RESUMEN

We describe truncation and SAR studies to identify a pentapeptide that binds Cbl tyrosine kinase binding domain with a higher affinity than the parental peptide. The pentapeptide has an alternative binding mode that allows occupancy of a previously uncharacterized groove. A peptide library was used to map the binding site and define the interface landscape. Our results suggest that the pentapeptide is an ideal starting point for the development of inhibitors against Cbl driven diseases.

Asunto(s)

Modelos Moleculares , Oligopéptidos/química , Proteína Oncogénica v-cbl/química , Proteínas Tirosina Quinasas/química , Sitios de Unión , Proteína Oncogénica v-cbl/metabolismo , Biblioteca de Péptidos , Unión Proteica , Proteínas Tirosina Quinasas/metabolismo , Relación Estructura-Actividad , Termodinámica

RESUMEN

The casitas B-lineage lymphoma (Cbl) proteins play an important role in regulating signal transduction pathways by functioning as E3 ubiquitin ligases. The Cbl proteins contain a conserved tyrosine kinase binding (TKB) domain that binds more than a dozen proteins, including protein tyrosine kinases (PTKs), in a phosphorylation-dependent manner. The cell surface expression levels of the PTKs are regulated by Cbl-mediated ubiquitination, internalization, and degradation. Dysfunction in this signaling cascade has resulted in prolonged activation of the PTKs and, therefore, has been implicated in inflammatory diseases and various cancers. Due to this negative regulatory function, Cbl has been largely ignored as a therapeutic target. However, recent studies, such as the identification of (i) gain of function c-Cbl mutations in subsets of myeloid cancer and (ii) c-Cbl as a prostate basal cell marker that correlates with poor clinical outcome, suggest otherwise. Here we report the development of a competitive high-throughput fluorescence polarization assay in a 384-well format to identify inhibitors of Cbl(TKB). The high-throughput screen readiness of the assay was demonstrated by screening the Prestwick Chemical Library.

Asunto(s)

Inhibidores Enzimáticos/química , Polarización de Fluorescencia/métodos , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-cbl/metabolismo , Bases de Datos Factuales , Ensayos Analíticos de Alto Rendimiento/métodos , Fosfopéptidos/química , Unión Proteica , Proteínas Tirosina Quinasas/química , Proteínas Proto-Oncogénicas c-cbl/antagonistas & inhibidores , Ubiquitinación

RESUMEN

Venezuelan equine encephalitis virus (VEEV) is a prototypical enveloped ssRNA virus of the family Togaviridae. To better understand alphavirus assembly, we analyzed newly formed nucleocapsid particles (termed pre-viral nucleocapsids) isolated from infected cells. These particles were intermediates along the virus assembly pathway, and ultimately bind membrane-associated viral glycoproteins to bud as mature infectious virus. Purified pre-viral nucleocapsids were spherical with a unimodal diameter distribution. The structure of one class of pre-viral nucleocapsids was determined with single particle reconstruction of cryo-electron microscopy images. These studies showed that pre-viral nucleocapsids assembled into an icosahedral structure with a capsid stoichiometry similar to the mature nucleocapsid. However, the individual capsomers were organized significantly differently within the pre-viral and mature nucleocapsids. The pre-viral nucleocapsid structure implies that nucleocapsids are highly plastic and undergo glycoprotein and/or lipid-driven rearrangements during virus self-assembly. This mechanism of self-assembly may be general for other enveloped viruses.

Asunto(s)

Virus de la Encefalitis Equina Venezolana/química , Virus de la Encefalitis Equina Venezolana/fisiología , Encefalomielitis Equina Venezolana/virología , Ensamble de Virus , Alphavirus/química , Alphavirus/aislamiento & purificación , Alphavirus/fisiología , Alphavirus/ultraestructura , Animales , Cricetinae , Virus de la Encefalitis Equina Venezolana/aislamiento & purificación , Virus de la Encefalitis Equina Venezolana/ultraestructura , Humanos , Modelos Moleculares , Nucleocápside/química , Nucleocápside/aislamiento & purificación , Nucleocápside/ultraestructura

RESUMEN

BRCT(BRCA1) plays a major role in DNA repair pathway, and does so by recognizing the conserved sequence pSXXF in its target proteins. Remarkably, tetrapeptides containing pSXXF motif bind with high specificity and micromolar affinity. Here, we have characterized the binding interactions of pSXXF tetrapeptides using NMR spectroscopy and calorimetry. We show that BRCT is dynamic and becomes structured on binding, that pSer and Phe residues dictate overall binding, and that the binding affinities of the tetrapeptides are intimately linked to structural and dynamic changes both in the BRCT(BRCA1) and tetrapeptides. These results provide critical insights for designing high-affinity BRCT(BRCA1) inhibitors.

