been reported, the exact molecular mechanism(s) of signaling and the pathways affected are not well understood. Here, the transcriptomic response of Jurkat T cells to H2O2 was investigated to determine global effects on gene expression. With a low H2O2 concentration (10 µM) that did not induce an oxidative stress response or cell death, extensive changes in gene expression occurred after 4 h (6803 differentially expressed genes). Of the genes with a greater then 2-fold change in expression, 85% were upregulated suggesting that in a physiological setting H2O2 predominantly activates gene expression. Pathway analysis identified gene expression signatures associated with FOXO and NTRK signaling. These signatures were associated with an overlapping set of transcriptional regulators. Overall, our results provide a snapshot of gene expression changes in response to H2O2, which, along with further studies, will lead to new insights into the specific pathways that are activated in response to endogenous production of H2O2, and the molecular mechanisms of H2O2 signaling.
Assuntos
Peróxido de Hidrogênio , Transdução de Sinais , Animais , Humanos , Peróxido de Hidrogênio/farmacologia , Células Jurkat , Oxidantes/farmacologia , Estresse Oxidativo , Expressão Gênica , MamíferosRESUMO
GP1bß is a component of the von Willebrand factor (vWF) receptor complex that is necessary for platelet formation and activation. A novel frameshift variant in GP1BB has been identified in a family with macrothrombocytopenia. The variant leads to a protein that is 101 amino acids longer than wild type with loss of the transmembrane domain. As there is no defect in platelet aggregation, the family are classified as heterozygous carriers of a Bernard-Soulier syndrome-related mutation. The levels of the vWF receptor on platelets are reduced to 50% of the controls, with the presence of large platelets but normal platelet aggregation demonstrating that decreased vWF receptor expression impacts proplatelet formation but not platelet function.
Assuntos
Mutação da Fase de Leitura/genética , Complexo Glicoproteico GPIb-IX de Plaquetas/genética , Glicoproteínas da Membrana de Plaquetas/metabolismo , Trombocitopenia/genética , Feminino , Humanos , Masculino , Agregação PlaquetáriaRESUMO
Tumour necrosis factor (TNF) receptor associated factor (TRAF) family members share a common domain architecture, but play non-redundant physiological roles in cell signalling. At the N terminus, most TRAFs have a RING domain, followed by a series of Zinc finger (ZF) domains. The RING domain of TRAF6 dimerizes, and the RING homodimer together with the first ZF assembles ubiquitin chains that form a platform which facilitates activation of downstream kinases. The RING dimer interface is conserved amongst TRAF proteins, suggesting that functional heterodimers could be possible. Here we report the structure of the TRAF5-TRAF6 RING heterodimer, which accounts for the stability of the heterodimer as well as its ability to assemble ubiquitin chains. We also show that the RING domain of TRAF6 heterodimerizes with TRAF3 and TRAF2, and demonstrate that the linker helix and first ZF of TRAF2 can cooperate with TRAF6 to promote chain assembly. Collectively our results suggest that TRAF RING homo- and hetero-dimers have the potential to bridge interaction of nearby TRAF trimers and modulate TRAF-mediated signalling.
Assuntos
Ligação Proteica , Ubiquitina/química , Ubiquitinação , Dimerização , Humanos , Domínios e Motivos de Interação entre Proteínas , Receptores do Fator de Necrose Tumoral/química , Receptores do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais , Fator 2 Associado a Receptor de TNF/genética , Fator 2 Associado a Receptor de TNF/metabolismo , Fator 3 Associado a Receptor de TNF/metabolismo , Fator 5 Associado a Receptor de TNF/metabolismo , Fator 6 Associado a Receptor de TNF , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases , Dedos de ZincoRESUMO
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 U937Assuntos
Citocromos c/genética , Predisposição Genética para Doença , Variação Genética , Trombocitopenia/genética , Alelos , Substituição de Aminoácidos , Citocromos c/química , Estudos de Associação Genética , Genótipo , Humanos , Modelos Moleculares , Mutação , Fenótipo , Contagem de Plaquetas , Conformação Proteica , Relação Estrutura-Atividade , Trombocitopenia/sangue , Trombocitopenia/diagnósticoRESUMO
The peroxidase activity of cytochrome c is proposed to contribute to apoptosis by peroxidation of cardiolipin in the mitochondrial inner membrane. However, cytochrome c heme is hexa-coordinate with a methionine (Met80) on the distal side, stopping it from acting as an efficient peroxidase. The first naturally occurring variant of cytochrome c discovered, G41S, has higher peroxidase activity than wild-type. To understand the basis for this increase and gain insight into the peroxidase activity of wild-type, we have studied wild-type, G41S and the unnatural variant G41T. Through a combined kinetic and mass spectrometric analysis, we have shown that hydrogen peroxide specifically oxidizes Met80 to the sulfoxide. In the absence of substrate this can be further oxidized to the sulfone, leading to a decrease in peroxidase activity. Peroxidase activity can be correlated with the proportion of sulfoxide present and if fully in that form, all variants have the same activity without a lag phase caused by activation of the protein.
