RESUMO
Listeria monocytogenes is the causative agent of listeriosis, a very serious food-borne human disease. The analysis of the proteins coded by the L. monocytogenes genome reveals the presence of two eukaryotic-type Ser/Thr-kinases (lmo1820 and lmo0618) and a Ser/Thr-phosphatase (lmo1821). Protein phosphorylation regulates enzyme activities and protein interactions participating in physiological and pathophysiological processes in bacterial diseases. However in the case of L. monocytogenes there is scarce information about biochemical properties of these enzymes, as well as the physiological processes that they modulate. In the present work the catalytic domain of the protein coded by lmo1820 was produced as a functional His(6)-tagged Ser/Thr-kinase, and was denominated PrkA. PrkA was able to autophosphorylate specific Thr residues within its activation loop sequence. A similar autophosphorylation pattern was previously reported for Ser/Thr-kinases from related prokaryotes, whose role in kinase activity and substrate recruitment was demonstrated. We studied the kinase interactome using affinity chromatography and proteomic approaches. We identified 62 proteins that interact, either directly or indirectly, with the catalytic domain of PrkA, including proteins that participate in carbohydrates metabolism, cell wall metabolism and protein synthesis. Our results suggest that PrkA could be involved in the regulation of a variety of fundamental biological processes.
Assuntos
Listeria monocytogenes/enzimologia , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Proteômica , Proteínas de Bactérias , Domínio Catalítico , Humanos , Metabolismo , Fosforilação , Proteômica/métodos , Especificidade por SubstratoRESUMO
The five muscarinic acetylcholine receptors (M(1)-M(5)) are differentially expressed in the brain. M(2) and M(4) are coupled to inhibition of stimulated adenylyl cyclase, while M(1), M(3) and M(5) are mainly coupled to the phosphoinositide pathway. We studied the muscarinic receptor regulation of adenylyl cyclase activity in the rat hippocampus, compared to the striatum and amygdala. Basal and forskolin-stimulated adenylyl cyclase activity was higher in the striatum but the muscarinic inhibition was much lower. Highly selective muscarinic toxins MT1 and MT2-affinity order M(1) > or = M(4) >> others-and MT3-highly selective M(4) antagonist-did not show significant effects on basal or forskolin-stimulated cyclic AMP production but, like scopolamine, counteracted oxotremorine inhibition. Since MTs have negligible affinity for M(2), M(4) would be the main subtype responsible for muscarinic inhibition of forskolin-stimulated enzyme. Dopamine stimulated a small fraction of the enzyme (3.1% in striatum, 1.3% in the hippocampus). Since MT3 fully blocked muscarinic inhibition of dopamine-stimulated enzyme, M(4) receptor would be responsible for this regulation.
Assuntos
Hipocampo/enzimologia , Antagonistas Muscarínicos/farmacologia , Neostriado/enzimologia , Receptor Muscarínico M4/efeitos dos fármacos , Adenilil Ciclases/metabolismo , Tonsila do Cerebelo/efeitos dos fármacos , Tonsila do Cerebelo/fisiologia , Animais , Colforsina/farmacologia , AMP Cíclico/biossíntese , Dopamina/farmacologia , Relação Dose-Resposta a Droga , Hipocampo/efeitos dos fármacos , Masculino , Agonistas Muscarínicos/farmacologia , Neostriado/efeitos dos fármacos , Oxotremorina/farmacologia , Ratos , Ratos Wistar , Receptor Muscarínico M4/agonistas , Receptor Muscarínico M4/antagonistas & inibidoresRESUMO
All five subtypes of muscarinic acetylcholine receptors (mAChR; M(1)-M(5)) are expressed in the hippocampus, where they are involved both in cognitive functions and in synaptic plasticity, such as long-term potentiation (LTP). Muscarinic toxins (MTs) are small proteins from mamba snake venoms that display exquisite discrimination between mAChRs. MT1 acts as an agonist at M(1) and an antagonist at M(4) receptors, with similar affinities for both. MT3, the most selective antagonist available for M(4) receptors, infused into the CA1 region immediately after training caused amnesia in the rat, indicating the participation of M(4) receptors in memory consolidation. Our goal was to investigate the participation of M(4) receptor in neurotransmission at the hippocampal Schaffer collaterals-CA1 synapses. Two different preparations were used: 1) field potential recordings in freshly prepared rat hippocampal slices with high-frequency stimulation to induce potentiation and 2) whole-cell voltage clamp in cultured hippocampal organotypic slices with paired stimuli. In preparation 1, a dose of MT3 that was previously shown to cause amnesia blocked LTP; the nonselective antagonist scopolamine blocked LTP without affecting basal transmission, although it was depressed with higher concentration. In preparation 2, basal transmission was decreased and LTP induction was prevented by an MT3 concentration that would bind mainly to M(4) receptors. Although M(1) receptors appeared to modulate transmission positively at these excitatory synapses, M(1) activation concomitant with M(4) blockade (by MT1) only allowed a brief, short-term potentiation. Accordingly, M(4) blockade by MT3 strongly supports a permissive role of M(4) receptors and suggests their necessary participation in synaptic plasticity at these synapses.
