RESUMO
The centromere/kinetochore interaction is responsible for the pairing and segregation of replicated chromosomes in eukaryotes. Centromere DNA is portrayed as scarcely conserved, repetitive in nature, quickly evolving and protein-binding competent. Among primates, the major class of centromeric DNA is the pancentromeric α-satellite, made of arrays of 171 bp monomers, repeated in a head-to-tail pattern. α-satellite sequences can either form tandem heterogeneous monomeric arrays or assemble in higher-order repeats (HORs). Gorilla centromere DNA has barely been characterized, and data are mainly based on hybridizations of human alphoid sequences. We isolated and finely characterized gorilla α-satellite sequences and revealed relevant structure and chromosomal distribution similarities with other great apes as well as gorilla-specific features, such as the uniquely octameric structure of the suprachromosomal family-2 (SF2). We demonstrated for the first time the orthologous localization of alphoid suprachromosomal families-1 and -2 (SF1 and SF2) between human and gorilla in contrast to chimpanzee centromeres. Finally, the discovery of a new 189 bp monomer type in gorilla centromeres unravels clues to the role of the centromere protein B, paving the way to solve the significance of the centromere DNA's essential repetitive nature in association with its function and the peculiar evolution of the α-satellite sequence.
Assuntos
Autoantígenos/genética , Proteína B de Centrômero/genética , Centrômero/genética , Proteínas Cromossômicas não Histona/genética , DNA Satélite/genética , Gorilla gorilla/genética , Animais , Sequência de Bases , Linhagem Celular , Proteína Centromérica A , Mapeamento Cromossômico , DNA Satélite/isolamento & purificação , Humanos , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Pan troglodytes/genética , Reação em Cadeia da Polimerase , Pongo pygmaeus/genética , Sequências Repetitivas de Ácido Nucleico , Análise de Sequência de DNA , Homologia de Sequência do Ácido NucleicoRESUMO
Celiac disease (CD) is characterized by abnormally high concentrations of certain peptides in the small bowel. These peptides can be grouped in 'toxic' and 'immunogenic' classes, which elicit an innate immune response and an HLA-mediated adaptive response, respectively. It is not clear on which molecular mechanisms responses to these different classes are based, but the 31-43 (P31-43) and the 56-68 (P56-68) A-gliadin fragments are usually adopted as sequence representatives of toxic and immunogenic peptides, respectively. Here we report fluorescence experiments aiming to mimic the interaction of these peptides with the cell membrane surface by using sodium dodecyl sulphate (SDS) as a membrane-mimetic medium. We show that P31-43 is able to bind SDS micelles in a way that resembles mixed micelle formation. On the other hand, no binding at all could be detected for P56-68. This different behaviour could be related to the paracellular or transcellular route through which gluten peptides may cross the intestinal epithelium, and open new insights into the pathogenetic mechanisms of CD.
Assuntos
Membrana Celular/metabolismo , Gliadina , Fragmentos de Peptídeos , Doença Celíaca/metabolismo , Membrana Celular/química , Corantes/química , Detergentes/química , Gliadina/química , Gliadina/imunologia , Gliadina/toxicidade , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Micelas , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/toxicidade , Dodecilsulfato de Sódio/químicaRESUMO
The effect of porins, major hydrophobic outer membrane proteins purified from Salmonella typhimurium, on human blood coagulation was investigated. It was found that micromolar concentrations of porins accelerated markedly human blood coagulation in vitro. Using appropriate experiments, data were obtained showing that the main target of the porin-induced procoagulant effect was thrombin. A possible binding of porins with thrombin has been suggested to be the basis of this effect. The implications of this finding in the pathogenesis of the disseminated intravascular coagulation syndrome (DIC) occurring during the Gram-negative septic shock is discussed.
