RESUMEN
The basic 7S globulin (Bg7S) is one of the major globulins of soybean seeds. Despite its dual subunit composition and oligomeric assembly, Bg7S has a compact 3D structure (PDB: 3AUP) which is stabilized by a network of inter- and intra-chain disulphide bridges. Bg7S shares several structural elements with a number of homologous proteins from other seeds, whose function is still uncertain. In this work, Bg7S native conformation was probed by using the proteolytic enzyme trypsin. In spite of the presence of many arginine and lysine residues, the protein resulted extremely recalcitrant to in vitro enzymatic cleavage. Indeed, only two scissile bonds located near the C- and N-termini of the large and small subunits, respectively, were cleaved. The partially cleaved products were stable even at prolonged incubation times. Although the generated small peptide fragments were not covalently bound to the remnant of the main chains, they were held in place, as assessed by denaturing and non-denaturing chromatographic approaches. Moreover, both the already observed pH-dependent association/dissociation behaviour of the protein and its insulin binding capacity were preserved both at neutral and acidic pH values. These results are in line with the growing view that the degradation of seed proteins, either storage and non-storage, may be a controlled process related to specific functionalities.
Asunto(s)
Globulinas/química , Glycine max/química , Técnicas de Sonda Molecular , Semillas/química , Proteínas de Soja/química , Tripsina/química , Sitios de Unión , Modelos Químicos , Modelos Moleculares , Sondas Moleculares/química , Unión Proteica , Conformación ProteicaRESUMEN
Cowpea seed ß-vignin, a vicilin-like globulin, proved to exert various health favourable effects, including blood cholesterol reduction in animal models. The need of a simple scalable enrichment procedure for further studies for tailored applications of this seed protein is crucial. A chromatography-independent fractionation method allowing to obtain a protein preparation with a high degree of homogeneity was used. Further purification was pursued to deep the molecular characterisation of ß-vignin. The results showed: (i) differing glycosylation patterns of the two constituent polypeptides, in agreement with amino acid sequence features; (ii) the seed accumulation of a gene product never identified before; (iii) metal binding capacity of native protein, a property observed only in few other legume seed vicilins.
Asunto(s)
Globulinas/química , Globulinas/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Vigna/química , Glicosilación , Metales/metabolismo , Semillas/químicaRESUMEN
Aurora kinases have emerged as potential targets in cancer therapy, and several drugs are currently undergoing preclinical and clinical validation. Whether clinical resistance to these drugs can arise is unclear. We exploited a hypermutagenic cancer cell line to select mutations conferring resistance to a well-studied Aurora inhibitor, ZM447439. All resistant clones contained dominant point mutations in Aurora B. Three mutations map to residues in the ATP-binding pocket that are distinct from the "gatekeeper" residue. The mutants retain wild-type catalytic activity and were resistant to all of the Aurora inhibitors tested. Our studies predict that drug-resistant Aurora B mutants are likely to arise during clinical treatment. Furthermore, because the plasticity of the ATP-binding pocket renders Aurora B insensitive to multiple inhibitors, our observations indicate that the drug-resistant Aurora B mutants should be exploited as novel drug targets.
Asunto(s)
Benzamidas/farmacología , Resistencia a Antineoplásicos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Quinazolinas/farmacología , Alelos , Aurora Quinasa B , Aurora Quinasas , Benzamidas/química , Ciclo Celular , Línea Celular Tumoral , Humanos , Modelos Moleculares , Fosforilación , Mutación Puntual , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/química , Quinazolinas/químicaRESUMEN
The demonstration that the small synthetic molecule reversine [2-(4-morpholinoanilino)-N6-cyclohexyladenine] promotes the dedifferentiation of committed cells into multipotent progenitor-type cells has raised hopes on the exploitation of this small chemical tool for the generation of stem cells. Here, we show that reversine causes a failure in cytokinesis and induces polyploidization. These effects of reversine are due to the inhibition of Aurora A and B, two related kinases that are implicated in several aspects of mitosis and that are frequently amplified and overexpressed in human tumors. Reversine inhibits the phosphorylation of histone H3, a direct downstream target of Aurora kinases. Similarly to the Aurora kinase inhibitor VX-680, which has recently entered phase II clinical trials for cancer treatment, reversine inhibited colony formation of leukemic cells from patients with acute myeloid leukemia but was significantly less toxic than VX-680 on cells from healthy donors. The crystal structure of the reversine-Aurora B kinase complex shows that reversine is a novel class of ATP-competitive Aurora kinase inhibitors. Thus, although our studies raise serious doubts on the application of reversine in regenerative medicine, they support the paradigm that reversine might be a useful agent in cancer chemotherapy.