Assuntos
Citocromo-c Peroxidase/química , Citocromo-c Peroxidase/metabolismo , Metionina/química , Safrol/análogos & derivados , Apoptose/fisiologia , Cardiolipinas/fisiologia , Ativação Enzimática , Glicina/química , Glicina/genética , Heme/química , Humanos , Peróxido de Hidrogênio/química , Concentração de Íons de Hidrogênio , Ferro/química , Ferro/metabolismo , Cinética , Mutação , Safrol/química , Sulfonas/químicaRESUMO
Mutations in the cytochrome c gene (CYCS) cause autosomal dominant thrombocytopenia by an unknown mechanism. While attempting to generate megakaryoblastic cell lines exogenously expressing cytochrome c variants, we discovered that endogenous cytochrome c expression increased both upon induction of differentiation with the phorbol ester phorbol 12-myristate 13-acetate (PMA), and as cell density increased. A concomitant increase in cytochrome c oxidase subunit II in response to PMA, but not cell higher cell density, suggests upregulation of the mitochondrial respiratory chain may be a specific feature of differentiation. These results highlight the likely importance of cytochrome c in both differentiating and proliferating cells, and illustrate the unsuitability of megakaryoblastic lines for modeling CYCS-associated thrombocytopenia.
Assuntos
Diferenciação Celular , Citocromos c/metabolismo , Células Progenitoras de Megacariócitos/enzimologia , Regulação para Cima , Diferenciação Celular/efeitos dos fármacos , Células HeLa , Humanos , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Apoptosis signal-regulating kinases (ASK1-3) are apical kinases of the p38 and JNK MAP kinase pathways. They are activated by diverse stress stimuli, including reactive oxygen species, cytokines, and osmotic stress; however, a molecular understanding of how ASK proteins are controlled remains obscure. Here, we report a biochemical analysis of the ASK1 kinase domain in conjunction with its N-terminal thioredoxin-binding domain, along with a central regulatory region that links the two. We show that in solution the central regulatory region mediates a compact arrangement of the kinase and thioredoxin-binding domains and the central regulatory region actively primes MKK6, a key ASK1 substrate, for phosphorylation. The crystal structure of the central regulatory region reveals an unusually compact tetratricopeptide repeat (TPR) region capped by a cryptic pleckstrin homology domain. Biochemical assays show that both a conserved surface on the pleckstrin homology domain and an intact TPR region are required for ASK1 activity. We propose a model in which the central regulatory region promotes ASK1 activity via its pleckstrin homology domain but also facilitates ASK1 autoinhibition by bringing the thioredoxin-binding and kinase domains into close proximity. Such an architecture provides a mechanism for control of ASK-type kinases by diverse activators and inhibitors and demonstrates an unexpected level of autoregulatory scaffolding in mammalian stress-activated MAP kinase signaling.
Assuntos
MAP Quinase Quinase Quinase 5/química , MAP Quinase Quinase Quinase 5/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Humanos , MAP Quinase Quinase 6/química , MAP Quinase Quinase 6/genética , MAP Quinase Quinase 6/metabolismo , MAP Quinase Quinase Quinase 5/genética , Modelos Biológicos , Modelos Moleculares , Mutação , Fosforilação , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Transdução de Sinais , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Thrombocytopenia Cargeeg is a rare autosomal dominant disorder and one of three thrombocytopenias caused by mutation of cytochrome c (Online Mendelian Inheritance in Man entry THC4). Our previous observations of platelet-like structures in the marrow space and early platelet production in vitro suggested that the low platelet phenotype in Thrombocytopenia Cargeeg subjects is caused by premature release of platelets into non-vascular regions of the bone marrow. We now show that two processes of platelet release occur in Thrombocytopenia Cargeeg subjects. Circulating platelets have a normal marginal microtubule coil, and cultured megakaryocytes derived from peripheral blood cells of Thrombocytopenia Cargeeg subjects form proplatelets normally and release platelets containing a marginal microtubule coil, consistent with effective platelet release via the proplatelet mechanism. In contrast, platelet-like structures within the extravascular bone marrow space have the dimensions of platelets but lack the marginal microtubule coil, suggesting abnormal proplatelet-independent platelet release. The mechanism of extravascular platelet release remains unclear. The failure to recapitulate this mechanism in vitro implies that the phenotype is not simply an intrinsic property of CYCS mutation-carrying megakaryocytes, but is dependent on the interaction between these cells and their environment.
