RESUMEN
Cell migration is important during early animal embryogenesis. Cell migration and cell shape are controlled by actin assembly and dynamics, which depend on capping proteins, including the barbed-end heterodimeric actin capping protein (CP). CP activity can be regulated by capping-protein-interacting (CPI) motif proteins, including CARMIL (capping protein Arp2/3 myosin-I linker) family proteins. Previous studies of CARMIL3, one of the three highly conserved CARMIL genes in vertebrates, have largely been limited to cells in culture. Towards understanding CARMIL function during embryogenesis in vivo, we analyzed zebrafish lines carrying mutations of carmil3. Maternal-zygotic mutants showed impaired endodermal migration during gastrulation, along with defects in dorsal forerunner cell (DFC) cluster formation, which affected the morphogenesis of Kupffer's vesicle (KV). Mutant KVs were smaller, contained fewer cells and displayed decreased numbers of cilia, leading to defects in left/right (L/R) patterning with variable penetrance and expressivity. The penetrance and expressivity of the KV phenotype in carmil3 mutants correlated well with the L/R heart positioning defect at the end of embryogenesis. This in vivo animal study of CARMIL3 reveals its new role during morphogenesis of the vertebrate embryo. This role involves migration of endodermal cells and DFCs, along with subsequent morphogenesis of the KV and L/R asymmetry.
Asunto(s)
Tipificación del Cuerpo , Movimiento Celular , Embrión no Mamífero/embriología , Desarrollo Embrionario , Proteínas de Microfilamentos/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Animales , Proteínas de Microfilamentos/genética , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
The heterodimeric actin capping protein (CP) is regulated by a set of proteins that contain CP-interacting (CPI) motifs. Outside of the CPI motif, the sequences of these proteins are unrelated and distinct. The CPI motif and surrounding sequences are conserved within a given protein family, when compared to those of other CPI-motif protein families. Using biochemical assays with purified proteins, we compared the ability of CPI-motif-containing peptides from different protein families (a) to bind to CP, (b) to allosterically inhibit barbed-end capping by CP, and (c) to allosterically inhibit interaction of CP with V-1, another regulator of CP. We found large differences in potency among the different CPI-motif-containing peptides, and the different functional assays showed different orders of potency. These biochemical differences among the CPI-motif peptides presumably reflect interactions between CP and CPI-motif peptides involving amino acid residues that are conserved but are not part of the strictly defined consensus, as it was originally identified in comparisons of sequences of CPI motifs across all protein families [Hernandez-Valladares, M., et al. (2010) Structural characterization of a capping protein interaction motif defines a family of actin filament regulators. Nat. Struct. Mol. Biol. 17, 497-503; Bruck, S., et al. (2006) Identification of a Novel Inhibitory Actin-capping Protein Binding Motif in CD2-associated Protein. J. Biol. Chem. 281, 19196-19203]. These biochemical differences may be important for conserved distinct functions of CPI-motif protein families in cells with respect to the regulation of CP activity and actin assembly near membranes.
Asunto(s)
Proteína CapZ/química , Proteína CapZ/metabolismo , Actinas/química , Actinas/metabolismo , Regulación Alostérica , Secuencias de Aminoácidos , Animales , Proteína CapZ/genética , Dimerización , Eucariontes/clasificación , Eucariontes/genética , Eucariontes/metabolismo , Humanos , Cinética , Péptidos/química , Péptidos/metabolismo , Filogenia , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de ProteínasRESUMEN
Actin assembly is important for cell motility. The ability of actin subunits to join or leave filaments via the barbed end is critical to actin dynamics. Capping protein (CP) binds to barbed ends to prevent subunit gain and loss and is regulated by proteins that include V-1 and CARMIL. V-1 inhibits CP by sterically blocking one binding site for actin. CARMILs bind at a distal site and decrease the affinity of CP for actin, suggested to be caused by conformational changes. We used hydrogen-deuterium exchange with mass spectrometry (HDX-MS) to probe changes in structural dynamics induced by V-1 and CARMIL binding to CP. V-1 and CARMIL induce changes in both proteins' binding sites on the surface of CP, along with a set of internal residues. Both also affect the conformation of CP's ßß subunit "tentacle," a second distal actin-binding site. Concerted regulation of actin assembly by CP occurs through allosteric couplings between CP modulator and actin binding sites.