Asunto(s)
Antineoplásicos/farmacología , Leucemia Mieloide Aguda/enzimología , Morfolinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Purinas/farmacología , Aurora Quinasa B , Aurora Quinasas , Sitios de Unión/efectos de los fármacos , Diferenciación Celular , Línea Celular Tumoral , Proliferación Celular , Relación Dosis-Respuesta a Droga , Células HeLa , Histonas/metabolismo , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Modelos Moleculares , Morfolinas/química , Morfolinas/metabolismo , Fosforilación , Poliploidía , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Purinas/química , Purinas/metabolismoRESUMEN
Seed proteome analysis by 2D IEF/SDS-PAGE techniques is challenging for the intrinsic difficulties related to quantitative disparity of the seed proteins, i.e. storage and non-storage proteins, their polymorphic nature, the extensive post-translational modifications and the paucity of deposited primary structures available. Conversely, 2D maps of seed proteomes can be extremely useful for a number of fundamental and applied investigations. In this work, we have used a combination of two experimental approaches to identify the main protein components of an emerging protein-rich legume seed, that is white lupin seed (Lupinus albus, L.). One is the canonical proteomic approach including 2D electrophoretic separation and mass spectrometry of selected trypsin-digested polypeptides; the other approach is a group comparative 2D electrophoretic analysis of cotyledonary protein families. To this second purpose, the three main families of lupin seed proteins, namely alpha-conglutins, the 11S globulin fraction, beta-conglutins, the 7S globulin fraction, and gamma-conglutin, a basic 7S protein, were isolated by conventional biochemical techniques and their 2D reference maps were compared with the total protein map. With the first approach 37 out of 40 spots, making up about 35% of total spot volumes in the 2D map, were found to belong to the main seed protein families. Thanks to cDNA-deduced lupin storage protein sequences, determined on purpose and deposited, most of the identification statistical parameters were very good. Moreover, it was possible to identify several endogenously proteolysed subunits in the map. The second comparative approach, beside confirming these attributions, allowed to allocate 124 polypeptides within the three main lupin protein families. These two approaches proved to be mutually validating and their combined use was effective for the establishment of a seed proteome map even in the case of sequence and protein post-translational processing lack of information. The results obtained also extend our knowledge of the seed storage protein polymorphism of white lupin.
Asunto(s)
Electroforesis en Gel Bidimensional/métodos , Lupinus/metabolismo , Proteínas de Plantas/análisis , Proteoma/análisis , Semillas/metabolismo , Secuencia de Aminoácidos , Cromatografía Liquida , Electroforesis en Gel de Poliacrilamida , Datos de Secuencia Molecular , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en TándemRESUMEN
Bowman-Birk serine protease inhibitors are a family of small plant proteins, whose physiological role has not been ascertained as yet, while chemopreventive anticarcinogenic properties have repeatedly been claimed. In this work we present data on the isolation of a lentil (Lens culinaris, L., var. Macrosperma) seed trypsin inhibitor (LCTI) and its functional and structural characterization. LCTI is a 7448 Da double-headed trypsin/chymotrypsin inhibitor with dissociation constants equal to 0.54 nM and 7.25 nM for the two proteases, respectively. The inhibitor is, however, hydrolysed by trypsin in a few minutes timescale, leading to a dramatic loss of its affinity for the enzyme. This is due to a substantial difference in the kon and k*on values (1.1 microM-1.s-1 vs. 0.002 microM-1.s-1), respectively, for the intact and modified inhibitor. A similar behaviour was not observed with chymotrypsin. The twenty best NMR structures concurrently showed a canonical Bowman-Birk inhibitor (BBI) conformation with two antipodal beta-hairpins containing the inhibitory domains. The tertiary structure is stabilized by ion pairs and hydrogen bonds involving the side chain and backbone of Asp10-Asp26-Arg28 and Asp36-Asp52 residues. At physiological pH, the final structure results in an asymmetric distribution of opposite charges with a negative electrostatic potential, centred on the C-terminus, and a highly positive potential, surrounding the antitryptic domain. The segment 53-55 lacks the anchoring capacity found in analogous BBIs, thus rendering the protein susceptible to hydrolysis. The inhibitory properties of LCTI, related to the simultaneous presence of two key amino acids (Gln18 and His54), render the molecule unusual within the natural Bowman-Birk inhibitor family.