Assuntos
Hipocampo/fisiologia , Neurônios/fisiologia , Receptor Muscarínico M4/fisiologia , Sinapses/fisiologia , Transmissão Sináptica , Animais , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intercelular , Potenciação de Longa Duração/efeitos dos fármacos , Masculino , Antagonistas Muscarínicos/toxicidade , Neurônios/efeitos dos fármacos , Neurotoxinas/toxicidade , Técnicas de Patch-Clamp , Peptídeos/toxicidade , Ratos , Ratos Wistar , Receptor Muscarínico M4/antagonistas & inibidores , Escopolamina/toxicidade , Sinapses/efeitos dos fármacosRESUMO
The aggregation of alpha-synuclein (AS) is a critical step in the etiology of Parkinson's disease (PD) and other neurodegenerative synucleinopathies. Protein-metal interactions play a critical role in AS aggregation and might represent the link between the pathological processes of protein aggregation and oxidative damage. Our previous studies established a hierarchy in AS-metal ion interactions, where Cu(II) binds specifically to the protein and triggers its aggregation under conditions that might be relevant for the development of PD. In this work, we have addressed unresolved structural details related to the binding specificity of Cu(II) through the design of site-directed and domain-truncated mutants of AS and by the characterization of the metal-binding features of its natural homologue beta-synuclein (BS). The structural properties of the Cu(II) complexes were determined by the combined application of nuclear magnetic resonance, electron paramagnetic resonance, UV-vis, circular dichroism spectroscopy, and matrix-assisted laser desorption ionization mass spectrometry (MALDI MS). Two independent, noninteracting copper-binding sites with significantly different affinities for the metal ion were detected in the N-terminal regions of AS and BS. MALDI MS provided unique evidence for the direct involvement of Met1 as the primary anchoring residue for Cu(II) in both proteins. Comparative spectroscopic analysis of the two proteins allowed us to deconvolute the Cu(II) binding modes and unequivocally assign the higher-affinity site to the N-terminal amino group of Met1 and the lower-affinity site to the imidazol ring of the sole His residue. Through the use of competitive chelators, the affinity of the first equivalent of bound Cu(II) was accurately determined to be in the submicromolar range for both AS and BS. Our results prove that Cu(II) binding in the C-terminal region of synucleins represents a nonspecific, very low affinity process. These new insights into the bioinorganic chemistry of PD are central to an understanding of the role of Cu(II) in the fibrillization process of AS and have implications for the molecular mechanism by which BS might inhibit AS amyloid assembly.
Assuntos
Cobre/química , Metaloproteínas/química , alfa-Sinucleína/química , beta-Sinucleína/química , Sequência de Aminoácidos , Sítios de Ligação , Dicroísmo Circular , Espectroscopia de Ressonância de Spin Eletrônica , Cinética , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Alinhamento de Sequência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The cholinergic system plays a crucial role in learning and memory. Modulatory mechanisms of this system in the acquisition and consolidation processes have been extensively studied, but their participation in the memory retrieval process is still poorly understood. Conventional pharmacological agents are not highly selective for particular muscarinic acetylcholine receptor subtypes. Muscarinic toxins (MTs) that are highly selective for muscarinic receptors were extracted from the venom of the mamba snake, like the toxin MT3, selective for the M4 receptor subtype. These toxins are useful tools in studies of the specific functions of the M4 mediated transmission. The M4 receptor selective antagonist MT3, given into the dorsal hippocampus before the test, have enhanced the memory retrieval of an inhibitory avoidance task in rats. MT3 had no effect in the habituation to a new environment, including basic motor parameters, meaning that the effect in he inhibitory avoidance is purely cognitive. Our results suggest an endogenous negative modulation of the cholinergic muscarinic system upon the retrieval of previously consolidated aversive memories, hereby shown by the facilitatory effect of MT3.