Assuntos
Plaquetas/patologia , Citocromos c/genética , Megacariócitos/patologia , Mutação , Trombocitopenia/patologia , Medula Óssea/patologia , Células Cultivadas , Humanos , Microtúbulos , Trombocitopenia/genéticaRESUMO
Peroxiredoxin 1 is a member of the ubiquitous peroxiredoxin family of thiol peroxidases that catalyse the reduction of peroxides. In recent years eukaryotic peroxiredoxins have emerged as a critical component of cellular redox signalling, particularly in response to alterations in production of hydrogen peroxide. Peroxiredoxins are exquisitely sensitive to oxidation by hydrogen peroxide making them key peroxide sensing enzymes within cells. Evidence gathered over the last decade suggests that in addition to sensing the redox signal, peroxiredoxins have a major role in transducing this signal to downstream signalling proteins, ultimately contributing to regulation of diverse cellular processes including proliferation, differentiation and apoptosis. In this review we present the three current models for the sensing and signal transducing roles of peroxiredoxins, with a specific focus on mammalian peroxiredoxin 1. The evidence for each mechanism is discussed and areas for future work are identified.
Assuntos
Peróxido de Hidrogênio/metabolismo , Peroxirredoxinas/metabolismo , Animais , Apoptose , Catálise , Diferenciação Celular , Proliferação de Células , Humanos , Cinética , Oxirredução , Peroxirredoxina III/metabolismo , Multimerização Proteica , Processamento de Proteína Pós-Traducional , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Ratos , Transdução de SinaisRESUMO
The naturally occurring human cytochrome c variant (G41S) is associated with a mild autosomal dominant thrombocytopenia (Thrombocytopenia Cargeeg) caused by dysregulation of platelet production. The molecular basis of the platelet production defect is unknown. Despite high conservation of cytochrome c between human and mouse (91.4% identity), introducing the G41S mutation into mouse cytochrome c in a knockin mouse (CycsG41S/G41S) did not recapitulate the low platelet phenotype of Thrombocytopenia Cargeeg. While investigating the cause of this disparity we found a lack of conservation of the functional impact of cytochrome c mutations on caspase activation across species. Mutation of cytochrome c at residue 41 has distinct effects on the ability of cytochrome c to activate caspases depending on the species of both the cytochrome c and its binding partner Apaf-1. In contrast to our previous results showing the G41S mutation increases the ability of human cytochrome c to activate caspases, here we find this activity is decreased in mouse G41S cytochrome c. Additionally unlike wildtype human cytochrome c, G41S cytochrome c is unable to activate caspases in Xenopus embryo extracts. Taken together these results demonstrate a previously unreported species-specific component to the interaction of cytochrome c with Apaf-1. This suggests that the electrostatic interaction between cytochrome c and Apaf-1 is not the sole determinant of binding, with additional factors controlling binding specificity and affinity. These results have important implications for studies of the effects of cytochrome c mutations on the intrinsic apoptosis pathway.
Assuntos
Citocromos c/genética , Sequência de Aminoácidos , Animais , Apoptose , Caspases/metabolismo , Citocromos c/metabolismo , Ativação Enzimática , Técnicas de Introdução de Genes , Hematopoese , Humanos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Mutação , Contagem de Plaquetas , Especificidade da EspécieRESUMO
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
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.