Asunto(s)
Lens (Planta)/química , Semillas/química , Inhibidor de la Tripsina de Soja de Bowman-Birk/química , Inhibidor de la Tripsina de Soja de Bowman-Birk/metabolismo , Secuencia de Aminoácidos , Cristalografía por Rayos X , Lens (Planta)/metabolismo , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Semillas/metabolismo , Soluciones , Inhibidor de la Tripsina de Soja de Bowman-Birk/aislamiento & purificaciónRESUMEN
Neuroserpin (NS) is a serpin inhibitor of tissue plasminogen activator (tPA) in the brain. The polymerisation of NS pathologic mutants is responsible for a genetic dementia known as familial encephalopathy with neuroserpin inclusion bodies (FENIB). So far, a pharmacological treatment of FENIB, i.e. an inhibitor of NS polymerisation, remains an unmet challenge. Here, we present a biophysical characterisation of the effects caused by embelin (EMB a small natural compound) on NS conformers and NS polymerisation. EMB destabilises all known NS conformers, specifically binding to NS molecules with a 1:1 NS:EMB molar ratio without unfolding the NS fold. In particular, NS polymers disaggregate in the presence of EMB, and their formation is prevented. The NS/EMB complex does not inhibit tPA proteolytic activity. Both effects are pharmacologically relevant: firstly by inhibiting the NS polymerisation associated to FENIB, and secondly by potentially antagonizing metastatic processes facilitated by NS activity in the brain.
Asunto(s)
Benzoquinonas/metabolismo , Neuropéptidos/metabolismo , Multimerización de Proteína , Serpinas/metabolismo , Benzoquinonas/química , Dicroismo Circular , Humanos , Cinética , Ligandos , Espectrometría de Masas/métodos , Neuropéptidos/química , Unión Proteica , Conformación Proteica , Estabilidad Proteica , Serpinas/química , Activador de Tejido Plasminógeno/antagonistas & inhibidores , NeuroserpinaRESUMEN
The 11S storage globulin of white lupin seeds binds to a metal affinity chromatography matrix. Two unusual stretches of contiguous histidine residues, reminiscent of the multiple histidines forming metal binding motifs, at the C-terminal end of 11S globulin acidic chains were hypothesized as candidate elements responsible for the binding capacity. To prove this, the protein was incubated with a lupin seed endopeptidase previously shown to cleave at twin arginine motifs, recurrent in the sequence region of interest. Upon incubation with this enzyme, the loss of metal binding capacity paralleled that of the anti-his-tag reactive polypeptides. The recovered small proteolytic fragment was analyzed by mass spectrometry and N-terminal sequencing and found to correspond to the 24-mer region cleaved off at twin arginine residues and containing the natural his-tag-like region. Similarly, when lupin seeds were germinated for a few days, the his-tag containing 11S globulin chain was converted to a form devoid of such region, suggesting that this mechanism is a part of the natural degradatory process of the protein. The hypothesis that the ordered and controlled dismantling of storage proteins may generate peptide fragments with potential functional roles in plant ontogenesis is presented and discussed.
Asunto(s)
Globulinas/metabolismo , Lupinus/metabolismo , Proteínas de Plantas/metabolismo , Semillas/metabolismo , Secuencia de Aminoácidos , Arginina/química , Arginina/metabolismo , Germinación , Globulinas/química , Lupinus/química , Lupinus/crecimiento & desarrollo , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas de Plantas/química , Proteolisis , Semillas/química , Semillas/crecimiento & desarrolloRESUMEN
This work describes the in vitro interaction between a lupin seed protein, namely, conglutin gamma, and insulin. The binding to an insulin-immobilized matrix occurs in the pH range from 7.5 to 4.2 and is strongly affected by ionic strength, suggesting that it is driven primarily by electrostatic interactions. The quantitative parameters of the binding were determined by surface plasmon resonance. On the basis of the conditions required for the interaction to take place and the quantitative binding parameters, it appeared that the interaction is specific, despite the fact that the origin of the two protein molecules is completely different. The effect of the oral administration of conglutin gamma on the glycemic levels of rats subjected to glucose overloading was a statistically significant reduction in glycemia comparable to that of metformin, a well-known glucose lowering drug. These findings represent the first molecular evidence of the possible use of a legume protein in the control of glycemia.
