RESUMEN
Technological advancements in recent years have promoted a marked progress in understanding the genetic basis of phenotypes. In line with these advances, genomics has changed the paradigm of biological questions in full genome-wide scale (genome-wide), revealing an explosion of data and opening up many possibilities. On the other hand, the vast amount of information that has been generated points the challenges that must be overcome for storage (Moore's law) and processing of biological information. In this context, bioinformatics and computational biology have sought to overcome such challenges. This review presents an overview of bioinformatics and its use in the analysis of biological data, exploring approaches, emerging methodologies, and tools that can give biological meaning to the data generated.
Asunto(s)
Biología Computacional , Secuencia de Aminoácidos , Secuencia de Bases , Bases de Datos Genéticas , Humanos , Modelos Moleculares , Sistemas de Lectura Abierta , Medicina de Precisión , Proteínas/química , Proteínas/genética , Alineación de Secuencia , Análisis de Secuencia de ADNRESUMEN
Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of nucleosides and deoxynucleosides, generating ribose 1-phosphate and the purine base, which is an important step of purine catabolism pathway. The lack of such an activity in humans, owing to a genetic disorder, causes T-cell impairment, and thus drugs that inhibit human PNP activity have the potential of being utilized as modulators of the immunological system to treat leukemia, autoimmune diseases, and rejection in organ transplantation. Besides, the purine salvage pathway is the only possible way for apicomplexan parasites to obtain the building blocks for RNA and DNA synthesis, which makes PNP from these parasites an attractive target for drug development against diseases such as malaria. Hence, a number of research groups have made efforts to elucidate the mechanism of action of PNP based on structural and kinetic studies. It is conceivable that the mechanism may be different for PNPs from diverse sources, and influenced by the oligomeric state of the enzyme in solution. Furthermore, distinct transition state structures can make possible the rational design of specific inhibitors for human and apicomplexan enzymes. Here, we review the current status of these research efforts to elucidate the mechanism of PNP-catalyzed chemical reaction, focusing on the mammalian and Plamodium falciparum enzymes, targets for drug development against, respectively, T-Cell- and Apicomplexan parasites-mediated diseases.
Asunto(s)
Apicomplexa/enzimología , Sistemas de Liberación de Medicamentos/métodos , Infecciones por Protozoos/enzimología , Purina-Nucleósido Fosforilasa/metabolismo , Linfocitos T/enzimología , Animales , Apicomplexa/patogenicidad , Humanos , Infecciones por Protozoos/tratamiento farmacológico , Infecciones por Protozoos/parasitología , Purina-Nucleósido Fosforilasa/antagonistas & inhibidores , Linfocitos T/parasitologíaRESUMEN
Considerable interest is currently focused on fish haemoglobins in order to identify the structural basis for their diversity of functional behavior. Hoplosternum littorale is a catfish that presents bimodal gill (water)/gut (air)-breathing, which allows this species to survive in waters with low oxygen content. The hemolysate of this fish showed the presence of two main haemoglobins, cathodic and anodic. This work describes structural features analyzed here by integration of molecular modeling with small angle X-ray scattering. Here is described a molecular model for the cathodic haemoglobin in the unliganded and liganded states. The models were determined by molecular modeling based on the high-resolution crystal structure of fish haemoglobins. The structural models for both forms of H. littorale haemoglobin were compared to human haemoglobin.
Asunto(s)
Bagres/sangre , Hemoglobinas/química , Secuencia de Aminoácidos , Animales , Hemo/química , Hemo/metabolismo , Hemoglobinas/metabolismo , Histidina/química , Histidina/metabolismo , Modelos Moleculares , Oxígeno/metabolismo , Estructura Cuaternaria de Proteína , Homología de Secuencia de Aminoácido , Difracción de Rayos X/métodosRESUMEN
The molecular structure of human uropepsin, an aspartic proteinase from the urine produced in the form of pepsinogen A in the gastric mucosa, has been determined by molecular replacement using human pepsin as the search model. Crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 50.99, b = 75.56, c = 89.90 A. Crystallographic refinement led to an R factor of 0.161 at 2.45 A resolution. The positions of 2437 non-H protein atoms in 326 residues have been determined and the model contains 143 water molecules. The structure is bilobal, consisting of two predominantly beta-sheet lobes which, as observed in other aspartic proteinases, are related by a pseudo-twofold axis. A model of the uropepsin-pepstatin complex has been constructed based on the high-resolution crystal structure of pepsin complexed with pepstatin.