Asunto(s)
Proteínas de Capping de la Actina/metabolismo , Medición de Intercambio de Deuterio , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de Microfilamentos/metabolismo , Regulación Alostérica , Animales , Ratones , Unión Proteica , Multimerización de Proteína , Estructura Secundaria de Proteína , SolventesRESUMEN
We determine and compare the crystal structure of two proteases belonging to the subtilisin superfamily: S41, a cold-adapted serine protease produced by Antarctic bacilli, at 1.4 A resolution and Sph, a mesophilic serine protease produced by Bacillus sphaericus, at 0.8 A resolution. The purpose of this comparison was to find out whether multiple calcium ion binding is a molecular factor responsible for the adaptation of S41 to extreme low temperatures. We find that these two subtilisins have the same subtilisin fold with a root mean square between the two structures of 0.54 A. The final models for S41 and Sph include a calcium-loaded state of five ions bound to each of these two subtilisin molecules. None of these calcium-binding sites correlate with the high affinity known binding site (site A) found for other subtilisins. Structural analysis of the five calcium-binding sites found in these two crystal structures indicate that three of the binding sites have two side chains of an acidic residue coordinating the calcium ion, whereas the other two binding sites have either a main-chain carbonyl, or only one acidic residue side chain coordinating the calcium ion. Thus, we conclude that three of the sites are of high affinity toward calcium ions, whereas the other two are of low affinity. Because Sph is a mesophilic subtilisin and S41 is a psychrophilic subtilisin, but both crystal structures were found to bind five calcium ions, we suggest that multiple calcium ion binding is not responsible for the adaptation of S41 to low temperatures.
Asunto(s)
Bacillus/enzimología , Subtilisinas/química , Animales , Calcio/metabolismo , Dominio Catalítico , Frío , Cristalografía por Rayos X , Modelos Moleculares , Unión Proteica , Subtilisinas/metabolismoRESUMEN
In this article, we describe for the first time the high-resolution crystal structure of a phenylalanine tRNA synthetase from the pathogenic bacterium Staphylococcus haemolyticus. We demonstrate the subtle yet important structural differences between this enzyme and the previously described Thermus thermophilus ortholog. We also explain the structure-activity relationship of several recently reported inhibitors. The native enzyme crystals were of poor quality--they only diffracted X-rays to 3-5A resolution. Therefore, we have executed a rational surface mutagenesis strategy that has yielded crystals of this 2300-amino acid multidomain protein, diffracting to 2A or better. This methodology is discussed and contrasted with the more traditional domain truncation approach.
Asunto(s)
Proteínas Bacterianas/química , Fenilalanina-ARNt Ligasa/química , Staphylococcus haemolyticus/enzimología , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X/métodos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Mutagénesis , Fenilalanina-ARNt Ligasa/antagonistas & inhibidores , Fenilalanina-ARNt Ligasa/metabolismo , Ingeniería de Proteínas/métodos , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Staphylococcus haemolyticus/genéticaRESUMEN
Dandruff and seborrheic dermatitis (D/SD) are common hyperproliferative scalp disorders with a similar etiology. Both result, in part, from metabolic activity of Malassezia globosa and Malassezia restricta, commensal basidiomycete yeasts commonly found on human scalps. Current hypotheses about the mechanism of D/SD include Malassezia-induced fatty acid metabolism, particularly lipase-mediated breakdown of sebaceous lipids and release of irritating free fatty acids. We report that lipase activity was detected in four species of Malassezia, including M. globosa. We isolated lipase activity by washing M. globosa cells. The isolated lipase was active against diolein, but not triolein. In contrast, intact cells showed lipase activity against both substrates, suggesting the presence of at least another lipase. The diglyceride-hydrolyzing lipase was purified from the extract, and much of its sequence was determined by peptide sequencing. The corresponding lipase gene (LIP1) was cloned and sequenced. Confirmation that LIP1 encoded a functional lipase was obtained using a covalent lipase inhibitor. LIP1 was differentially expressed in vitro. Expression was detected on three out of five human scalps, as indicated by reverse transcription-PCR. This is the first step in a molecular description of lipid metabolism on the scalp, ultimately leading toward a test of its role in D/SD etiology.
