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1.
Genes Brain Behav ; 15(7): 660-8, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27324142

RESUMO

Preliminary studies suggest that lithium (Li) response might be associated with some circadian gene polymorphisms, we therefore performed a pharmacogenetic study on the core clock genes in two independent samples suffering from bipolar disorder (BD) and thoroughly characterized for their Li response. Two independent Caucasian samples (165 and 58 bipolar patients) treated with Li were selected from samples recruited in a French multicenter study and assessed for their Li response using the Alda scale. The two samples were genotyped using the Human660 (H660) and OmniExpress (OE) BeadChips and gene-based association analyses of 22 core clock genes were conducted. In the first sample (H660 chip), the RAR-related orphan receptor-a gene (RORA) and the Peroxisome Proliferator-Activated Receptor Gamma, Coactivator 1 Alpha gene (PPARGC1A or PGC-1α) were significantly associated with the Li response (empirical P-value = 0.0015 and 0.04, respectively), and remained significant only for RORA after Bonferroni correction. In the second sample (OE chip), PPARGC1A was significantly associated with the Li response (empirical P-value = 0.04), and did not remain significant after Bonferroni correction. PPARGC1A is a master regulator of mitochondrial function and a key component of the endogenous clock that stimulates the expression of Bmal1 and Rev-erb-alpha through coactivation of RORA. Although the observed associations deserve further replication and investigation, our results suggest genetic associations between Li response and these two close biological partners: PPARGC1A and RORA involved in circadian rhythms and bioenergetics processes in Li response.


Assuntos
Transtorno Bipolar/tratamento farmacológico , Transtorno Bipolar/genética , Ritmo Circadiano/genética , Compostos de Lítio/uso terapêutico , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Adulto , Transtorno Bipolar/metabolismo , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Proteínas de Choque Térmico/genética , Humanos , Masculino , Pessoa de Meia-Idade , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Polimorfismo de Nucleotídeo Único , Fatores de Transcrição/genética
2.
Neuroscience ; 137(2): 473-82, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16289835

RESUMO

3,4-Methylenedioxymethamphetamine (ecstasy), a widely used recreational drug with psychoactive properties, induces both serotonin and dopamine release in the brain. However, little is known about its intracellular effects. We previously showed that 3,4-methylenedioxymethamphetamine rewarding effects in mice were dependent upon extracellular signal-regulated kinase activation and that dorsal striatum was a critical region for mediating extracellular signal-regulated kinase-dependent Egr1 3,4-methylenedioxymethamphetamine-induced transcription. Here, we extend these findings by showing that 3,4-methylenedioxymethamphetamine is indeed able to activate extracellular signal-regulated kinase within this structure. To identify genes regulated by acute 3,4-methylenedioxymethamphetamine in the mice dorsal striatum, and selectively controlled by this kinase, we performed microarray experiments by using a selective inhibitor of extracellular signal-regulated kinase activation, SL327. Of the approximately 24,000 genes from the microarray, 27 showed altered expression after exposure to 3,4-methylenedioxymethamphetamine, and among these, 59% were partially or totally inhibited by SL327 pretreatment. Our results showed that the genes regulated by 3,4-methylenedioxymethamphetamine encode proteins that belong to transcription factors family, signaling pathways (phosphatases, cytoskeleton regulation), and synaptic functions. These early changes, and especially those controlled by extracellular signal-regulated kinase activation might play significant roles in the expression of many of the behaviors that occur following 3,4-methylenedioxymethamphetamine taking.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , N-Metil-3,4-Metilenodioxianfetamina/farmacologia , Neostriado/efeitos dos fármacos , Neostriado/enzimologia , Neurônios/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Animais , Proteínas do Citoesqueleto/genética , Ativação Enzimática/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica/fisiologia , Alucinógenos/farmacologia , Masculino , Camundongos , Proteínas do Tecido Nervoso/genética , Plasticidade Neuronal/efeitos dos fármacos , Plasticidade Neuronal/fisiologia , Neurônios/enzimologia , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Transdução de Sinais/genética , Ativação Transcricional/fisiologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologia
3.
J Mol Biol ; 305(1): 151-65, 2001 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-11114254