Assuntos
Coagulação Sanguínea , Coagulação Intravascular Disseminada/fisiopatologia , Porinas/metabolismo , Salmonella typhimurium/metabolismo , Trombina/metabolismo , Antitrombina III/metabolismo , Antitrombinas/metabolismo , Coagulação Intravascular Disseminada/etiologia , Humanos , Tempo de Tromboplastina Parcial , Peptídeo Hidrolases/metabolismo , Porinas/farmacologia , Porinas/fisiologia , Tempo de Protrombina , Choque Séptico/metabolismo , Choque Séptico/fisiopatologia , Síndrome , Tempo de Coagulação do Sangue TotalRESUMO
This paper proposes to assess hydrogen-bonding contributions to the protein stability, using a set of model proteins for which both X-ray structures and calorimetric unfolding data are known. Pertinent thermodynamic quantities are first estimated according to a recent model of protein energetics based on the dissolution of alkyl amides. Then it is shown that the overall free energy of hydrogen-bond formation accounts for a hydrogen-bonding propensity close to helix-forming tendencies previously found for individual amino acids. This allows us to simulate the melting curve of an alanine-rich helical 50-mer with good precision. Thereafter, hydrogen-bonding enthalpies and entropies are expressed as linear combinations of backbone-backbone, backbone-side-chain, side-chain-backbone, and side-chain-side-chain donor-acceptor contributions. On this basis, each of the four components shows a different free energy versus temperature trend. It appears that structural preference for side-chain-side-chain hydrogen bonding plays a major role in stabilizing proteins at elevated temperatures.
Assuntos
Proteínas de Plantas/química , Dobramento de Proteína , Tripsina/química , Humanos , Ligação de Hidrogênio , Muramidase/química , Mioglobina/química , Desnaturação Proteica , Termodinâmica , Inibidores da Tripsina , alfa-Amilases/antagonistas & inibidoresRESUMO
We highlight conditions under which coincidence of van't Hoff and calorimetric enthalpies can be experimentally verified for reactions of biochemical interest. First, we clarify that, often, chemical equations in condensed phase do not explicitly contain information on all processes involved. Second, we underline that the accuracy of electrochemical methods is much higher than that of other non-calorimetric techniques. Electrochemical data on the binding of ethidium ion to DNA are re-examined to verify that the entropy evaluated as the temperature derivative of the free energy agrees in full with the calorimetric one. Third, we point out that unfolding or self-association enthalpies of redox proteins can be reliably obtained by electromotive force measurements, taking advantage of their linkage to redox enthalpies. Thermodynamic cycles coupling biochemical transformations to redox systems are briefly discussed.
Assuntos
Calorimetria , Eletroquímica/métodos , Termodinâmica , Fenômenos Bioquímicos , Grupo dos Citocromos c/química , DNA/química , Etídio/química , Heme/química , Indicadores e Reagentes/química , Modelos Teóricos , Oxirredução , Oxirredutases/química , Dobramento de Proteína , TemperaturaRESUMO
The position of unfolding curves of oligomeric proteins depends on the protein concentration. The extent of this dependence is analyzed here in terms of the midpoint concentration, i.e., the denaturant concentration at which the fractions of folded and unfolded protein are equal. Reexamination of published data highlights that the midpoint concentration decreases as the protein concentration becomes lower, as expected. Moreover, there are differences between urea and guanidine hydrochloride, as well as discrepancies between the linear extrapolation model and the denaturant binding model. These discrepancies could be used to choose the denaturation model that best fits experimental data. The equations used can be applied to any oligomeric system to check the validity of the two-state assumption.
Assuntos
Oligopeptídeos/química , Dobramento de Proteína , Modelos Químicos , Conformação Proteica , Desnaturação ProteicaRESUMO
The interaction between the 1-47 N-terminus fragment of the cholecystokinin receptor and the nonsulfated cholecystokinin octapeptide, CCK8, is monitored by fluorescence emission. Quenching of the fluorescence intensities is observed on binding. Dissociation constants calculated by these data are in the same submicromolar range as found for the binding of linear CCK8 analogues to B-type receptors. Although detailed structural information cannot be obtained, fluorescence emission is more sensitive than other techniques and permits fast detection of receptor-ligand interaction.
