RESUMO
Necroptosis is a recently described Caspase 8-independent method of cell death that denotes organized cellular necrosis. The roles of RIP1 and RIP3 in mediating hepatocyte death from acute liver injury are incompletely defined. Effects of necroptosis blockade were studied by separately targeting RIP1 and RIP3 in diverse murine models of acute liver injury. Blockade of necroptosis had disparate effects on disease outcome depending on the precise etiology of liver injury and component of the necrosome targeted. In ConA-induced autoimmune hepatitis, RIP3 deletion was protective, whereas RIP1 inhibition exacerbated disease, accelerated animal death, and was associated with increased hepatocyte apoptosis. Conversely, in acetaminophen-mediated liver injury, blockade of either RIP1 or RIP3 was protective and was associated with lower NLRP3 inflammasome activation. Our work highlights the fact that diverse modes of acute liver injury have differing requirements for RIP1 and RIP3; moreover, within a single injury model, RIP1 and RIP3 blockade can have diametrically opposite effects on tissue damage, suggesting that interference with distinct components of the necrosome must be considered separately.
Assuntos
Apoptose/genética , Proteínas Ativadoras de GTPase/antagonistas & inibidores , Hepatite Autoimune/genética , Fígado/lesões , Proteína Serina-Treonina Quinases de Interação com Receptores/antagonistas & inibidores , Acetaminofen , Animais , Apoptose/efeitos dos fármacos , Apoptose/imunologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Caspase 8/metabolismo , Quimiocina CCL2/sangue , Concanavalina A , Modelos Animais de Doenças , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Hepatócitos/patologia , Interleucina-6/sangue , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR , Necrose/genética , Espécies Reativas de Oxigênio/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Fator de Necrose Tumoral alfa/sangueRESUMO
The formation of a complex between beta-catenin and members of the TCF/LEF family of high-mobility group proteins is a key regulatory event in the wnt-signaling pathway, essential for embryonal development as well as the growth of normal and malignant colon epithelium. We have characterized the binding of TCF4 to human beta-catenin by steady-state intrinsic fluorescence quenching experiments, surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). Binding studies in solution and in heterogeneous phase showed that TCF4 binds reversibly to beta-catenin with an affinity (KB) of 3(+/-1) 10(8) M(-1). Site-directed mutagenesis, together with calorimetric measurements, revealed that residue D16 in TCF4 plays a crucial role in high-affinity binding. Mutation of this residue to alanine resulted in a decrease of KB by two orders of magnitude as well as a significant reduction in binding enthalpy. Binding of TCF4 to beta-catenin gave rise to a large negative enthalpy change at 25 degrees C (-29.7 kcal/mol). Binding enthalpies were strongly temperature dependent, which resulted in the determination of a large heat capacity change upon binding of -1.5 kcal/(mol K). The molecular events that take place upon complex formation are discussed using the measured thermodynamic data together with the crystal structure of the beta-catenin arm repeat region/TCF complex.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Termodinâmica , Transativadores , Fatores de Transcrição/metabolismo , Sítios de Ligação , Dicroísmo Circular , Clonagem Molecular , Proteínas do Citoesqueleto/química , Primers do DNA/química , Fluorescência , Glutationa Transferase/metabolismo , Humanos , Modelos Moleculares , Reação em Cadeia da Polimerase , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Ressonância de Plasmônio de Superfície/métodos , Fatores de Transcrição TCF , Proteína 2 Semelhante ao Fator 7 de Transcrição , Fatores de Transcrição/química , Fatores de Transcrição/genética , beta CateninaRESUMO
The mitochondrial outer membrane enzyme kynurenine 3-hydroxylase (K3H) is an NADPH-dependent flavin mono-oxygenase involved in the tryptophan pathway, where it catalyzes the hydroxylation of kynurenine. K3H was transiently expressed in COS-1 cells as a glutathione S-transferase (GST) fusion protein, and the pure recombinant protein (rec-K3H) was obtained with a specific activity of about 2000 nmol.min-1.mg-1. Rec-K3H was shown to have an optimum pH at 7.5, to use NADPH more efficiently than NADH, and to contain one molecule of non-covalently bound FAD per molecule of enzyme. The mechanism of the rec-K3H-catalyzed reaction was investigated by overall initial-rate measurements, and a random mechanism in which combination of the enzyme with one substrate does not influence its affinity for the other is proposed. Further kinetic studies revealed that K3H activity was inhibited by both pyridoxal phosphate and Cl-, and that NADPH-catalyzed oxidation occurred even in the absence of kynurenine if 3-hydroxykynurenine was present, suggesting an uncoupling effect of 3-hydroxykynurenine with peroxide formation. This observation could be of clinical interest, as peroxide formation could explain the neurotoxicity of 3-hydroxykynurenine in vivo.