Asunto(s)
Proteínas Fúngicas/metabolismo , Regulación Enzimológica de la Expresión Génica , Lipasa/genética , Lipasa/metabolismo , Malassezia/enzimología , Cuero Cabelludo/microbiología , Clonación Molecular , Diglicéridos/química , Regulación Fúngica de la Expresión Génica , Glicéridos/química , Humanos , Lípidos/química , Modelos Biológicos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Trioleína/químicaRESUMEN
In this article we describe the application of structural biology methods to the discovery of novel potent inhibitors of methionine aminopeptidases. These enzymes are employed by the cells to cleave the N-terminal methionine from nascent peptides and proteins. As this is one of the critical steps in protein maturation, it is very likely that inhibitors of these enzymes may prove useful as novel antibacterial agents. Involvement of crystallography at the very early stages of the inhibitor design process resulted in serendipitous discovery of a new inhibitor class, the pyrazole-diamines. Atomic-resolution structures of several inhibitors bound to the enzyme illuminate a new mode of inhibitor binding.
Asunto(s)
Bacterias/enzimología , Inhibidores de Proteasas/farmacología , Aminopeptidasas/química , Aminopeptidasas/aislamiento & purificación , Bacterias/efectos de los fármacos , Proteínas Bacterianas/farmacología , Cristalización , Cristalografía por Rayos X , Cinética , Espectroscopía de Resonancia Magnética , Metionil Aminopeptidasas , Modelos Moleculares , Inhibidores de Proteasas/química , Conformación Proteica , Teoría CuánticaRESUMEN
Protein tyrosine phosphatases (PTPs) play roles in many biological processes and are considered to be important targets for drug discovery. As inhibitor development has proven challenging, crystal structure-based design will be very helpful to advance inhibitor potency and selectivity. Successful application of protein crystallography to drug discovery heavily relies on high-quality crystal structures of the protein of interest complexed with pharmaceutically interesting ligands. It is very important to be able to produce protein-ligand crystals rapidly and reproducibly for as many ligands as necessary. This study details our efforts to engineer the catalytic domain of human protein tyrosine phosphatase beta (HPTPbeta-CD) with properties suitable for rapid-turnaround crystallography. Structures of apo HPTPbeta-CD and its complexes with several novel small-molecule inhibitors are presented here for the first time.
Asunto(s)
Dominio Catalítico , Diseño de Fármacos , Ingeniería de Proteínas , Proteínas Tirosina Fosfatasas/química , Sitios de Unión , Cristalografía por Rayos X , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Estructura Secundaria de Proteína , Relación Estructura-ActividadRESUMEN
This communication details the synthesis, biological activity, and binding mode of a novel class of 2-benzimidazole substituted pyrimidines. The most potent analogs disclosed showed low nanomolar activity for the inhibition of Lck kinase and a representative analog was co-crystallized with Hck (a structurally related member of the Src family kinases).
Asunto(s)
Bencimidazoles/química , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/química , Enlace de Hidrógeno , Concentración 50 Inhibidora , Interleucina-2/metabolismo , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/síntesis química , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Estructura Molecular , Inhibidores de Proteínas Quinasas/química , Proteínas Proto-Oncogénicas c-hck/química , Proteínas Proto-Oncogénicas c-hck/metabolismo , Relación Estructura-ActividadRESUMEN
Utilizing modeling information from a recently resolved structure of human HIF-1alpha prolyl hydroxylase (EGLN1) and structure-based design, a novel series of imidazo[1,2-a]pyridine derivatives was prepared. The activity of these compounds was determined in a human EGLN1 assay and a limited SAR was developed.
