RESUMEN
Monoclonal antibodies combine specificity and high affinity binding with excellent pharmacokinetic properties and are rapidly being developed for a wide range of drug targets including clinically important potassium ion channels. Nonetheless, while therapeutic antibodies come with great promise, K+ channels represent particularly difficult targets for biologics development for a variety of reasons that include their dynamic structures and relatively small extracellular loops, their high degree of sequence conservation (leading to immune tolerance), and their generally low-level expression in vivo. The process is made all the more difficult when large numbers of antibody candidates must be screened for a given target, or when lead candidates fail to cross-react with orthologous channels in animal disease models due to their highly selective binding properties. While the number of antibodies targeting potassium channels in preclinical or clinical development is still modest, significant advances in the areas of protein expression and antibody screening are converging to open the field to an avalanche of new drugs. Here, the opportunities and constraints associated with the discovery of antibodies against K+ channels are discussed, with an emphasis on novel technologies that are opening the field to exciting new possibilities for biologics development.
Asunto(s)
Anticuerpos Monoclonales , Canales de Potasio , Animales , Anticuerpos Monoclonales/uso terapéuticoRESUMEN
A transmission-blocking vaccine targeting the sexual stages of Plasmodium species could play a key role in eradicating malaria. Multiple studies have identified the P. falciparum proteins Pfs25 and Pfs48/45 as prime targets for transmission-blocking vaccines. Although significant advances have been made in recombinant expression of these antigens, they remain difficult to produce at large scale and lack strong immunogenicity as subunit antigens. We linked a self-assembling protein, granule lattice protein 1 (Grl1p), from the ciliated protozoan, Tetrahymena thermophila, to regions of the ectodomains of either Pfs25 or Pfs48/45. We found that resulting protein chimera could be produced in E. coli as nanoparticles that could be readily purified in soluble form. When produced in the E. coli SHuffle strain, fusion to Grl1p dramatically increased solubility of target antigens while at the same time directing the formation of particles with diameters centering on 38 and 25â¯nm depending on the antigen. In a number of instances, co-expression with chaperone proteins and induction at a lower temperature further increased expression and solubility. Based on Western blotting and ELISA analysis, Pfs25 and Pfs48/45 retained their transmission-blocking epitopes within E. coli-derived particles, and the particles themselves elicited strong antibody responses in rabbits when given with an aluminum-based adjuvant. Antibodies against Pfs25-containing nanoparticles blocked parasite transmission in standard membrane-feeding assays. In conclusion, fusion to Grl1p can act as a solubility enhancer for proteins with limited solubility while retaining correct folding, which may be useful for applications such as the production of vaccines and other biologics.
Asunto(s)
Anticuerpos Antiprotozoarios/biosíntesis , Proteínas de Unión al Calcio/genética , Vacunas contra la Malaria/genética , Malaria Falciparum/prevención & control , Glicoproteínas de Membrana/genética , Plasmodium falciparum/química , Proteínas Protozoarias/genética , Tetrahymena thermophila/química , Animales , Antígenos de Protozoos/administración & dosificación , Antígenos de Protozoos/química , Antígenos de Protozoos/genética , Antígenos de Protozoos/inmunología , Bioensayo , Proteínas de Unión al Calcio/administración & dosificación , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/inmunología , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Inmunogenicidad Vacunal , Vacunas contra la Malaria/administración & dosificación , Malaria Falciparum/inmunología , Malaria Falciparum/parasitología , Glicoproteínas de Membrana/administración & dosificación , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/inmunología , Mosquitos Vectores/parasitología , Nanopartículas , Plasmodium falciparum/inmunología , Pliegue de Proteína , Proteínas Protozoarias/administración & dosificación , Proteínas Protozoarias/química , Proteínas Protozoarias/inmunología , Conejos , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Solubilidad , Tetrahymena thermophila/inmunologíaRESUMEN
Integral membrane proteins (MPs) are important drug targets across most fields of medicine, but historically have posed a major challenge for drug discovery due to difficulties in producing them in functional forms. We review the state of the art in drug discovery strategies using recombinant multipass MPs, and outline methods to successfully express, stabilize, and formulate them for small-molecule and monoclonal antibody therapeutics development. Advances in structure-based drug design and high-throughput screening are allowing access to previously intractable targets such as ion channels and transporters, propelling the field towards the development of highly specific therapies targeting desired conformations.
