RESUMEN
Nuclear receptors (NRs) are an important group of ligand-dependent transcriptional factors. Presently, no natural or synthetic ligand has been identified for a large group of orphan NRs. Small molecules to target these orphan NRs will provide unique resources for uncovering regulatory systems that impact human health and to modulate these pathways with drugs. The orphan NR tailless (TLX, NR2E1), a transcriptional repressor, is a major player in neurogenesis and Neural Stem Cell (NSC) derived brain tumors. No chemical probes that modulate TLX activity are available, and it is not clear whether TLX is druggable. To assess TLX ligand binding capacity, we created homology models of the TLX ligand binding domain (LBD). Results suggest that TLX belongs to an emerging class of NRs that lack LBD helices α1 and α2 and that it has potential to form a large open ligand binding pocket (LBP). Using a medium throughput screening strategy, we investigated direct binding of 20,000 compounds to purified human TLX protein and verified interactions with a secondary (orthogonal) assay. We then assessed effects of verified binders on TLX activity using luciferase assays. As a result, we report identification of three compounds (ccrp1, ccrp2 and ccrp3) that bind to recombinant TLX protein with affinities in the high nanomolar to low micromolar range and enhance TLX transcriptional repressive activity. We conclude that TLX is druggable and propose that our lead compounds could serve as scaffolds to derive more potent ligands. While our ligands potentiate TLX repressive activity, the question of whether it is possible to develop ligands to de-repress TLX activity remains open.
Asunto(s)
Didrogesterona/farmacología , Piperazinas/farmacología , Pirazoles/farmacología , Receptores Citoplasmáticos y Nucleares/fisiología , Activación Transcripcional/efectos de los fármacos , Secuencia de Aminoácidos , Sitios de Unión , Factor de Transcripción COUP II/antagonistas & inhibidores , Factor de Transcripción COUP II/fisiología , Receptor beta de Estrógeno/antagonistas & inhibidores , Receptor beta de Estrógeno/fisiología , Genes Reporteros , Células HeLa , Humanos , Concentración 50 Inhibidora , Ligandos , Luciferasas de Renilla/biosíntesis , Luciferasas de Renilla/genética , Modelos Moleculares , Datos de Secuencia Molecular , Receptores Nucleares Huérfanos , Unión Proteica , Receptores Citoplasmáticos y Nucleares/antagonistas & inhibidores , Receptores Citoplasmáticos y Nucleares/química , Receptor alfa X Retinoide/antagonistas & inhibidores , Receptor alfa X Retinoide/fisiología , Transcripción Genética/efectos de los fármacosRESUMEN
There is a significant need for antibodies that can bind targets with greater affinity. Here we describe a novel strategy employing chemical semisynthesis to produce symmetroadhesins: antibody-like molecules having nonprotein hinge regions that are more flexible and extendible and are capable of two-handed binding. Native chemical ligation was carried out under mild, non-denaturing conditions to join a ligand binding domain (Aß peptide) to an IgG1 Fc dimer via discrete oxyethylene oligomers of various lengths. Two-handed Aß-Fc fusion proteins were obtained in quantitative yield and shown by surface plasmon resonance to bind an anti-Aß antibody with a K(D) at least two orders of magnitude greater than the cognate Aß peptide. MALDI-TOF MS analysis confirmed the protein/nonprotein/protein structure of the two-handed molecules, demonstrating its power to characterize complex protein-nonprotein hybrids by virtue of desorption/ionization mediated by peptide sequences contained therein. We anticipate many applications for symmetroadhesins that combine the target specificity of antibodies with the novel physical, chemical and biological properties of nonprotein hinges.
Asunto(s)
Anticuerpos Monoclonales/química , Nanopartículas/química , Secuencia de Aminoácidos , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Humanos , Cinética , Espectrometría de Masas , Datos de Secuencia Molecular , Péptidos/química , Docilidad , Unión Proteica , Conformación Proteica , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Proteínas Recombinantes de Fusión/síntesis química , Proteínas Recombinantes de Fusión/química , Resonancia por Plasmón de Superficie , Tripsina/metabolismoRESUMEN
Clathrin-coated vesicle formation is responsible for membrane traffic to and from the endocytic pathway during receptor-mediated endocytosis and organelle biogenesis, influencing how cells relate to their environment. Generating these vesicles involves self-assembly of clathrin molecules into a latticed coat on membranes that recruits receptors and organizes protein machinery necessary for budding. Here we define a molecular mechanism regulating clathrin lattice formation by obtaining structural information from co-crystals of clathrin subunits. Low resolution X-ray diffraction data (7.9-9.0 A) was analyzed using a combination of molecular replacement with an energy-minimized model and noncrystallographic symmetry averaging. Resulting topological information revealed two conformations of the regulatory clathrin light chain bound to clathrin heavy chain. Based on protein domain positions, mutagenesis, and biochemical assays, we identify an electrostatic interaction between the clathrin subunits that allows the observed conformational variation in clathrin light chains to alter the conformation of the clathrin heavy chain and thereby regulates assembly.