Asunto(s)
Procolágeno-Prolina Dioxigenasa/antagonistas & inhibidores , Piridinas/farmacología , Humanos , Prolina Dioxigenasas del Factor Inducible por Hipoxia , Relación Estructura-ActividadRESUMEN
The synthesis and structure-activity relationships of a novel series of N-sulfonyl-2-indole carboxamides that bind to peroxisome proliferator-activated receptor gamma (PPAR-gamma) are reported. Chemical optimization of the series led to the identification of 4q (IC(50)=50 nM) as a potent binding agent of PPAR-gamma. Also reported is preliminary cell based data suggesting the use of these compounds in the treatment of osteoporosis.
Asunto(s)
Amidas/farmacología , Diseño de Fármacos , Indoles/farmacología , Osteoporosis/tratamiento farmacológico , PPAR gamma/metabolismo , Células 3T3 , Amidas/síntesis química , Animales , Indoles/síntesis química , RatonesRESUMEN
A new series of potent 8-hydroxyquinolines was designed based on the newly resolved X-ray crystal structure of EGLN-1. Both alkyl and aryl 8-hydroxyquinoline-7-carboxyamides were good HIF-1alpha prolyl hydroxylase (EGLN) inhibitors. In subsequent VEGF induction assays, these exhibited potent VEGF activity. In addition, this class of compounds did show the ability to stabilize HIF-1alpha.
Asunto(s)
Diseño de Fármacos , Oxiquinolina/análogos & derivados , Oxiquinolina/farmacología , Procolágeno-Prolina Dioxigenasa/antagonistas & inhibidores , Prolina Dioxigenasas del Factor Inducible por Hipoxia , Oxiquinolina/síntesis química , Oxiquinolina/química , Procolágeno-Prolina Dioxigenasa/química , Relación Estructura-ActividadRESUMEN
Recently resolved X-ray crystal structure of HIF-1alpha prolyl hydroxylase was used to design and develop a novel series of pyrazolopyridines as potent HIF-1alpha prolyl hydroxylase inhibitors. The activity of these compounds was determined in a human EGLN-1 assay. Structure-based design aided in optimizing the potency of the initial lead (2, IC(50) of 11 microM) to a potent (11l, 190 nM) EGLN-1 inhibitor. Several of these analogs were potent VEGF inducers in a cell-based assay. These pyrazolopyridines were also effective in stabilizing HIF-1alpha.
Asunto(s)
Diseño de Fármacos , Procolágeno-Prolina Dioxigenasa/antagonistas & inhibidores , Pirazoles/síntesis química , Pirazoles/farmacología , Piridinas/síntesis química , Piridinas/farmacología , Línea Celular , Humanos , Prolina Dioxigenasas del Factor Inducible por HipoxiaRESUMEN
Structure-guided de novo drug design led to the identification of a novel series of substituted pyridine derivatives as HIF-1alpha prolyl hydroxylase inhibitors. Pyridine carboxyamide derivatives bearing a substituted aryl group at the 5-position of the pyridine ring show appreciable activity, while constraining the side chain by placing a pyrazole carboxylic acid generated a potent lead series with consistent activity against EGLN-1.
Asunto(s)
Diseño de Fármacos , Procolágeno-Prolina Dioxigenasa/antagonistas & inhibidores , Piridinas/síntesis química , Piridinas/farmacología , Humanos , Prolina Dioxigenasas del Factor Inducible por HipoxiaRESUMEN
A series of C-2, C-8, and N-9 trisubstituted purine based inhibitors of TNF-alpha production are described. The most potent analogs showed low nanomolar activity against LPS-induced TNF-alpha production in a THP-1 cell based assay. The SAR of the series was optimized with the aid of X-ray co-crystal structures of these inhibitors bound with mutated p38 (mp38).