Asunto(s)
Descubrimiento de Drogas , Proteínas de la Membrana , Anticuerpos Monoclonales , Diseño de Fármacos , Humanos , Canales IónicosRESUMEN
We describe a cysteine-rich, membrane-penetrating, joint-targeting, and remarkably stable peptide, EgK5, that modulates voltage-gated KV1.3 potassium channels in T lymphocytes by a distinctive mechanism. EgK5 enters plasma membranes and binds to KV1.3, causing current run-down by a phosphatidylinositol 4,5-bisphosphate-dependent mechanism. EgK5 exhibits selectivity for KV1.3 over other channels, receptors, transporters, and enzymes. EgK5 suppresses antigen-triggered proliferation of effector memory T cells, a subset enriched among pathogenic autoreactive T cells in autoimmune disease. PET-CT imaging with 18F-labeled EgK5 shows accumulation of the peptide in large and small joints of rodents. In keeping with its arthrotropism, EgK5 treats disease in a rat model of rheumatoid arthritis. It was also effective in treating disease in a rat model of atopic dermatitis. No signs of toxicity are observed at 10-100 times the in vivo dose. EgK5 shows promise for clinical development as a therapeutic for autoimmune diseases.
RESUMEN
Proteins containing a classical NLS are transported into the nucleus by the import receptor importin beta, which binds to cargoes via the adaptor importin alpha. The import complex is translocated through the nuclear pore complex by interactions of importin beta with a series of nucleoporins. Previous studies have defined a nucleoporin binding region in the NH2-terminal half of importin beta. Here we report the identification of a second nucleoporin binding region in its COOH-terminal half. Although the affinity of the COOH-terminal region for nucleoporins is dramatically weaker than that of the NH2-terminal region, sets of mutations that perturb the nucleoporin binding of either region reduce the nuclear import activity of importin beta to a similar extent ( approximately 50%). An importin beta mutant with a combination of mutations in the NH2- and COOH-terminal regions is completely inactive for nuclear import. Thus, importin beta possesses two nucleoporin binding sites, both of which are important for its nuclear import function.
Asunto(s)
Transporte Activo de Núcleo Celular/fisiología , Células Eucariotas/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Poro Nuclear/metabolismo , beta Carioferinas/metabolismo , Transporte Activo de Núcleo Celular/genética , Aminoácidos/metabolismo , Sitios de Unión/genética , Ácidos Carboxílicos/metabolismo , Regulación hacia Abajo/genética , Células HeLa , Humanos , Poro Nuclear/genética , Mutación Puntual/genética , Estructura Terciaria de Proteína/genética , Transporte de Proteínas/genética , beta Carioferinas/genéticaRESUMEN
Previous work has shown that the transport of some small protein cargoes through the nuclear pore complex (NPC) can occur in vitro in the absence of nucleoside triphosphate hydrolysis. We now demonstrate that in the importin alpha/beta and transportin import pathways, efficient in vitro transport of large proteins, in contrast to smaller proteins, requires hydrolyzable GTP and the small GTPase Ran. Morphological and biochemical analysis indicates that the presence of Ran and GTP allows large cargo to efficiently cross central regions of the NPC. We further demonstrate that this function of RanGTP at least partly involves its direct binding to importin beta and transportin. We suggest that RanGTP functions in these pathways to promote the transport of large cargo by enhancing the ability of import complexes to traverse diffusionally restricted areas of the NPC.
Asunto(s)
Transporte Activo de Núcleo Celular/fisiología , Núcleo Celular/metabolismo , Guanosina Trifosfato/metabolismo , Poro Nuclear/metabolismo , Proteína de Unión al GTP ran/metabolismo , Sitios de Unión , Guanosina Trifosfato/análogos & derivados , Células HeLa , Humanos , Peso Molecular , Poro Nuclear/ultraestructura , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo , alfa Carioferinas/genética , alfa Carioferinas/metabolismo , beta Carioferinas/genética , beta Carioferinas/metabolismo , Proteína de Unión al GTP ran/genéticaRESUMEN
The initial steps of spliceosomal small nuclear ribonucleoprotein (snRNP) maturation take place in the cytoplasm. After formation of an Sm-core and a trimethylguanosine (TMG) cap, the RNPs are transported into the nucleus via the import adaptor snurportin1 (SPN) and the import receptor importin-beta. To better understand this process, we identified SPN residues that are required to mediate interactions with TMG caps, importin-beta, and the export receptor, exportin1 (Xpo1/Crm1). Mutation of a single arginine residue within the importin-beta binding domain (IBB) disrupted the interaction with importin-beta, but preserved the ability of SPN to bind Xpo1 or TMG caps. Nuclear transport assays showed that this IBB mutant is deficient for snRNP import but that import can be rescued by addition of purified survival of motor neurons (SMN) protein complexes. Conserved tryptophan residues outside of the IBB are required for TMG binding. However, SPN can be imported into the nucleus without cargo. Interestingly, SPN targets to Cajal bodies when U2 but not U1 snRNPs are imported as cargo. SPN also relocalizes to Cajal bodies upon treatment with leptomycin B. Finally, we uncovered an interaction between the N- and C-terminal domains of SPN, suggesting an autoregulatory function similar to that of importin-alpha.