Asunto(s)

Proteína BRCA1/antagonistas & inhibidores , Proteína BRCA1/química , Diseño de Fármacos , Oligopéptidos/química , Secuencias de Aminoácidos , Humanos , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Conformación Proteica , Termodinámica

RESUMEN

Finding specific small-molecule inhibitors of protein-protein interactions remains a significant challenge. Recently, attention has grown toward "hot spot" interactions where binding is dominated by a limited number of amino acid contacts, theoretically offering an increased opportunity for disruption by small molecules. Inhibitors of the interaction between BRCT (the C-terminal portion of BRCA1, a key tumor suppressor protein with various functions) and phosphorylated proteins (Abraxas/BACH1/CtIP), implicated in DNA damage response and repair pathways, should prove to be useful in studying BRCA1's role in cancer and in potentially sensitizing tumors to chemotherapeutic agents. We developed and miniaturized to a 1536-well format and 3-mul final volume a pair of fluorescence polarization (FP) assays using fluorescein- and rhodamine-labeled pBACH1 fragment. To minimize the effect of fluorescence artifacts and to increase the overall robustness of the screen, the 75,552 compound library members all were assayed against both the fluorescein- and rhodamine-labeled probe-protein complexes in separate but interleaved reactions. In addition, every library compound was tested over a range of concentrations following the quantitative high-throughput screening (qHTS) paradigm. Analyses of the screening results led to the selection and subsequent confirmation of 16 compounds active in both assays. Faced with a traditionally difficult protein-protein interaction assay, by performing two-fluorophore qHTS, we were able to confidently select a number of actives for further studies.

Asunto(s)

Proteína BRCA1/química , Proteína BRCA1/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Colorantes Fluorescentes/metabolismo , Fosfoproteínas/metabolismo , Relación Dosis-Respuesta a Droga , Estabilidad de Medicamentos , Almacenaje de Medicamentos , Concentración 50 Inhibidora , Miniaturización , Preparaciones Farmacéuticas/química , Unión Proteica , Bibliotecas de Moléculas Pequeñas

Asunto(s)

Proteína BRCA1/química , Fosfopéptidos/química , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/química , Proteínas del Grupo de Complementación de la Anemia de Fanconi/química , Polarización de Fluorescencia , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Unión Proteica , Estructura Terciaria de Proteína , Termodinámica

RESUMEN

The C-terminus region of the 1863 residue early onset of breast cancer gene 1 (BRCA1) nuclear protein contains a tandem globular carboxy terminus domain termed BRCT. The BRCT repeats in BRCA1 are phosphoserine- and/or phosphothreonine-specific binding modules. The interaction of the BRCT(BRCA1) domains with phosphorylated BRCA1-associated carboxyl terminal helicase (BACH1) is cell cycle regulated and is essential for DNA damage-induced checkpoint control during the transition from the G(2) phase to the M phase of the cell cycle. Development of a competitive, homogeneous, high-throughput fluorescence polarization (FP) assay to identify small molecule inhibitors of BRCT(BRCA1)-BACH1 interaction is reported here. The FP assay was used for measuring binding affinities and inhibition constants of BACH1 peptides and small molecule inhibitors of BRCT(BRCA1) domains, respectively. A fluorescently labeled wild-type BACH1 decapeptide (BDP1) containing the critical phosphoserine, a phenylalanine at (P+3), and a GST-BRCT fusion protein were used to establish the FP assay. BDP1 has a dissociation constant (K(d)) of 1.58+/-0.01microM and a dynamic range (DeltamP) of 164.9+/-1.9. The assay tolerates 20% dimethyl sulfoxide, which enables screening poorly soluble compounds. Under optimized conditions, a Z' factor of 0.87 was achieved in a 384-well format for high-throughput screening.