Assuntos
Aprendizagem da Esquiva/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Inibição Psicológica , Antagonistas Muscarínicos/administração & dosagem , Peptídeos/administração & dosagem , Animais , Comportamento Animal/efeitos dos fármacos , Relação Dose-Resposta a Droga , Comportamento Exploratório/efeitos dos fármacos , Hipocampo/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Rememoração Mental/efeitos dos fármacos , Ratos , Ratos Wistar , Tempo de Reação/efeitos dos fármacosRESUMO
The identification of phosphorylation sites in proteins provides a powerful tool to study signal transduction pathways and to establish interaction networks involving signaling elements. Using different strategies to identify phosphorylated residues, we report here mass spectrometry studies of the entire intracellular regions of four 'receptor-like' protein kinases from Mycobacterium tuberculosis (PknB, PknD, PknE, and PknF), each consisting of an N-terminal kinase domain and a juxtamembrane region of varying length (26-100 residues). The enzymes were observed to incorporate different numbers of phosphates, from five in PknB up to 11 in PknD or PknE, and all detected sites were dephosphorylated by the cognate mycobacterial phosphatase PstP. Comparison of the phosphorylation patterns reveals two recurrent clusters of pThr/pSer residues, respectively, in their activation loops and juxtamembrane regions, which have a distinct effect on kinase activity. All studied kinases have at least two conserved phosphorylated residues in their activation loop and mutations of these residues in PknB significantly decreased the kinase activity, whereas deletion of the entire juxtamembrane regions in PknB and PknF had little effect on their activities. These results reinforce the hypothesis that mycobacterial kinase regulation includes a conserved activation loop mechanism, and suggest that phosphorylation sites in the juxtamembrane region might be involved in putative kinase-mediated signaling cascades.
Assuntos
Mycobacterium tuberculosis/enzimologia , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Membrana Celular/enzimologia , Primers do DNA , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosforilação , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Cytochrome c-dependent electron transfer and apoptosome activation require protein-protein binding, which are mainly directed by conformational and specific electrostatic interactions. Cytochrome c contains four highly conserved tyrosine residues, one internal (Tyr67), one intermediate (Tyr48), and two more accessible to the solvent (Tyr74 and Tyr97). Tyrosine nitration by biologically-relevant intermediates could influence cytochrome c structure and function. Herein, we analyzed the time course and site(s) of tyrosine nitration in horse cytochrome c by fluxes of peroxynitrite. Also, a method of purifying each (nitrated) cytochrome c product by cation-exchange HPLC was developed. A flux of peroxynitrite caused the time-dependent formation of different nitrated species, all less positively charged than the native form. At low accumulated doses of peroxynitrite, the main products were two mononitrated cytochrome c species at Tyr97 and Tyr74, as shown by peptide mapping and mass spectrometry analysis. At higher doses, all tyrosine residues in cytochrome c were nitrated, including dinitrated (i.e., Tyr97 and Tyr67 or Tyr74 and Tyr67) and trinitrated (i.e., Tyr97, Tyr74, and Tyr67) forms of the protein, with Tyr67 well represented in dinitrated species and Tyr48 being the least prone to nitration. All mono-, di-, and trinitrated cytochrome c species displayed an increased peroxidase activity. Nitrated cytochrome c in Tyr74 and Tyr67, and to a lesser extent in Tyr97, was unable to restore the respiratory function of cytochrome c-depleted mitochondria. The nitration pattern of cytochrome c in the presence of tetranitromethane (TNM) was comparable to that obtained with peroxynitrite, but with an increased relative nitration yield at Tyr67. The use of purified and well-characterized mono- and dinitrated cytochrome c species allows us to study the influence of nitration of specific tyrosines in cytochrome c functions. Moreover, identification of cytochrome c nitration sites in vivo may assist in unraveling the chemical nature of proximal reactive nitrogen species.