Asunto(s)
Carioferinas/metabolismo , Proteínas de Unión a Caperuzas de ARN/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , beta Carioferinas/metabolismo , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Animales , Cuerpos Enrollados/metabolismo , Secuencia Conservada , Ácidos Grasos Insaturados/farmacología , Células HeLa , Humanos , Datos de Secuencia Molecular , Neuronas Motoras/metabolismo , Mutación , Unión Proteica , Estructura Terciaria de Proteína , Ribonucleoproteína Nuclear Pequeña U2/metabolismo , Homología de Secuencia de Aminoácido , beta Carioferinas/genética , Proteína Exportina 1RESUMEN
Identifying monoclonal antibodies that block human voltage-gated ion channels (VGICs) is a challenging endeavor exacerbated by difficulties in producing recombinant ion channel proteins in amounts that support drug discovery programs. We have developed a general strategy to address this challenge by combining high-level expression of recombinant VGICs in Tetrahymena thermophila with immunization of phylogenetically diverse species and unique screening tools that allow deep-mining for antibodies that could potentially bind functionally important regions of the protein. Using this approach, we targeted human Kv1.3, a voltage-gated potassium channel widely recognized as a therapeutic target for the treatment of a variety of T-cell mediated autoimmune diseases. Recombinant Kv1.3 was used to generate and recover 69 full-length anti-Kv1.3 mAbs from immunized chickens and llamas, of which 10 were able to inhibit Kv1.3 current. Select antibodies were shown to be potent (IC50<10 nM) and specific for Kv1.3 over related Kv1 family members, hERG and hNav1.5.
Asunto(s)
Anticuerpos Monoclonales , Descubrimiento de Drogas/métodos , Canal de Potasio Kv1.3/antagonistas & inhibidores , Animales , Camélidos del Nuevo Mundo , Pollos , Humanos , Proteínas Recombinantes , Tetrahymena thermophilaRESUMEN
In eukaryotic cells, proteins and RNAs are transported between the nucleus and the cytoplasm by nuclear import and export receptors. Over the past decade, small molecules that inhibit the nuclear export receptor CRM1 have been identified, most notably leptomycin B. However, up to now no small molecule inhibitors of nuclear import have been described. Here we have used our automated confocal nanoscanning and bead picking method (CONA) for on-bead screening of a one-bead one-compound library to identify the first such import inhibitor, karyostatin 1A. Karyostatin 1A binds importin ß with high nanomolar affinity and specifically inhibits importin α/ß mediated nuclear import at low micromolar concentrations in vitro and in living cells, without perturbing transportin mediated nuclear import or CRM1 mediated nuclear export. Surface plasmon resonance binding experiments suggest that karyostatin 1A acts by disrupting the interaction between importin ß and the GTPase Ran. As a selective inhibitor of the importin α/ß import pathway, karyostatin 1A will provide a valuable tool for future studies of nucleocytoplasmic trafficking.
Asunto(s)
Transporte Activo de Núcleo Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos/métodos , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , beta Carioferinas/antagonistas & inhibidores , beta Carioferinas/metabolismo , Células HeLa , Humanos , Unión Proteica/efectos de los fármacos , beta Carioferinas/química , Proteína de Unión al GTP ran/metabolismoRESUMEN
In conjugating Tetrahymena thermophila, massive DNA elimination occurs upon the development of the new somatic genome from the germ line genome. Small, approximately 28-nucleotide scan RNAs (scnRNAs) and Twi1p, an Argonaute family member, mediate H3K27me3 and H3K9me3 histone H3 modifications, which lead to heterochromatin formation and the excision of the heterochromatinized germ line-limited sequences. In our search for new factors involved in developmental DNA rearrangement, we identified two Twi1p-interacting proteins, Wag1p and CnjBp. Both proteins contain GW (glycine and tryptophan) repeats, which are characteristic of several Argonaute-interacting proteins in other organisms. Wag1p and CnjBp colocalize with Twi1p in the parental macronucleus early in conjugation and in the new developing macronucleus during later developmental stages. Around the time DNA elimination occurs, Wag1p forms multiple nuclear bodies in the developing macronuclei that do not colocalize with heterochromatic DNA elimination structures. Analyses of DeltaWAG1, DeltaCnjB, and double DeltaWAG1 DeltaCnjB knockout strains revealed that WAG1 and CnjB genes need to be deleted together to inhibit the downregulation of specific scnRNAs, the formation of DNA elimination structures, and DNA excision. Thus, Wag1p and CnjBp are two novel players with overlapping functions in RNA interference-mediated genome rearrangement in Tetrahymena.