Assuntos
Receptores da Colecistocinina/química , Receptores da Colecistocinina/metabolismo , Sincalida/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Dicroísmo Circular , Humanos , Técnicas In Vitro , Cinética , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Receptor de Colecistocinina A , Receptor de Colecistocinina B , Sincalida/química , Espectrometria de Fluorescência , Sulfatos/químicaRESUMO
The protein SV-IV, a major protein secreted from the rat seminal vesicle epithelium, is a basic protein with immunomodulatory, anti-inflammatory, and procoagulant activity. Predictions suggested that this protein is very flexible, with its three tyrosyl residues presumably located in water-exposed segments of the primary structure. The solution behaviour of the protein was investigated by two types of spectroscopic techniques. Modifications of the spectral characteristics of tyrosyl residues induced by changes of protein concentration were demonstrated by absorption and fluorescence experiments. In addition, secondary structure rearrangements associated with a possible self-association equilibrium were highlighted by far-UV CD spectra. The equilibrium, confirmed by chromatographic techniques, appears to control some biological properties of the protein.
Assuntos
Proteínas/química , Glândulas Seminais/química , Animais , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Técnicas In Vitro , Masculino , Estrutura Quaternária de Proteína , Proteínas/fisiologia , Ratos , Espectrometria de FluorescênciaRESUMO
The transglutaminase from rat coagulating gland secretion has been proposed as a new member of the transglutaminase family. Its basal activity is about 11-fold lower than those of other transglutaminases (e.g., the cytosolic tissue transglutaminase), but reaches levels comparable to those of other transglutaminases on addition of specific surfactant agents. There is no study devoted to understanding the molecular basis of this apparently anomalous activation, which is maximal at approximately 1.5 mM sodium dodecyl sulfate. We provide evidence that in the presence of this detergent modifications of the intrinsic fluorescence as well as energy transfer of the protein fluorescence to a micellar probe parallel the activation of the enzyme. As the sodium dodecyl sulfate concentration inducing maximal activation equals the critical micellar concentration, the biological activity of this transglutaminase appears to be modulated by the binding of micellar aggregates. In fact, the enzyme is modified by posttranslational modifications consisting of some lipid tails. At least two of these tails could act as aggregation nuclei of the enzyme with detergents. This behavior is different from that typical of molecular forms purified from other sources.
Assuntos
Próstata/enzimologia , Dodecilsulfato de Sódio/farmacologia , Transglutaminases/efeitos dos fármacos , Animais , Ativação Enzimática , Fluorescência , Masculino , Modelos Químicos , Processamento de Proteína Pós-Traducional , RatosRESUMO
The molecular properties of the human eukaryotic initiation factor 5A precursor and its site directed Lys50-->Arg variant have been investigated and compared. Structure perturbation methods were used to gain information about the protein architecture in solution. Intrinsic and extrinsic spectroscopic probes strategically located in the protein matrix detected the independent unfolding of two molecular regions. Three cysteines out of four were titrated in the native protein and the peculiar presence of a tyrosinate band at neutral pH was detected. At alkaline pH only two tyrosines out of three were titratable in the native protein, with an apparent pK of about 9.9. Native protein and its Lys50-->Arg variant reacted in a similar fashion to guanidine and to pH variation, but differently to thermal stress. The complex thermal unfolding of both proteins indicated the presence of intermediates. Spectroscopic data showed that these intermediates are differently structured. Consequently, the two proteins seem to have different unfolding pathways.
Assuntos
Arginina/química , Lisina/química , Fatores de Iniciação de Peptídeos/química , Precursores de Proteínas/química , Proteínas de Ligação a RNA , Substituição de Aminoácidos , Sequência de Bases , Dicroísmo Circular , Primers do DNA , Corantes Fluorescentes , Guanidina/química , Humanos , Mutagênese Sítio-Dirigida , Naftalenossulfonatos , Fatores de Iniciação de Peptídeos/genética , Conformação Proteica , Desnaturação Proteica , Precursores de Proteínas/genética , Espectrofotometria Ultravioleta , Fator de Iniciação de Tradução Eucariótico 5ARESUMO
The effect of additives on micellar aggregation is treated using the same framework developed for macromolecular binding. This procedure, hinging on the pseudo-phase transition model, allows us to evaluate the binding constant of any added substance to a micellar aggregate as well as the number of particles bound per amphiphilic unit. The analysis of published literature data confirms that the model provides a general description of micellar aggregation in the presence of added ingredients, regardless of their ionic or non-ionic nature. Original data from our laboratory are well described by the suggested macromolecular binding approach. The dependence of experimental trends on micellization modifiers is also briefly discussed. Copyright 1998 Academic Press.