Asunto(s)

Proteína BRCA1/antagonistas & inhibidores , Proteína BRCA1/química , Polarización de Fluorescencia/métodos , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Proteína BRCA1/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Proteínas del Grupo de Complementación de la Anemia de Fanconi/metabolismo , Femenino , Humanos , Cinética , Pruebas de Sensibilidad Microbiana , Péptidos/síntesis química , Péptidos/química , Péptidos/metabolismo , Estructura Terciaria de Proteína/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Titulación a Punto Final de Prueba Cutánea

RESUMEN

When expressed in Escherichia coli, the recombinant coat protein (rCP) of Sesbania mosaic virus (SeMV) was shown to self-assemble into T = 3 capsids encapsidating CP mRNA and 23S rRNA derived from the host. Expression of CP-P53A, in which a conserved proline at position 53 in the beta-annulus was substituted by alanine (CP-P53A), also produced similar capsids. Purified rCP and CP-P53A particles were crystallized and X-ray crystal structures of their mutant capsids were determined to resolutions of 3.6 and 4.1 A, respectively. As in the native viral CP, the CPs in these recombinant capsids adopt the jelly-roll beta-sandwich fold. The amino-terminal residues of the C subunits alone are ordered and form the beta-annulus structure at the quasi-sixfold axes. A characteristic bend in the beta-annulus remains unaffected in CP-P53A. The quasi-threefold interfaces of the capsids harbour calcium ions coordinated by ligands from the adjacent threefold-related subunits in a geometry that is analogous to that observed in the native capsid. Taken together with studies on deletion and substitution mutants of SeMV CP, these results suggest the possibility that the beta-annulus and nucleic acid-mediated interactions may be less important for the assembly of sobemoviruses than previously envisaged.

Asunto(s)

Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Cápside/química , Virus del Mosaico/química , Virus del Mosaico/genética , Mutación , Calcio/metabolismo , Biología Computacional , Cristalografía por Rayos X , Escherichia coli/metabolismo , Eliminación de Gen , Ligandos , Modelos Moleculares , Modelos Estadísticos , Péptidos/química , Prolina/química , Unión Proteica , Conformación Proteica , Estructura Secundaria de Proteína , ARN Mensajero/metabolismo , ARN Ribosómico 23S/química , Proteínas Recombinantes/química , Programas Informáticos

RESUMEN

Sesbania mosaic virus (SeMV) capsids are stabilized by protein-protein, protein-RNA and calcium-mediated protein-protein interactions. The N-terminal random domain of SeMV coat protein (CP) controls RNA encapsidation and size of the capsids and has two important motifs, the arginine-rich motif (ARM) and the beta-annulus structure. Here, mutational analysis of the arginine residues present in the ARM to glutamic acid was carried out. Mutation of all the arginine residues in the ARM almost completely abolished RNA encapsidation, although the assembly of T=3 capsids was not affected. A minimum of three arginine residues was found to be essential for RNA encapsidation. The mutant capsids devoid of RNA were less stable to thermal denaturation when compared to wild-type capsids. The results suggest that capsid assembly is entirely mediated by CP-dependent protein-protein inter-subunit interactions and encapsidation of genomic RNA enhances the stability of the capsids. Because of the unique structural ordering of beta-annulus segment at the icosahedral 3-folds, it has been suggested as the switch that determines the pentameric and hexameric clustering of CP subunits essential for T=3 capsid assembly. Surprisingly, mutation of a conserved proline within the segment that forms the beta-annulus to alanine, or deletion of residues 48-53 involved in hydrogen bonding interactions with residues 54-58 of the 3-fold related subunit or deletion of all the residues (48-59) involved in the formation of beta-annulus did not affect capsid assembly. These results suggest that the switch for assembly into T=3 capsids is not the beta-annulus. The ordered beta-annulus observed in the structures of many viruses could be a consequence of assembly to optimize intersubunit interactions.

Asunto(s)

Secuencia de Aminoácidos , Arginina/química , Proteínas de la Cápside , Cápside , Virus del Mosaico/genética , Conformación Proteica , Cápside/química , Cápside/ultraestructura , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Cristalografía por Rayos X , Análisis Mutacional de ADN , Modelos Moleculares , Datos de Secuencia Molecular , Virus del Mosaico/química , Mutagénesis Sitio-Dirigida , Estructura Cuaternaria de Proteína , ARN Viral/química , ARN Viral/genética , Alineación de Secuencia