Assuntos
Oxigenases de Função Mista/metabolismo , Animais , Células COS , Cloretos/metabolismo , Cloretos/farmacologia , Flavina-Adenina Dinucleotídeo/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Íons , Cinética , Cinurenina/análogos & derivados , Cinurenina/metabolismo , Quinurenina 3-Mono-Oxigenase , Oxigenases de Função Mista/antagonistas & inibidores , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/isolamento & purificação , Peso Molecular , NAD/metabolismo , NADP/metabolismo , NADPH Oxidases/metabolismo , Oxirredução , Peróxidos/metabolismo , Fosfato de Piridoxal/metabolismo , Fosfato de Piridoxal/farmacologia , Proteínas Recombinantes de Fusão/antagonistas & inibidores , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato , Termodinâmica , TransfecçãoRESUMO
Kynureninase [E.C.3.7.1.3.] is one of the enzymes involved in the biosynthesis of NAD cofactors from tryptophan through the kynurenine pathway. By tryptic and CNBr digestion of purified rat liver kynureninase, we obtained about 28% of the amino acid sequence of the enzyme. The rat kynureninase cDNA, isolated by means of reverse-transcribed polymerase chain reaction and hybridization screening, codes for a polypeptide of 464 amino acids. Northern blot analysis revealed the synthesis of a 2.0 kb rat kynureninase mRNA. A cDNA encoding human liver kynureninase was also isolated. The deduced amino acid sequence is 85% identical to that of the rat protein. COS-1 cells were transfected with both cDNAs. The Km values of the rat enzyme, for L-kynurenine and DL-3-hydroxykynurenine, were 440 +/- 20 microM and 32 +/- 5 microM and of the human enzyme 440 /- 20 microM and 49 +/- 6 microM, respectively. Interestingly, COS-1 cells transfected with the cDNA coding for rat kynureninase also display cysteine-conjugate beta-lyase activity.
Assuntos
Liases de Carbono-Enxofre , Hidrolases/genética , Fígado/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Células COS , Clonagem Molecular , Expressão Gênica/genética , Humanos , Hidrolases/química , Hidrolases/metabolismo , Cinética , Cinurenina/análogos & derivados , Cinurenina/metabolismo , Liases/metabolismo , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , RNA Mensageiro/genética , Ratos , Alinhamento de Sequência , Análise de Sequência , Transfecção/genéticaAssuntos
Encéfalo/enzimologia , Liases , Transaminases/biossíntese , Transaminases/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Clonagem Molecular , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Biossíntese de Proteínas , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia de Sequência do Ácido Nucleico , Transaminases/metabolismo , Transcrição Gênica , TransfecçãoRESUMO
A new mutein of interleukin-6, called delta 22-IL-6 Cys 3,4, characterized by the deletion of the first 22 amino acids at the N-terminal end and by the substitution of the first two cysteines (Cys23 and Cys29) with serine residues, was produced in Escherichia coli and was found to maintain the structural and functional properties of the human native form. A partially purified preparation still showed in isoelectric focusing a minor acidic component (pI 6.10) and a more basic component (pI 6.70), the native form having a pI of 6.56. This preparation was further fractionated in a multi-compartment electrolyser with isoelectric membranes, which allowed the collection of the more alkaline species for characterization. Mass spectra of the pI 6.70 form gave an additional mass of 32 atomic mass units (amu), suggesting the addition of two oxygen atoms (a potential oxidation of two methionine residues to sulphoxide). However, the five methionine residues in this higher pI form were identified after enzymatic hydrolysis and peptide mapping and were found to be in a reduced state. In addition, the pI 6.70 form was quickly converted into the native form by mild reductive treatment. On digestion and fingerprinting, the peptide from residues 50 to 65 of the pI 6.70 species (containing the only two cysteine residues of the molecule) exhibited a more hydrophobic behaviour in reversed-phase high-performance liquid chromatography and retained a mass increase of 32 amu. These experimental findings more likely suggest the addition of an extra sulphur atom to the only disulphide bridge to give an unusual protein trisulphide molecule.