RESUMO

Aqualysin I, a thermostable homologue of subtilisin, requires its propeptide (ProA) to function as an intramolecular chaperone (IMC). To decipher the mechanisms through which propeptides can initiate protein folding, we characterized ProA in terms of its sequence, structure and function. Our results show that, in contrast to ProS (propeptide of subtilisin), ProA can fold spontaneously, reversibly and cooperatively into a stable monomeric alpha-beta conformation, even when isolated from its cognate protease-domain. ProA displays an indiscernible amount of tertiary structure with a considerable solvent-accessible hydrophobic surface, but is not a classical molten-globule folding intermediate. Moreover, despite showing only 21 % sequence identity with ProS, ProA can not only inhibit enzymatic activity with a magnitude tenfold greater than ProS, but can also chaperone subtilisin folding, albeit with a lower efficiency. The structure of ProA complexed with subtilisin is different from that of isolated ProA. Hence, additional interactions seem necessary to induce ProA into a compact structure. Our results also suggest that: (a) propeptides that are potent inhibitors are not necessarily better IMCs; (b) propeptides within the subtilase family appear polymorphic and; (c) the intrinsic instability within propeptides may be necessary for rapid activation of the cognate protein.


Assuntos
Precursores Enzimáticos/química , Precursores Enzimáticos/metabolismo , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Dobramento de Proteína , Serina Endopeptidases/química , Serina Endopeptidases/metabolismo , Sequência de Aminoácidos , Bacillus subtilis/enzimologia , Domínio Catalítico , Cromatografia em Gel , Dicroísmo Circular , Clonagem Molecular , Diálise , Precursores Enzimáticos/genética , Precursores Enzimáticos/isolamento & purificação , Cinética , Modelos Moleculares , Chaperonas Moleculares/genética , Chaperonas Moleculares/isolamento & purificação , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Desnaturação Proteica , Renaturação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Serina Endopeptidases/genética , Serina Endopeptidases/isolamento & purificação , Espectrometria de Fluorescência , Subtilisinas/antagonistas & inibidores , Subtilisinas/química , Subtilisinas/genética , Subtilisinas/isolamento & purificação , Subtilisinas/metabolismo , Thermus/enzimologia
4.
Proteins ; 39(4): 365-71, 2000 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-10813818

RESUMO

Based on the recently described three-dimensional model of the 507-749 region of neprilysin, which contains the catalytic site of the enzyme, experiments were performed to improve the proposed topology of its large and hydrophobic S(')(1) subsite. Docking studies, site-directed mutagenesis, and biochemical studies were combined. The mutations of various residues proposed to be part of the S(')(1) subsite (F563A, F564A, M579A, F716A, and I718A) did not induce major structural reorganization of the active site as demonstrated by the slight modification of the enzyme activity. The mutations were also analyzed by measuring the inhibitory potencies of thiol inhibitors containing P(')(1) moieties of increasing sizes. These results combined with molecular modeling studies support the proposed topology of the S(')(1) subsite. This, and the critical role of F563 and M579 in inhibitor binding, could facilitate the synthesis of new potent and selective inhibitors.


Assuntos
Modelos Moleculares , Neprilisina/química , Animais , Células COS , Expressão Gênica , Mutagênese Sítio-Dirigida , Neprilisina/antagonistas & inibidores , Neprilisina/genética , Neprilisina/metabolismo
5.
J Mol Biol ; 285(5): 1911-5, 1999 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-9925774

RESUMO

The zinc metalloendopeptidase, thermolysin (EC 3.4.24.27) produced by Bacillus thermoproteolyticus serves as a model of important physiological enzymes such as neprilysin, angiotensin converting enzyme and endothelin converting enzyme. Thermolysin is synthesised as a pre-proenzyme, with an N-terminal prosequence of 204 residues and a mature sequence of 316 residues. The prosequence facilitates the folding of the denatured mature sequence in vitro and the cleavage of the peptide bond linking the pro and mature sequences occurs by an autocatalytic, intramolecular process. With the aim to study the role of the prosequence in vivo and to produce active mutants for structural studies, the mature sequence of thermolysin has now been expressed in Escherichia coli, either alone or with the prosequence as an independent polypeptide, i.e. in trans form. In addition, the mature sequence of an inactive mutant in which Glu143 involved in the catalytic process was replaced by Ala has also been expressed in trans with the prosequence. The results show that the pro-sequence is required to obtain active thermolysin and that a covalent link with the mature sequence is not necessary for the correct folding of the protease in vivo. Moreover, when expressed in E. coli (in trans with the prosequence), the yield of correctly folded E143A mutant was similar to that of the wild-type protease, whereas no mature enzyme was detected when it was expressed as a pre-proenzyme in Bacillus subtilis. These results demonstrate that the thermolysin prosequence acts as an intramolecular chaperone in vivo and open the way to structural studies of catalytic site mutants produced in large quantities in E. coli.