RESUMEN

Sesbania mosaic virus particles consist of 180 coat protein subunits of 29kDa organized on a T=3 icosahedral lattice. N-terminal deletion mutants of coat protein that lack 36 (CP-NDelta36) and 65 (CP-NDelta65) residues from the N terminus, when expressed in Escherichia coli, produced similar T=1 capsids of approximate diameter 20nm. In contrast to the wild-type particles, these contain only 60 copies of the truncated protein subunits (T=1). CP-NDelta65 lacks the "beta-annulus" believed to be responsible for the error-free assembly of T=3 particles. Though the CP-NDelta36 mutant has the beta-annulus segment, it does not form a T=3 capsid, presumably because it lacks an arginine-rich motif found close to the amino terminus. Both CP-NDelta36 and CP-NDelta65 T=1 capsids retain many key features of the T=3 quaternary structure. Calcium binding geometries at the coat protein interfaces in these two particles are also nearly identical. When the conserved aspartate residues that coordinate the calcium, D146 and D149 in the CP-NDelta65, were mutated to asparagine (CP-NDelta65-D146N-D149N), the subunits assembled into T=1 particles but failed to bind calcium ions. The structure of this mutant revealed particles that were slightly expanded. The analysis of the structures of these mutant capsids suggests that although calcium binding contributes substantially to the stability of T=1 particles, it is not mandatory for their assembly. In contrast, the presence of a large fraction of the amino-terminal arm including sequences that precede the beta-annulus and the conserved D149 appear to be indispensable for the error-free assembly of T=3 particles.

Asunto(s)

Proteínas de la Cápside/química , Proteínas de la Cápside/genética , Virus del Mosaico/química , Virus del Mosaico/genética , Sitios de Unión , Calcio/metabolismo , Proteínas de la Cápside/metabolismo , Cristalografía por Rayos X , Fabaceae/virología , Modelos Moleculares , Virus del Mosaico/fisiología , Mutagénesis Sitio-Dirigida , Estructura Cuaternaria de Proteína , Subunidades de Proteína , Electricidad Estática , Ensamble de Virus

RESUMEN

Sesbania mosaic virus (SeMV) capsids are stabilized by RNA-protein, protein-protein and calcium-mediated protein-protein interactions. The removal of calcium has been proposed to be a prerequisite for the disassembly of the virus. The crystal structure of native T=3 SeMV capsid revealed that residues D146 and D149 from one subunit and Y205, N267 and N268 of the neighboring subunit form the calcium-binding site (CBS). The CBS environment is found to be identical even in the recombinant CP-NDelta65 T=1 capsids. Here, we have addressed the role of calcium and the residues involved in calcium co-ordination in the assembly and stability of T=3 and T=1 capsids by mutational analysis. Deletion of N267 and N268 did not affect T=3 or T=1 assembly, although the capsids were devoid of calcium, suggesting that assembly does not require calcium ions. However, the stability of the capsids was reduced drastically. Site-directed mutagenesis revealed that either a single mutation (D149N) or a double mutation (D146N-D149N) of SeMV coat protein affected drastically both the assembly and stability of T=3 capsids. On the other hand, the D146N-D149N mutation in CP-NDelta65 did not affect the assembly of T=1 capsid, although their stability was reduced considerably. Since the major difference between the T=3 and T=1 capsids is the absence of the N-terminal arginine-rich motif (N-ARM) and the beta-annulus from the subunits forming the T=1 capsids, it is possible that D149 initiates the N-ARM-RNA interactions that lead to the formation of the beta-annulus, which is essential for T=3 capsid assembly.

Asunto(s)

Proteínas de la Cápside/química , Virus del Mosaico/química , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión/genética , Calcio/metabolismo , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Proteínas de la Cápside/ultraestructura , Cristalografía por Rayos X , ADN Viral/genética , Fabaceae/virología , Microscopía Electrónica , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Virus del Mosaico/genética , Virus del Mosaico/fisiología , Virus del Mosaico/ultraestructura , Mutagénesis Sitio-Dirigida , Desnaturalización Proteica , Subunidades de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Termodinámica , Tripsina , Ensamble de Virus

RESUMEN

Sesbania mosaic virus (SeMV) polyprotein was shown to undergo proteolytic processing when expressed in E. coli. Mutational analysis of the proposed catalytic triad residues (H181, D216, and S284) present in the N-terminal serine protease domain of the polyprotein showed that the protease was indeed responsible for this processing. Analysis of the cleavage site mutants confirmed the cleavage between protease-viral protein genome linked (VPg) and VPg-RNA-dependent RNA polymerase (RdRP) at E(325)-T(326) and E(402)-T(403) sites, respectively. An additional suboptimal cleavage at E(498)-S(499) site was also identified which resulted in the further processing of RdRP to 10- and 52-kDa proteins. Thus, the protease has both E-T and E-S specificities. The polyprotein has a domain arrangement of protease-VPg-p10-RdRP, which is cleaved by the protease. The purified serine protease was also active in trans and cleaved the polyprotein at the same specific sites. These results demonstrate that the serine protease domain is responsible for the processing of SeMV polyprotein both in cis and in trans.