Asunto(s)
Glucemia/efectos de los fármacos , Hiperglucemia/tratamiento farmacológico , Insulina/química , Proteínas de Plantas/uso terapéutico , Administración Oral , Animales , Cromatografía de Afinidad , Masculino , Proteínas de Plantas/química , Unión Proteica , Ratas , Resonancia por Plasmón de SuperficieRESUMEN
The susceptibility to trypsin of conglutin gamma, a lupin seed glycoprotein affected by this enzyme only when in a non-native conformation, was used to study the effect of Zn(2+) and other metal ions on the structural dynamics of the protein. When acid-treated trypsin-susceptible conglutin gamma was incubated at neutral pH in the presence of Zn(2+), it became resistant to tryptic attack, contrary to the protein treated in the absence of Zn(2+). The time course of this refolding event has been quantitatively evaluated by SDS-PAGE. Amino acid sequencing of the major polypeptide fragments, produced by trypsin before completion of the refolding process, indicated that only a few cleavable bonds were accessible to the enzyme. This suggested that the presence of metal ions affected the pathway of degradation of the protein, by inducing its folding. Among the other metal ions tested, Ni(2+) also promoted the adoption of a trypsin-resistant conformation of conglutin gamma, whereas Mn(2+) and Ca(2+) had only much lower effects. The relevance of these findings for a deeper understanding of the in vivo degradation of plant food proteins and how it is affected by metal ions are discussed.
Asunto(s)
Endopeptidasas/farmacología , Metales/farmacología , Proteínas de Plantas/química , Desnaturalización Proteica , Pliegue de Proteína , Tripsina/farmacología , Resistencia a Medicamentos , Electroforesis en Gel de Poliacrilamida , Concentración de Iones de Hidrógeno , Cinética , Níquel/farmacología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Proteínas de Plantas/metabolismo , Análisis de Secuencia de ProteínaRESUMEN
The Aurora family is a well conserved and well characterized group of serine-threonine kinases involved in the normal progression of mitosis. The deregulation of Aurora kinases impairs spindle assembly, checkpoint function and cell division. To date, many small molecules that compete with ATP for binding to Aurora kinases have been developed and characterized. Here, the first structure of the Xenopus laevis Aurora B-INCENP complex bound to the clinically relevant small molecule barasertib was determined. The binding properties of this inhibitor to the Aurora B active site are analyzed and reported. An unexpected crystal-packing contact in the Aurora B-INCENP structure coordinated by an ATP analogue is also reported, in which the INCENP C-terminus occupies the substrate-binding region, resembling the protein kinase A inhibitory mechanism.
Asunto(s)
Antineoplásicos/química , Aurora Quinasa B/química , Proteínas Cromosómicas no Histona/química , Organofosfatos/química , Quinazolinas/química , Proteínas de Xenopus/química , Adenilil Imidodifosfato/química , Secuencia de Aminoácidos , Animales , Dominio Catalítico , Cristalografía por Rayos X , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Homología Estructural de Proteína , Xenopus laevisRESUMEN
Sakacin A was purified to homogeneity through simple chromatographic procedures from cultures of Lactobacillus sakei DSMZ 6333 grown on a low-cost medium. The highly purified protein dissipated both transmembrane potential (ΔΨ) and transmembrane pH gradient (ΔpH) in Listeria cells in a very intense, rapid, and energy-dependent fashion. On a slower timescale, purified sakacin A also showed a lytic activity toward isolated cell walls of Listeria. Mass spectrometry was used to analyze the products of sakacin A action on cell walls, evidencing that sakacin A acts on various types of bonds within peptoglycans.
Asunto(s)
Antibacterianos/farmacología , Bacteriocinas/farmacología , Pared Celular/efectos de los fármacos , Listeria/efectos de los fármacos , Fuerza Protón-Motriz/efectos de los fármacos , Antibacterianos/aislamiento & purificación , Antibacterianos/metabolismo , Bacteriocinas/aislamiento & purificación , Bacteriocinas/metabolismo , Pared Celular/química , Cromatografía/métodos , Medios de Cultivo/química , Humanos , Lactobacillus/metabolismo , Espectrometría de Masas , Peptidoglicano/análisisRESUMEN
The occurrence of twin-arginine motifs (-R-R-) in the amino acid sequences of animal pro-proteins frequently defines the cleavage site(s) for their structural/functional maturation. No information is available on the presence and possible biological meaning of these motifs in the seed storage proteins. In this work, a novel endopeptidase activity with cleavage specificity to twin-arginine pairs has been detected in mature dry Lupinus albus seeds. The endopeptidase was tested with a number of endogenous and exogenous protein substrates, which were selected according to the presence of one or more twin-arginine residue motifs in their amino acid sequences. The observed hydrolysis patterns were limited and highly specific. Partial proteolysis led to stable polypeptide fragments that were characterized by 1- and 2-D electrophoresis. Selected polypeptides were submitted to N-terminal amino acid sequencing and mass spectrometry analyses. These approaches, supported by bioinformatic analysis of the available sequences, allowed the conclusion that the polypeptide cleavage events had occurred at the peptide bonds comprised between twin-arginine residue pairs with all tested protein substrates. The endopeptidase activity was inhibited by 4-(2-AminoEthyl)Benzene-Sulphonyl Fluoride hydrochloride (AEBSF), leupeptin, and serine proteinase protein inhibitors, while it was not affected by pepstatin, trans-Epoxysuccinyl-L-leucylamido(4-guanidino)butane (E64), and ethylenediaminetetraacetic acid (EDTA), thus qualifying the Arg-Arg cleaving enzyme as a serine endopeptidase. The structural features of storage proteins from lupin and other legume seeds strongly support the hypothesis that the occurrence of an endopeptidase activity cleaving -R-R- bonds may be functional to facilitate their degradation at germination and possibly generate polypeptide fragments with specific biological activity.