Assuntos
Precursores Enzimáticos/metabolismo , Escherichia coli/genética , Proteínas Recombinantes/genética , Termolisina/genética , Termolisina/metabolismo , Western Blotting , Divisão Celular/genética , Precursores Enzimáticos/genética , Escherichia coli/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica , Vetores Genéticos/genética , Isopropiltiogalactosídeo/farmacologia , Chaperonas Moleculares/metabolismo , Mutagênese Sítio-Dirigida , Mutação , Proteínas Recombinantes/metabolismo
6.
FEBS Lett ; 438(3): 215-9, 1998 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-9827548

RESUMO

Important homologies in the topology of the catalytic site and the mechanism of action of thermolysin and neprilysin have been evidenced by site-directed mutagenesis. The determination of differences in transition state stabilization between these peptidases could facilitate the design of specific inhibitors. Thus, two residues of thermolysin which could be directly (Tyr157) or indirectly (Asp226) involved in the stabilization of the transition state and their putative counterparts in neprilysin (Tyr625 and Asp709) have been mutated. The results show that Tyr157 is important for thermolysin activity while Tyr625 has no functional role in neprilysin. Conversely, the mutation of Asp226 induced a slight perturbation of thermolysin activity while Asp709 in neprilysin seems crucial in neprilysin catalysis. Taken together these data seem to indicate differences in the transition state mode of stabilization in the two peptidases.


Assuntos
Neprilisina/química , Neprilisina/metabolismo , Termolisina/química , Termolisina/metabolismo , Sequência de Aminoácidos , Ácido Aspártico , Bacillus subtilis/enzimologia , Domínio Catalítico , Estabilidade Enzimática , Glicopeptídeos/farmacologia , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Inibidores de Proteases/farmacologia , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Tiorfano/farmacologia , Tirosina
7.
J Biol Chem ; 273(10): 5697-701, 1998 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-9488701

RESUMO

Thermolysin, an extracellular zinc endopeptidase from Bacillus thermoproteolyticus, is synthesized as a pre-proenzyme and the prosequence has been shown to assist the refolding of the denatured enzyme in vitro and to inhibit enzyme activity (O'Donohue, M. J., and Beaumont, A. (1996) J. Biol. Chem. 271, 26477-26481). To determine whether prosequence cleavage from the mature enzyme is autocatalytic and if so, whether it is an intermolecular or intramolecular process, N-terminal histidine-tagged prothermolysin was expressed in Escherichia coli. Although partial processing to mature enzyme occurred, most of the proenzyme was recovered intact from inclusion bodies. This was then solubilized in guanidinium hydrochloride, immobilized on a cobalt-containing resin, and after dialysis against renaturation buffer, was quantitatively transformed to mature enzyme. However, when a mutation was introduced into the mature sequence to inactivate thermolysin, the proenzyme was not processed either in vivo or in vitro. In addition, mutated prothermolysin was not processed by exogenous thermolysin under a variety of experimental conditions. The results demonstrate that thermolysin maturation can proceed via an autocatalytic intramolecular pathway.


Assuntos
Bacillus/enzimologia , Precursores de Proteínas/metabolismo , Termolisina/biossíntese , Catálise , Ativação Enzimática/fisiologia , Escherichia coli/genética , Expressão Gênica/genética , Mutagênese Sítio-Dirigida/genética , Dobramento de Proteína , Precursores de Proteínas/genética , Processamento de Proteína Pós-Traducional/fisiologia , Proteínas Recombinantes/metabolismo , Termolisina/metabolismo
8.
Biochemistry ; 37(2): 686-92, 1998 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-9425092

RESUMO

The molecular components ensuring the strict exopeptidase action of aminopeptidase N (APN) and related zinc aminopeptidases of the M1 family have not yet been clearly established. The specific recognition of the N-terminal amino acid of the substrates by the enzymes has been proposed to involve either the complexation of the free amino group by the catalytic zinc ion or an interaction with an anionic binding site, which could be constituted by an aspartate or glutamate residue. To investigate the existence of such an ionic binding site, site-directed mutagenesis experiments have been performed on acidic residues of pig APN. Given that aminopeptidases of the M1 family are likely to have a common mechanism of action, only strictly conserved residues were mutated. As compared to the wild-type enzyme, the mutation D220E led only to slight modifications in the kinetic parameters of the enzyme and in the Ki values of various inhibitors, indicating that this residue is not critically involved in the hydrolytic mechanism. In contrast, the mutations E350Q and E350D induced a large decrease in enzyme activity, essentially due to modifications in kcat, whereas the E350A mutation led to an almost completely inactive enzyme. Moreover, among the inhibitors tested, only those acting as transition state analogs showed significant increases in their Ki values. These data are in favor of E350 belonging to the "anionic binding site" in APN. A mechanism of action, derived from that of thermolysin, is proposed for these aminopeptidases, which explains the importance of E350 in transition state formation, rather than in the Michaelis complex.