RESUMO
We have compared the X-ray structures of 13 thermophilic proteins with their mesophilic homologues, in order to bring out differences in the stability of helices. The energy terms of a helix-coil transition algorithm were used to evaluate helix stability. Helices of thermophilic proteins are more stable than the mesophilic homologues in 69% of cases. This is due mainly to intrinsic helical propensities of amino acids, whereas minor effects are linked to main chain H-bonds, side chain-side chain interactions, capping motifs and charge-dipole effects. Furthermore, the frequency of 10 helix stabilizing factors recognized by appropriate sequence patterns was evaluated. The only factor occurring significantly in the thermostable proteins was the lack of beta branched residues. Other factors do not show a definite trend, although their occurrence in proteins is believed to be important for stability. This is discussed in the light of protein engineering applications.
Assuntos
Proteínas/química , Animais , Estrutura Secundária de Proteína , Termodinâmica , Difração de Raios XRESUMO
Like cyclosporin A, cyclolinopeptide A binds specifically bovine cyclophilin A, inhibiting its peptidyl-prolyl cis-trans isomerase activity. We describe here the protein interaction with several synthetic analogues of cyclolinopeptide A, which are either homodetic or disulphide bridged heterodetic cyclopeptides characterized by different ring dimensions, in terms of dissociation and inhibition constants evaluated by fluorescence and inhibition of the enzyme activity, respectively. Dissociation constants from fluorescence experiments are practically identical and about 20-fold lower than for cyclosporin A. On the other hand, inhibition constants differ from compound to compound and are higher than for cyclosporin A. This result is therefore difficult to rationalize, but we would suggest decoupling between binding and inhibitory ability of cyclopeptides. The Pro1 residue of cyclolinopeptide A seems to play a fundamental role in determining the inhibition of the rotamase activity of cyclophilin A, as the homodetic analogue lacking this residue does not show any inhibitory ability. Similarly, heterodetic analogues with a ring size smaller than 7 residues do not display inhibition. We presume that the sequence -Pro-Pro-Phe-Phe- and a ring size of 8 residues for homodetic cyclic peptides could be used as starting points in the targeted synthesis of cyclopeptides able to bind both cyclosporin A and calcineurin. The only peptide showing similar values of the dissociation and inhibition constant is cyclolinopeptide A. This compound can be considered a novel model for the molecular design of immunosuppressant drugs.
Assuntos
Peptídeos Cíclicos/química , Peptidilprolil Isomerase/química , Sequência de Aminoácidos , Animais , Bovinos , Dicroísmo Circular , Mimetismo Molecular , Conformação Proteica , Espectrometria de FluorescênciaRESUMO
BACKGROUND: Diabetic patients are considered less suitable than nondiabetic patients for beta-blocker therapy because of the risk for worsened glucose and lipid metabolism and more severe hypoglycemic attacks. OBJECTIVE: To compare the metabolic and cardiovascular effects of carvedilol with those of atenolol in diabetic patients with hypertension. DESIGN: Randomized, double-blind, 24-week trial. SETTING: University hospital clinic. PATIENTS: 45 patients with non-insulin-dependent diabetes mellitus and hypertension. INTERVENTION: After a 4- to 6-week run-in period during which placebo was given in a single-blind manner, patients were randomly assigned to carvedilol or atenolol. MEASUREMENTS: An oral glucose tolerance test; assessment of insulin sensitivity and hormonal responses to insulin hypoglycemia; and assessment of lipid levels, blood pressure, left ventricular mass, and lipid peroxidation. RESULTS: Changes in systolic and diastolic blood pressure and left ventricular mass index were similar with carvedilol and atenolol (P > 0.2). Fasting plasma glucose and insulin levels decreased with carvedilol and increased with atenolol. Responses to carvedilol were greater than those to atenolol, as follows: increase in total glucose disposal, 9.54 mumol/kg of body weight per minute (95% CI, 7 to 11.9 mumol/kg per minute); decrease in plasma glucose response to oral glucose, 61 mmol/L x 180 minutes (CI, -101 to -21 mmol/L x 180 minutes); decrease in insulin response to oral glucose, 6.2 nmol/L x 180 minutes (CI, -9.8 to -2.6 nmol/L x 180 minutes); decrease in triglyceride level, 0.56 mmol/L (CI, -0.75 to -0.37 mmol/L; P < 0.001); increase in high-density lipoprotein cholesterol level, 0.13 mmol/L (CI, 0.09 to 0.17 mmol/L; P < 0.001); and decrease in lipid peroxidation, 0.25 mumol/L (CI, -0.34 to -0.16 mumol/L). CONCLUSIONS: By improving glucose and lipid metabolism and reducing lipid peroxidation, carvedilol may offer advantages in patients with diabetes and hypertension.