Asunto(s)
Clatrina/metabolismo , Conformación Proteica , Secuencia de Aminoácidos , Animales , Bovinos , Clatrina/química , Vesículas Cubiertas por Clatrina/metabolismo , Vesículas Cubiertas por Clatrina/fisiología , Cristalografía por Rayos X , Humanos , Sustancias Macromoleculares , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Orgánulos/fisiología , Unión Proteica , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Electricidad EstáticaRESUMEN
Huntingtin-interacting protein 1 (HIP1) is an important link between the actin cytoskeleton and clathrin-mediated endocytosis machinery. HIP1 has also been implicated in the pathogenesis of Huntington's disease. The binding of HIP1 to actin is regulated through an interaction with clathrin light chain. Clathrin light chain binds to a flexible coiled-coil domain in HIP1 and induces a compact state that is refractory to actin binding. To understand the mechanism of this conformational regulation, a high-resolution crystal structure of a stable fragment from the HIP1 coiled-coil domain was determined. The flexibility of the HIP1 coiled-coil region was evident from its variation from a previously determined structure of a similar region. A hydrogen-bond network and changes in coiled-coil monomer interaction suggest that the HIP1 coiled-coil domain is uniquely suited to allow conformational flexibility.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
The transcription factor NFATp integrates multiple signal transduction pathways through coordinate binding with basic-region leucine zipper (bZIP) proteins and other transcription factors. The NFATp monomer, even in the absence of its activation domains, recruits bZIP proteins to canonical NFAT-bZIP composite DNA elements. By contrast, the NFATp dimer and its bZIP partner bind noncooperatively to the NFAT-bZIP element of the tumor necrosis factor (TNF) gene promoter. This observation raises the possibility that the function of the activation domains of NFATp is dimer-specific. Here, we determine the consensus DNA binding site of the NFATp dimer, describe monomer- and dimer-specific NFATp-DNA contact patterns, and demonstrate that NFATp dimerization and dimer-specific activation subdomains are required for transcriptional activation from the TNF NFAT-bZIP element. We also show that these NFATp subdomains interact with the coactivator CBP (CREB-binding protein), which is required for NFATp-dependent TNF gene transcription. Thus, the context-specific function of the activation domains of NFAT can be potentiated by DNA-directed dimerization.
Asunto(s)
Factores de Transcripción NFATC/metabolismo , Secuencia de Bases , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Proteína de Unión a CREB/metabolismo , Secuencia de Consenso , ADN/metabolismo , Dimerización , Ensayo de Cambio de Movilidad Electroforética , Factores de Transcripción NFATC/química , Factores de Transcripción NFATC/genética , Regiones Promotoras Genéticas , Estructura Terciaria de Proteína , Transcripción Genética , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
The huntingtin-interacting protein family members (Hip1 and Hip1R in mammals and Sla2p in yeast) link clathrin-mediated membrane traffic to actin cytoskeleton dynamics. Genetic data in yeast have implicated the light chain subunit of clathrin in regulating this link. To test this hypothesis, the biophysical properties of mammalian Hip1 and Hip1R and their interaction with clathrin light chain and actin were analyzed. The coiled-coil domains (clathrin light chain-binding) of Hip1 and Hip1R were found to be stable homodimers with no propensity to heterodimerize in vitro. Homodimers were also predominant in vivo, accounting for cellular segregation of Hip1 and Hip1R functions. Coiled-coil domains of Hip1 and Hip1R differed in their stability and flexibility, correlating with slightly different affinities for clathrin light chain and more markedly with effects of clathrin light chain binding on Hip protein-actin interactions. Clathrin light chain binding induced a compact conformation of both Hip1 and Hip1R and significantly reduced actin binding by their THATCH domains. Thus, clathrin is a negative regulator of Hip-actin interactions. These observations necessarily change models proposed for Hip protein function.