Asunto(s)
Purinas/química , Factor de Necrosis Tumoral alfa/química , Línea Celular , Química Farmacéutica , Cristalografía por Rayos X , Diseño de Fármacos , Humanos , Lipopolisacáridos/química , Modelos Químicos , Modelos Moleculares , Proteínas Quinasas p38 Activadas por Mitógenos/química , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
A new class of tumor necrosis factor alpha (TNF-alpha) synthesis inhibitors based on an N-2,4-pyrimidine-N-phenyl-N'-phenyl urea scaffold is described. Many of these compounds showed low-nanomolar activity against lipopolysaccharide stimulated TNF-alpha production. X-ray co-crystallization studies with mutated p38alpha showed that these trisubstituted ureas interact with the ATP-binding pocket in a pseudo-bicyclic conformation brought about by the presence of an intramolecular hydrogen bonding interaction.
Asunto(s)
Compuestos de Fenilurea/química , Compuestos de Fenilurea/farmacología , Pirimidinas/química , Pirimidinas/farmacología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Adenosina Trifosfato/química , Sitios de Unión , Cristalografía por Rayos X , Enlace de Hidrógeno , Lipopolisacáridos/antagonistas & inhibidores , Lipopolisacáridos/farmacología , Modelos Moleculares , Estructura Molecular , Compuestos de Fenilurea/clasificación , Pirimidinas/clasificación , Estereoisomerismo , Relación Estructura-Actividad , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
4-Aryl-5-pyrimidyl based cytokine synthesis inhibitors that contain a novel monocyclic, pyrazolone heterocyclic core are described. Many of these inhibitors showed low nanomolar activity against LPS-induced TNF-alpha production. One of the compounds (6e) was found to be efficacious in the rat iodoacetate (RIA) in vivo model of osteoarthritis. The X-ray crystal structure of a pyrazolone inhibitor cocrystallized with mutated p38 (mp38) is presented.
Asunto(s)
Citocinas/antagonistas & inhibidores , Citocinas/biosíntesis , Pirazolonas/síntesis química , Pirazolonas/farmacología , Animales , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Cinética , Lipopolisacáridos/farmacología , Modelos Moleculares , Conformación Molecular , Osteoartritis/prevención & control , Pirazolonas/química , Ratas , Relación Estructura-Actividad , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/biosíntesis , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
4-Aryl-3-pyridyl and 4-aryl-3-pyrimidinyl based tumor necrosis factor-alpha (TNF-alpha) inhibitors, which contain a novel isoxazolone five-membered heterocyclic core are described. Many showed sub-micromolar activity against lipopolysaccharide-induced TNF-alpha production.
Asunto(s)
Isoxazoles/síntesis química , Isoxazoles/farmacología , Pirimidinas/síntesis química , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Acetaldehído , Sitios de Unión , Línea Celular , Humanos , Lipopolisacáridos/farmacología , Modelos Moleculares , Estructura Molecular , Conformación Proteica , Pirimidinas/farmacología , Relación Estructura-Actividad , Factor de Necrosis Tumoral alfa/química , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidoresRESUMEN
Novel substituted [5,5]-bicyclic pyrzazolones are presented as inhibitors of tumor necrosis factor-alpha (TNF-alpha) production. Many of these compounds show low nanomolar activity against lipopolysaccaride (LPS)-induced TNF-alpha production in THP-1 cells. This class of molecules was co-crystallized with mutated p38, and several analogs showed good oral bioavailability in the rat. Oral activity of these compounds in the rat iodoacetate model for osteoarthritis is discussed.
Asunto(s)
Pirazolonas/química , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Administración Oral , Animales , Disponibilidad Biológica , Lipopolisacáridos/farmacología , Modelos Moleculares , Pirazolonas/administración & dosificación , Pirazolonas/farmacocinética , Pirazolonas/farmacología , Ratas , Relación Estructura-Actividad , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
2-Aryl-3-pyrimidinyl based tumor necrosis factor-alpha (TNF-alpha) inhibitors, which contain a novel bicyclic pyrazolone core, are described. Many showed low-nanomolar activity against lipopolysaccharide-induced TNF-alpha production in monocytic cells. Secondary screening data are presented for the pyrimidinyl bicyclic pyrazolones. Several of these analogues showed good oral bioavailability in rat and efficacy in the rat iodoacetate in vivo model.