Assuntos
Citocromos c/química , Citocromos c/metabolismo , Ácido Peroxinitroso/química , Tirosina/metabolismo , Sequência de Aminoácidos , Animais , Transporte de Elétrons , Cavalos , Mitocôndrias Cardíacas/enzimologia , Dados de Sequência Molecular , Peroxidase/química , Ácido Peroxinitroso/metabolismo , Cianeto de Potássio/química , Ratos , Cianeto de Sódio/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Tetranitrometano/química , Fatores de TempoRESUMO
Human recombinant copper-zinc superoxide dismutase (CuZnSOD) was inactivated by peroxynitrite, the product of the reaction between nitric oxide and superoxide. The concentration of peroxynitrite that decreased the activity by 50% (IC(50)) was approximately 100 microM at 5 microM CuZnSOD and the inactivation was higher at alkaline pH. Stopped-flow determinations showed that the second-order rate constant for the direct reaction of peroxynitrite with CuZnSOD was (9.4 +/- 1.0) x 10(3) M(-1) s(-1) per monomer at pH 7.5 and 37 degrees C. Addition of peroxynitrite (1 mM) to CuZnSOD (0.5 mM) in the presence of the spin trap 2-methyl-2-nitrosopropane led to the electron paramagnetic resonance detection of an anisotropic signal typical of a protein radical adduct. Treatment with Pronase revealed a nearly isotropic signal consistent with the formation of histidinyl radical. The effects of nitrite, hydrogen peroxide, bicarbonate, and mannitol on the inactivation were assessed. Considering the mechanism accepted for the reaction of CuZnSOD with hydrogen peroxide and the fact that CuZnSOD promotes the nitration of phenolics by peroxynitrite, we herein propose that peroxynitrite reacts with CuZnSOD leading to nitrogen dioxide plus a copper-bound hydroxyl radical species that reacts with histidine residues, forming histidinyl radical.
Assuntos
Inibidores Enzimáticos/farmacologia , Histidina/química , Ácido Peroxinitroso/farmacologia , Superóxido Dismutase/antagonistas & inibidores , Bicarbonatos/química , Relação Dose-Resposta a Droga , Espectroscopia de Ressonância de Spin Eletrônica , Radicais Livres , Humanos , Peróxido de Hidrogênio/química , Concentração de Íons de Hidrogênio , Concentração Inibidora 50 , Cinética , Manitol/química , Modelos Químicos , Nitritos/química , Compostos Nitrosos/química , Ácido Peroxinitroso/química , Proteínas Recombinantes/química , Superóxido Dismutase/químicaRESUMO
Trypanosoma cruzi, the causative agent of Chagas disease, has evolved particular mechanisms of gene regulation. Gene expression is regulated firstly at post-transcriptional level. This feature makes proteomic methods a promising tool for studying adaptative changes in these parasites. In this work we generated a reproducible method for protein analysis by two-dimensional electrophoresis coupled to mass spectrometry, and a protein map for T. cruzi. Western-blot analysis supported the identity of some of the proteins. This work points to proteomic approach as a powerful tool to study differential expression, stress response or drug resistance in T. cruzi.
Assuntos
Doença de Chagas/genética , Proteoma/genética , Proteínas de Protozoários/genética , Trypanosoma cruzi/genética , Animais , Western Blotting/métodos , Ciclo Celular/genética , Doença de Chagas/metabolismo , Eletroforese em Gel Bidimensional/métodos , Expressão Gênica/genética , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Proteoma/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMO
MARCKS is an actin-modulating protein that can be phosphorylated in multiple sites by PKC and proline-directed kinases. We have previously described a phosphorylated form of this protein specific for differentiating chick neurons, detected with mAb 3C3. Here, we show that this antibody binds to MARCKS only when it is phosphorylated at Ser 25. These and previous data provide hints for a possible answer to the question of why this ubiquitous protein seems to be essential only for neural development.