Asunto(s)
Endopeptidasas/química , Secuencia de Aminoácidos , Sitios de Unión , Catálisis , Cristalización , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Pepstatinas/metabolismo , Conformación Proteica , Control de Calidad , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
The three-dimensional structure of human uropepsin complexed with pepstatin has been modelled using human pepsin as a template. Uropepsin is an aspartic proteinase from the urine, produced in the form of pepsinogen A in the gastric mucosa. The structure is bilobal, consisting of two predominantly beta-sheet lobes which, as observed in other aspartic proteinases, are related by a pseudo twofold axis. A structural comparison between binary complexes of pepsin:pepstatin and uropepsin:pepstatin is discussed.
Asunto(s)
Endopeptidasas/química , Modelos Moleculares , Pepstatinas/química , Sitios de Unión , Humanos , Enlace de Hidrógeno , Conformación Proteica , Control de Calidad , Especificidad por SustratoRESUMEN
Considerable interest is currently focused on fish haemoglobins in order to identify the structural basis for their diversity of functional behaviour. The armored catfish Liposarcus anisitsi presents accessorial air breathing through a modified stomach, which allows this species to survive in waters with low oxygen content. The analysis of its haemolysate has shown the presence of four main haemoglobins, with this work focusing on haemoglobin IV (LaHb-IV). LaHb-IV was crystallized and X-ray diffraction data were collected to 2.4 A resolution using a synchrotron-radiation source. The crystal belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 52.6 (1), b = 104.8 (2), c = 113.9 (2) A; preliminary structural analysis revealed the presence of one tetramer in the asymmetric unit. The structure was determined using the standard molecular-replacement technique.
Asunto(s)
Hemoglobinas/química , Animales , Bagres , Cristalización , Cristalografía por Rayos X , Hemoglobinas/aislamiento & purificación , Modelos Moleculares , Conformación ProteicaRESUMEN
Mastoparans are tetradecapeptides found to be the major component of vespid venoms. These peptides present a wide spectrum of biological activities, such as mast cell degranulation, hemolytic activity and also reveals antimicrobial activity. A mastoparan toxin isolated from the venom of Anterhynchium flavomarginatum micado has been crystallized. At room temperature these crystals diffracted to 2.8 A resolution. However, upon cooling to cryogenic temperature around 85 K, the original resolution limit could be improved to 2.0 A. Crystals were determined to belong to the space group P3(1) (P3(2)). This is the first mastoparan to be crystallized and it will provide further insights in the conformational significance of mastoparan toxins, with respect to their potency and activity in G protein regulation.
Asunto(s)
Cristalografía por Rayos X/métodos , Proteínas de Insectos/química , Venenos de Avispas/química , Avispas/metabolismo , Animales , Frío , Cristalización , Proteínas de Insectos/aislamiento & purificación , Venenos de Avispas/aislamiento & purificaciónRESUMEN
Haemoglobin, the 'honorary enzyme' [Brunori (1999), Trends Biochem. Sci. 24, 158-161], constitutes a prime prototype for allosteric models. Here, the crystallization and preliminary X-ray analysis of haemoglobin I from the South American fish Brycon cephalus are reported. X-ray diffraction data have been collected to 2.5 A resolution using synchrotron radiation (LNLS). Crystals were determined to belong to the space group P6(1)22 and preliminary structural analysis revealed the presence of one dimer (alphabeta) in the asymmetric unit. The structure was determined using standard molecular-replacement techniques.