Asunto(s)

Poliproteínas/metabolismo , Virus ARN/enzimología , Serina Endopeptidasas/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Mutagénesis Sitio-Dirigida , Mutación , Poliproteínas/química , Virus ARN/metabolismo , ARN Polimerasa Dependiente del ARN/metabolismo , Serina Endopeptidasas/química , Serina Endopeptidasas/genética , Proteínas del Núcleo Viral/metabolismo

RESUMEN

The recombinant coat protein (CP) of Sesbania mosaic virus (SeMV; genus Sobemovirus) was found to self-assemble into capsids encapsidating 23S rRNA and CP mRNA in Escherichia coli. The CP lacking 22 amino acids from the N-terminus assembled into stable T = 3 capsids that appeared similar to SeMV, indicating that the N-terminal 22 amino acid residues are dispensable for T = 3 assembly. Two distinct capsids, T = 1 and pseudo T = 2, were observed when the N-terminal 36 amino acids encompassing the arginine-rich motif (N-ARM) were removed. Only T = 1 particles were observed upon deletion of 65 amino acids from the N-terminus, which also included the sequence element for the beta-annulus. These results reveal that N-ARM acts as a molecular switch in regulating T = 3 assembly. Formation of stable pseudo T = 2 particles shows that pentamers of AB dimers could nucleate assembly at icosahedral-5-folds. Capsids assembled from the N-terminally truncated proteins also encapsidated 23S rRNA and CP mRNA, suggesting the presence of sites outside the N-terminal 65 residues that may be involved in RNA--protein interactions.

Asunto(s)

Cápside/genética , Regulación Viral de la Expresión Génica , Polimorfismo Genético , Virus ARN/metabolismo , Virión/metabolismo , Ensamble de Virus , Secuencia de Aminoácidos , Cápside/química , Cápside/metabolismo , Cápside/ultraestructura , Escherichia coli/genética , Escherichia coli/metabolismo , Eliminación de Gen , Microscopía Electrónica , Modelos Moleculares , Virus ARN/genética , Virión/genética

RESUMEN

The complete nucleotide sequence of the Sesbania mosaic virus (SeMV) genomic RNA was determined by sequencing overlapping cDNA clones. The SeMV genome is 4149 nucleotides in length and encodes four potential overlapping open reading frames (ORFs). Comparison of the nucleotide sequence and the deduced amino acid sequence of the four ORFs of SeMV with that of other sobemoviruses revealed that SeMV was closest to southern bean mosaic virus Arkansas isolate (SBMV-Ark, 73% identity). The 5' non-coding regions of SeMV, SBMV and southern cowpea mosaic virus (SCPMV) are nearly identical. However ORF1 of SeMV which encodes for a putative movement protein of M(r) 18370 has only 34% identity with SBMV-Ark. ORF 2 encodes a polyprotein containing the serine protease, genome linked viral protein (VPg) and RNA dependent RNA polymerase domains and shows 78% identity with SBMV-Ark. The N-terminal amino acid sequence of VPg was found to be TLPPELSIIEIP, which mapped to the region 326-337 of ORF2 product and the cleavage site between the protease domain and VPg was identified to be E325-T326. The cleavage site between VPg and RNA dependent RNA polymerase was predicted to be E445-T446 based on the amino acid sequence analysis of the polyprotein from different sobemoviruses. ORF3 is nested within ORF2 in a--1 reading frame. The potential ribosomal frame shift signal and the downstream stem-loop structure found in other sobemoviruses are also conserved in SeMV RNA sequence, indicating that ORF3 might be expressed via--1 frame shifting mechanism. ORF4 encodes the coat protein of SeMV, which shows 76 and 66% identity with SBMV-Ark and SCPMV, respectively. Thus the comparison of the non-coding regions and the ORFs of SeMV with other sobemoviruses clearly revealed that it is not a strain of SBMV. Phylogenetic analysis of six different sobemoviruses, including SeMV, suggests that recombination event is not frequent in this group and that SeMV is a distinct member of the genus sobemovirus. The analysis also shows sobemoviruses infecting monocotyledons and dicotyledons fall into two distinct clusters.

Asunto(s)

Fabaceae/virología , Virus del Mosaico/genética , Sistemas de Lectura Abierta , Filogenia , Plantas Medicinales , ARN Viral/genética , Proteínas Virales/genética , Secuencia de Bases , Codón , Cartilla de ADN , Genoma Viral , Datos de Secuencia Molecular , Virus del Mosaico/clasificación , Virus del Mosaico/aislamiento & purificación , ARN Viral/química , Alineación de Secuencia