Assuntos
Antagonistas Adrenérgicos alfa/uso terapêutico , Antagonistas Adrenérgicos beta/uso terapêutico , Anti-Hipertensivos/uso terapêutico , Atenolol/uso terapêutico , Carbazóis/uso terapêutico , Diabetes Mellitus Tipo 2/complicações , Angiopatias Diabéticas/tratamento farmacológico , Hipertensão/tratamento farmacológico , Propanolaminas/uso terapêutico , Glicemia/metabolismo , Carvedilol , Diabetes Mellitus Tipo 2/sangue , Angiopatias Diabéticas/sangue , Método Duplo-Cego , Feminino , Humanos , Hipertensão/sangue , Hipertensão/complicações , Peroxidação de Lipídeos/efeitos dos fármacos , Lipídeos/sangue , Masculino , Pessoa de Meia-Idade , Método Simples-CegoRESUMO
In this article we generalize the use of a relationship based on the occurrence of some characteristic temperatures in protein unfolding, which were originally highlighted for proteins showing the unfolding enthalpy-entropy convergence. On this basis, we show how to dissect the unfolding Gibbs energy of globular proteins in terms of solid-like and liquid-like contributions, untangling the protein energetics by a scheme which does not suffer from excessive intricacy. We also provide an experimental estimate of unfavorable polar contributions to the protein stability, by which it seems possible to evaluate the number of buried residues in individual proteins. A comparison is assessed with the so-called hard sphere model of globular proteins. Results seem to reconcile the view that the protein interior is liquid-like with the observation that protein organization resembles an assembly of closely packed spheres.
Assuntos
Proteínas/química , Modelos Químicos , Estrutura Molecular , Conformação Proteica , Dobramento de Proteína , Termodinâmica , Água/químicaRESUMO
Chromatographic separation of alpha-crystallin incubated with [3H]-labelled galactose showed the radioactivity to be concentrated in the low molecular mass subunits (20 and 40 kDa). The effect of glycation on the structural organization of alpha-crystallin was evaluated by FPLC analysis of native (pH 6.8 and 8.2) and glycated protein in dissociating conditions. Results suggest that the glycation acts on the protein surface by altering its charge distribution.
Assuntos
Cristalinas/química , Galactose/química , Animais , Bovinos , Cromatografia Líquida , Reagentes de Ligações Cruzadas , Concentração de Íons de Hidrogênio , Ligação ProteicaRESUMO
This study is concerned with the structural characterization in solution of the glutamate dehydrogenase from the Archaeon Sulfolobus solfataricus. At neutral pH both alpha-helix and beta-sheet constitute the secondary structure of this enzyme, on the basis of circular dichroism. A complex, temperature dependent self-association equilibrium regulates the formation of the enzyme quaternary structure, which seems to be accompanied by a reversible structural change. At 25 degrees C the enzyme is mostly represented by monomeric subunits at concentrations lower than 0.02 mg/ml, while oligomers are predominant at concentrations higher than 0.12 mg/ml. The mid-point of the association curve shifts from 0.05 mg/ml at 25 degrees C to about 0.1 mg/ml at 45 degrees C. Only the oligomeric form appears to be temperature resistant. Monomeric and oligomeric enzyme show distinct behaviour on guanidine hydrochloride perturbation at neutral pH. The monomer denaturation, although complex, is reversible. Two fluorescent tryptophan classes are detectable in the monomer, monitoring the independent unfolding of two regions through a multistate transition. Instead, the oligomeric protein shows a complex denaturation pattern with the tendency to aggregate irreversibly at high denaturant concentration.