Asunto(s)
Hemoglobinas Anormales/química , Hemoglobinas , Adenosina Trifosfato/metabolismo , Animales , Cristalización , Cristalografía por Rayos X , Peces , Hemoglobinas Anormales/aislamiento & purificación , Hemoglobinas Anormales/metabolismo , Conformación ProteicaRESUMEN
Mastoparans are tetradecapeptides found to be the major component of vespid venoms. A mastoparan toxin isolated from the venom of Anterhynchium flavomarginatum micado has been crystallized and X-ray diffraction data collected to 2.7 A resolution using a synchrotron-radiation source. Crystals were determined to belong to the space group P6(2)22 (P6(4)22). This is the first mastoparan to be crystallized and will provide further insights into the conformational significance of mastoparan toxins with respect to their potency and activity in G-protein regulation.
Asunto(s)
Degranulación de la Célula , Mastocitos/citología , Venenos de Avispas/química , Cristalización , Cristalografía por Rayos X , Conformación ProteicaRESUMEN
The crystal structure of Piratoxin-I (PrTX-I) a Lys49 homologue isolated from the venom of Bothrops pirajai has been determined and refined at 2.8 A to a crystallographic residual of 19.7% (Rfree = 29.7%). Amino-acid sequence differences between catalytically active phospholipases and PrTX-I in the putative Ca2+-binding loop, specifically the substitutions Tyr28 --> Asn, Gly32 --> Leu and Asp49 --> Lys, result in an altered conformation of this loop. The analysis of the position of the epsilon-amino group of Lys49 in the PrTX-I structure indicates that it fills the site normally occupied by the calcium ion in the catalytically active phospholipases. In contrast to the homologous monomeric Lys49 variant from Agkistrodon piscivorus piscivorus (App), PrTX-I is present as a dimer in the crystalline state, as observed in the structures of myotoxin II from Bothrops asper and Bothropstoxin I from Bothrops jararacussu. The two molecules in the asymmetric unit in the crystal structure of PrTX-I are related by a nearly perfect two-fold symmetry axis, yet the dimeric structure is radically different from the dimeric structure of the phospholipase from Crotalus atrox. In the C. atrox structure the dimer interface occludes the active sites, whereas in the PrTX-I structure they are exposed to solvent.
Asunto(s)
Bothrops , Venenos de Crotálidos/química , Fosfolipasas A/química , Animales , Calcio/metabolismo , Venenos de Crotálidos/toxicidad , Cristalografía por Rayos X , Fosfolipasas A2 Grupo II , Datos de Secuencia Molecular , Músculo Esquelético/efectos de los fármacos , Fosfolipasas A/toxicidad , Fosfolipasas A2 , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas de ReptilesRESUMEN
Cathepsin D, a lysosomal aspartic protease, has been purified from porcine liver using a combination of pepstatin-A agarose and Affi-Gel Blue affinity chromatography, followed by size-exclusion chromatography. The purified protein consists of two polypeptide chains of 15 and 30 kDa, and has an isoelectric point of 6.8. Porcine liver cathepsin D has maximum activity at pH 2.5-3.0 as determined by its activity against hemoglobin, with a Kcat of 14.3 s-1 and a kcat/KM of 2.70 x 10(6) s-1M-1 as determined by the hydrolysis of a fluorogenic peptide substrate.
Asunto(s)
Catepsina D/aislamiento & purificación , Catepsina D/metabolismo , Cromatografía de Afinidad , Hígado/enzimología , Animales , Catepsina D/química , Electroforesis en Gel de Poliacrilamida , Hemoglobinas/metabolismo , Concentración de Iones de Hidrógeno , Punto Isoeléctrico , Cinética , Peso Molecular , Pepstatinas , Péptidos/metabolismo , Porcinos , TriazinasRESUMEN
Large single crystals of piratoxin I. a Lys49-PLA2 homologue with low enzymatic activity, have been obtained. The crystals belong to the orthorhombic system space group P2(1)2(1)2(1), and diffract X-rays to a resolution of 2.8 A. Preliminary analysis reveals the presence of two molecules in the crystallographic asymmetric unit.