Asunto(s)
Actinas/química , Proteínas de Unión al ADN/química , Proteínas de Transporte Vesicular/química , Proteínas Adaptadoras Transductoras de Señales , Animales , Clatrina/química , Citoesqueleto/metabolismo , Proteínas de Unión al ADN/metabolismo , Dimerización , Escherichia coli/metabolismo , Células HeLa , Humanos , Ratones , Proteínas de Microfilamentos , Modelos Biológicos , Unión Proteica , Estructura Terciaria de Proteína , Resonancia por Plasmón de Superficie , Proteínas de Transporte Vesicular/metabolismoRESUMEN
Clathrin-coated vesicles (CCVs) sort proteins at the plasma membrane, endosomes and trans Golgi network for multiple membrane traffic pathways. Clathrin recruitment to membranes and its self-assembly into a polyhedral coat depends on adaptor molecules, which interact with membrane-associated vesicle cargo. To determine how adaptors induce clathrin recruitment and assembly, we mapped novel interaction sites between these coat components. A site in the ankle domain of the clathrin triskelion leg was identified that binds a common site on the appendages of tetrameric [AP1 and AP2] and monomeric (GGA1) adaptors. Mutagenesis and modeling studies suggested that the clathrin-GGA1 appendage interface is nonlinear, unlike other peptide-appendage interactions, but overlaps with a sandwich domain binding site for accessory protein peptides, allowing for competitive regulation of coated vesicle formation. A novel clathrin box in the GGA1 hinge region was also identified and shown to mediate membrane recruitment of clathrin, while disruption of the clathrin-GGA1 appendage interaction did not affect recruitment. Thus, the distinct sites for clathrin-adaptor interactions perform distinct functions, revealing new aspects to regulation of CCV formation.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Clatrina/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/química , Proteínas Adaptadoras del Transporte Vesicular/genética , Animales , Sitios de Unión , Clatrina/química , Ratones , Modelos Moleculares , Mutación/genética , Células 3T3 NIH , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
The clathrin triskelion self-assembles into a lattice that coats transport vesicles participating in several key membrane traffic pathways. A new model of a clathrin lattice at approximately 8 angstrom resolution, generated by Fotin et al. (Nature 2004;432:573) confirmed the basic structural features of clathrin that were defined over many years of biochemical and structural analysis. In addition, new structural features of the clathrin trimerization domain were modelled for the first time, and the predictions correlated well with previous biochemical studies. A second model, placing auxilin within the lattice suggested a possible lattice contact targeted during lattice disassembly (Fotin et al. Nature 2004;432:649). This contact predicts interactions of the newly modelled trimerization domain with a newly defined extension of the clathrin triskelion, the ankle domain. These aspects of the new models were emphasized in the published reports describing them and in recent commentary (Brodsky, Nature 2004;432:568). Also emerging from the new models is a better picture of how the clathrin structure is distributed throughout the lattice, allowing the first predictions of interacting molecular interfaces contributing to contacts in the assembled lattice. The focus of this interchange is to emphasize these additional features revealed by the recently published models from Fotin and colleagues.
Asunto(s)
Clatrina/química , Clatrina/metabolismo , Citoesqueleto/química , Citoesqueleto/metabolismoRESUMEN
The clathrin triskelion self-assembles into a polyhedral coat surrounding membrane vesicles that sort receptor cargo to the endocytic pathway. A triskelion comprises three clathrin heavy chains joined at their C-termini, extending into proximal and distal leg segments ending in a globular N-terminal domain. In the clathrin coat, leg segments entwine into parallel and anti-parallel interactions. Here we define the contributions of segmental interactions to the clathrin assembly reaction and measure the strength of their interactions. Proximal and distal leg segments were found to lack sufficient affinity to form stable homo- or heterodimers under assembly conditions. However, chimeric constructs of proximal or distal leg segments, trimerized by replacement of the clathrin trimerization domain with that of the invariant chain protein, were able to self-assemble in reversible reactions. Thus clathrin assembly occurs because weak leg segment affinities are coordinated through trimerization, sharing a dependence on multiple weak interactions with other biopolymers. Such polymerization is sensitive to small environmental changes and is therefore compatible with cellular regulation of assembly, disassembly and curvature during formation of clathrin-coated vesicles.
Asunto(s)
Clatrina/biosíntesis , Estructura Cuaternaria de Proteína , Animales , Bovinos , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Clathrin light chain subunits (LCa and LCb) contribute to regulation of coated vesicle formation to sort proteins during receptor-mediated endocytosis and organelle biogenesis. LC binding to clathrin heavy chain (HC) was characterized by genetic and structural approaches. The core interactions were mapped to HC residues 1267-1522 (out of 1675) and LCb residues 90-157 (out of 228), using yeast two-hybrid assays. The C-termini of both subunits also displayed interactions extending beyond the core domains. Mutations to helix breakers within the LCb core disrupted HC association. Further suppressor mutagenesis uncovered compensatory mutations in HC (K1415E or K1326E) capable of rescuing the binding defects of LCb mutations W127R or W105R plus W138R, thereby pinpointing contacts between HC and LCb. Mutant HC K1415E also rescued loss of binding by LCa W130R, indicating that both LCs interact similarly with HC. Based on circular dichroism data, mapping and mutagenesis, LCa and LCb were represented as alpha-helices, aligned along the HC and, using molecular dynamics, a structural model of their interaction was generated with novel implications for LC control of clathrin assembly.