Assuntos
Antígenos CD13/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Antígenos CD13/genética , Células COS , Exopeptidases , Ácido Glutâmico/genética , Ácido Glutâmico/metabolismo , Modelos Químicos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Peptídeo Hidrolases/genética , Peptídeo Hidrolases/metabolismo , Inibidores de Proteases , Ligação Proteica , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Suínos
9.
Biochemistry ; 36(45): 13938-45, 1997 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-9374873

RESUMO

Neprilysin (neutral endopeptidase-24.11, EC 3.4.24.11) is a mammalian zinc-endopeptidase involved in the degradation of biologically active peptides. Although no atomic structure is available for this enzyme, site-directed mutagenesis studies have shown that its active site resembles closely that of the bacterial zinc-endopeptidase, thermolysin (EC 3.4.24.27). One active site residue of thermolysin, Arg-203, is involved in inhibitor binding by forming hydrogen bonds with the carbonyl group of a residue in the P1 position and also participates in a hydrogen bond network involving Asp-170. Sequence alignment data shows that Arg-717 of neprilysin could play a similar role to Arg-203 of thermolysin. This was investigated by site-directed mutagenesis with Arg-203 of thermolysin and Arg-717 of neprilysin being replaced by methionine residues. This led, in both cases, to decreases in kcat/Km values, of 122-fold for neprilysin and 2300-fold for thermolysin, essentially due to changes in kcat. The Ki values of several inhibitors were also increased for the mutated enzymes. In addition, the replacement of Asp-170 of thermolysin by Ala residue resulted in a decrease in kcat/Km of 220-fold. The results, coupled with a molecular modeling study, suggest that Arg-717 of neprilysin corresponds to Arg-203 of thermolysin and that in both enzymes a hydrogen bond network exists, involving His-142, Asp-170, and Arg-203 in thermolysin and His-583, Asp-650, and Arg-717 in neprilysin, which is crucial for hydrolytic activity.


Assuntos
Arginina/genética , Mutagênese Sítio-Dirigida , Neprilisina/genética , Termolisina/genética , Sequência de Aminoácidos , Arginina/metabolismo , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Ligação Competitiva , DNA Complementar/genética , Glicopeptídeos/metabolismo , Hidrólise , Modelos Moleculares , Dados de Sequência Molecular , Neprilisina/antagonistas & inibidores , Neprilisina/biossíntese , Neprilisina/metabolismo , Inibidores de Proteases/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Especificidade por Substrato , Termolisina/antagonistas & inibidores , Termolisina/biossíntese , Termolisina/metabolismo , Tiorfano/metabolismo
10.
Plant J ; 11(5): 983-92, 1997 May.
Artigo em Inglês | MEDLINE | ID: mdl-9193070

RESUMO

Synchronously dividing cell cultures of Catharanthus roseus were used to isolate cDNAs for two mitotic cyclins, named CYS and CYM. The deduced protein sequence of CYS is similar to that of A-type cyclins, and CYM belongs to the group of B-type cyclins. In a fashion similar to the pattern of expression seen for A-type and B-type cyclins in mammalian cells, CYS is expressed before CYM in C. roseus cells during the cell cycle. CYS mRNA accumulated at the onset of S phase and disappeared early in the G2 phase, whereas CYM mRNA was detected in the G2 and M phases of the cell cycle. Tobacco homologs of the two genes showed similar cell-cycle dependent expression patterns in synchronous cultures of tobacco BY2 cells. In both systems, CYS was expressed much earlier in the cell cycle than most other plant A-type cyclins, and hence CYS along with the soybean cyc1GM can be classified into a distinct subclass. The activities of CYM and CYS promoters during the cell cycle were analyzed in stably transformed tobacco BY2 cells. Cyclin promoter sequences of 0.5 kb could confer the typical cell-cycle-dependent expression to the beta-glucuronidase (GUS) reporter gene: the CYS promoter directed S-phase-specific expression, whereas the CYM promoter drove M-phase-specific expression. These results indicate the important role of transcriptional regulation in the oscillations of cyclin mRNA levels during the cell cycle.


Assuntos
Ciclo Celular/genética , Ciclinas/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Proteínas de Plantas/genética , Células Cultivadas , Ciclinas/biossíntese , Ciclinas/classificação , DNA Complementar/genética , Teste de Complementação Genética , Biblioteca Genômica , Dados de Sequência Molecular , Proteínas de Plantas/biossíntese , Proteínas de Plantas/classificação , Plantas Tóxicas , Regiões Promotoras Genéticas/genética , Saccharomyces cerevisiae/genética , Análise de Sequência de DNA , Homologia de Sequência , Tabaco/genética , Transcrição Genética
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