Assuntos
Apoptose , Caspases/metabolismo , Citocromos c/genética , Citocromos c/metabolismo , Metionina/metabolismo , Mutação Puntual , Substituição de Aminoácidos , Linhagem Celular Tumoral , Citocromos c/química , Ativação Enzimática , Glicina/química , Glicina/genética , Glicina/metabolismo , Humanos , Ferro/metabolismo , Metionina/química , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Oxirredução , Conformação ProteicaRESUMO
Hydrogen peroxide is widely viewed as the main second messenger in redox signaling, and it has been proposed that deactivation of the antioxidant peroxiredoxin (Prdx) enzymes allows free peroxide to accumulate and directly oxidize target proteins (the floodgate model). We assessed the role of cytosolic Prdxs 1 and 2 in peroxide-induced activation of the apoptosis signaling kinase 1 (ASK1)/p38 signaling pathway, in which oxidation of ASK1 is required for phosphorylation of p38. In response to peroxide, Prdx1 catalyzed oxidation of ASK1 to a disulfide-linked multimer, and this occurred via transient formation of a Prdx1-ASK1 mixed disulfide intermediate. Oxidation of ASK1 and phosphorylation of p38 were inhibited by knockdown of Prdx1, but also by overexpression of Prdx2. This suggests that these two cytosolic Prdxs have distinct roles in the cellular peroxide response and compete for available peroxide substrate. These data imply that Prdx1 can function as a peroxide receptor in response to extracellular H(2)O(2), receiving the peroxide signal and transducing it into a disulfide bond that is subsequently transmitted to the substrate, ASK1, resulting in p38 phosphorylation. Interestingly, in response to peroxide, Prdx1 and Prdx3 transiently formed reducible higher molecular weight complexes, suggesting that multiple proteins are targets for Prdx-mediated oxidation via a disulfide-exchange mechanism. This model of active peroxide signal distribution via disulfide exchange is consistent with Prdx function in yeast and explains how peroxides may trigger specific disulfide bond formation in mammalian cells.
Assuntos
Macrófagos/metabolismo , Peroxirredoxinas/metabolismo , Transdução de Sinais , Linhagem Celular , Dissulfetos/química , Dissulfetos/metabolismo , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Peróxido de Hidrogênio/metabolismo , Peróxido de Hidrogênio/farmacologia , MAP Quinase Quinase Quinase 5/genética , MAP Quinase Quinase Quinase 5/metabolismo , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Oxirredução , Peroxirredoxina III/genética , Peroxirredoxina III/metabolismo , Peroxirredoxinas/genética , Fosforilação , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Thrombocytopenia (TP) Cargeeg is a unique autosomal dominant disorder, affecting a seven-generation family, caused by cytochrome C (CYCS) mutation that dysregulates platelet formation. The CYCS mutation in this disorder is a glycine 41 replacement by serine, which yields a cytochrome C variant with enhanced apoptotic pathway activity in vitro. The deregulated apoptosis in this disorder affects megakaryocytes (MK) during platelet formation, leading to early and ectopic platelet release in the bone marrow (BM). Notably, the family has no other phenotypic indication of abnormal apoptosis, implying that cytochrome C activity is not a critical regulator of physiological apoptosis in most cells. The pathophysiology of this unique inherited TP, with unaltered platelet survival and normal MK content in the BM, has implications for physiological and pathological mechanisms altering MK apoptosis, with implications for other unexplained thrombocytopenic disorders.
Assuntos
Apoptose/genética , Citocromos c/genética , Mutação de Sentido Incorreto , Trombocitopenia/genética , Sequência de Bases , Feminino , Humanos , Masculino , Megacariócitos/metabolismo , Megacariócitos/patologia , Linhagem , Trombocitopenia/congênito , Trombocitopenia/fisiopatologiaRESUMO
The naturally occurring G41S mutation to human (Hs) cytochrome (cyt) c enhances apoptotic activity based upon previous in vitro and in vivo studies, but the molecular mechanism underlying this enhancement remains unknown. Here, X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and density functional theory (DFT) calculations have been used to identify the structural and electronic differences between wild-type (WT) and G41S Hs cyt c. S41 is part of the hydrogen bonding network for propionate 7 of heme pyrrole ring A in the X-ray structure of G41S Hs cyt c and, compared to WT, G41S Hs cyt c has increased spin density on pyrrole ring C and a faster electron self-exchange rate. DFT calculations illustrate an electronic mechanism where structural changes near ring A can result in electronic changes at ring C. Since ring C is part of the solvent-exposed protein surface, we propose that this heme electronic structure change may ultimately be responsible for the enhanced proapoptotic activity of G41S Hs cyt c.