Asunto(s)
Lupinus/enzimología , Proteínas de Plantas/metabolismo , Proteínas de Almacenamiento de Semillas/química , Proteínas de Almacenamiento de Semillas/metabolismo , Semillas/enzimología , Serina Endopeptidasas/metabolismo , Secuencias de Aminoácidos , Arginina/química , Arginina/metabolismo , Lupinus/química , Proteínas de Plantas/química , Proteolisis , Semillas/química , Serina Endopeptidasas/químicaRESUMEN
The timing and localization of events during mitosis are controlled by the regulated phosphorylation of proteins by the mitotic kinases, which include Aurora A, Aurora B, Nek2 (never in mitosis kinase 2), Plk1 (Polo-like kinase 1), and the cyclin-dependent kinase complex Cdk1/cyclin B. Although mitotic kinases can have overlapping subcellular localizations, each kinase appears to phosphorylate its substrates on distinct sites. To gain insight into the relative importance of local sequence context in kinase selectivity, identify previously unknown substrates of these five mitotic kinases, and explore potential mechanisms for substrate discrimination, we determined the optimal substrate motifs of these major mitotic kinases by positional scanning oriented peptide library screening (PS-OPLS). We verified individual motifs with in vitro peptide kinetic studies and used structural modeling to rationalize the kinase-specific selection of key motif-determining residues at the molecular level. Cross comparisons among the phosphorylation site selectivity motifs of these kinases revealed an evolutionarily conserved mutual exclusion mechanism in which the positively and negatively selected portions of the phosphorylation motifs of mitotic kinases, together with their subcellular localizations, result in proper substrate targeting in a coordinated manner during mitosis.
Asunto(s)
Evolución Molecular , Mitosis/fisiología , Proteínas Serina-Treonina Quinasas/metabolismo , Transducción de Señal/fisiología , Proteínas de Xenopus/metabolismo , Secuencias de Aminoácidos , Animales , Humanos , Biblioteca de Péptidos , Fosforilación/fisiología , Xenopus laevisRESUMEN
Aurora family kinases regulate important events during mitosis including centrosome maturation and separation, mitotic spindle assembly, and chromosome segregation. Misregulation of Aurora kinases due to genetic amplification and protein overexpression results in aneuploidy and may contribute to tumorigenesis. Here we report the discovery of new small molecule aminothiazole inhibitors of Aurora kinases with exceptional kinase selectivity and report a 1.7 A cocrystal structure with the Aurora B:INCENP complex from Xenopus laevis. The compounds recapitulate the hallmarks of Aurora kinase inhibition, including decreased histone H3 serine 10 phosphorylation, failure to complete cytokinesis, and endoreduplication.
Asunto(s)
Aminas/química , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Tiazoles/química , Tiazoles/farmacología , Animales , Aurora Quinasas , Cianatos/química , Modelos Moleculares , Estructura Molecular , Unión Proteica , Proteínas Serina-Treonina Quinasas/química , Sensibilidad y Especificidad , Relación Estructura-Actividad , Xenopus laevisRESUMEN
Aurora family serine/threonine kinases control mitotic progression, and their deregulation is implicated in tumorigenesis. Aurora A and Aurora B, the best-characterized members of mammalian Aurora kinases, are approximately 60% identical but bind to unrelated activating subunits. The structure of the complex of Aurora A with the TPX2 activator has been reported previously. Here, we report the crystal structure of Aurora B in complex with the IN-box segment of the inner centromere protein (INCENP) activator and with the small molecule inhibitor Hesperadin. The Aurora B:INCENP complex is remarkably different from the Aurora A:TPX2 complex. INCENP forms a crown around the small lobe of Aurora B and induces the active conformation of the T loop allosterically. The structure represents an intermediate state of activation of Aurora B in which the Aurora B C-terminal segment stabilizes an open conformation of the catalytic cleft, and a critical ion pair in the kinase active site is impaired. Phosphorylation of two serines in the carboxyl terminus of INCENP generates the fully active kinase.