Asunto(s)
Reordenamiento Génico , Genoma de Protozoos/genética , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Secuencias Repetitivas de Aminoácido , Tetrahymena thermophila/genética , Tetrahymena thermophila/metabolismo , Animales , Secuencia de Bases , Núcleo Celular/metabolismo , ADN Protozoario/metabolismo , Regulación hacia Abajo/genética , Técnicas de Inactivación de Genes , Inmunoprecipitación , Fenotipo , Unión Proteica , Transporte de Proteínas , Proteínas Protozoarias/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , ARN no Traducido/metabolismo , Tetrahymena thermophila/citologíaRESUMEN
Tetrahymena eliminates micronuclear-limited sequences from the developing macronucleus during sexual reproduction. Homology between the sequences to be eliminated and approximately 28-nucleotide small RNAs (scnRNAs) associated with an Argonaute family protein Twi1p likely underlies this elimination process. However, the mechanism by which Twi1p-scnRNA complexes identify micronuclear-limited sequences is not well understood. We show that a Twi1p-associated putative RNA helicase Ema1p is required for the interaction between Twi1p and chromatin. This requirement explains the phenotypes of EMA1 KO strains, including loss of selective down-regulation of scnRNAs homologous to macronuclear-destined sequences, loss of H3K9 and K27 methylation in the developing new macronucleus, and failure to eliminate DNA. We further demonstrate that Twi1p interacts with noncoding transcripts derived from parental and developing macronuclei and this interaction is greatly reduced in the absence of Ema1p. We propose that Ema1p functions in DNA elimination by stimulating base-pairing interactions between scnRNAs and noncoding transcripts in both parental and developing new macronuclei.
Asunto(s)
ADN Protozoario/fisiología , Proteínas Protozoarias , ARN Helicasas/fisiología , ARN Interferente Pequeño/farmacología , ARN no Traducido , Tetrahymena thermophila/genética , Animales , Northern Blotting , Cromatina/genética , Conjugación Genética , ARN Protozoario/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tetrahymena thermophila/metabolismoRESUMEN
Nucleocytoplasmic transport is mediated by shuttling receptors that recognize specific signals on protein or RNA cargoes and translocate the cargoes through the nuclear pore complex. Transport receptors appear to move through the nuclear pore complex by facilitated diffusion, involving repeated cycles of binding to and dissociation from nucleoporins with phenylalanine-glycine motifs. We discuss recent experimental approaches and results that have begun to provide molecular insight into the mechanisms by which transport complexes traverse the nuclear pore complex, and point out the significant gaps in understanding that remain.
Asunto(s)
Transporte Activo de Núcleo Celular/fisiología , Canales Iónicos/fisiología , beta Carioferinas/metabolismo , Animales , Transporte Biológico Activo , Núcleo Celular/metabolismo , Citoplasma/enzimología , Modelos Moleculares , Conformación Proteica , beta Carioferinas/químicaRESUMEN
Nuclear import of proteins containing a classical nuclear localization signal (NLS) involves NLS recognition by importin alpha, which associates with importin beta via the IBB domain. Other proteins, including parathyroid hormone-related protein (PTHrP), are imported into the nucleus by direct interaction with importin beta. We solved the crystal structure of a fragment of importin beta-1 (1-485) bound to the nonclassical NLS of PTHrP. The structure reveals a second extended cargo binding site on importin beta distinct from the IBB domain binding site. Using a permeabilized cell import assay we demonstrate that importin beta (1-485) can import PTHrP-coupled cargo in a Ran-dependent manner. We propose that this region contains a prototypical nuclear import receptor domain, which could have evolved into the modern importin beta superfamily.
Asunto(s)
Transporte Activo de Núcleo Celular/fisiología , Núcleo Celular/metabolismo , Hormonas Peptídicas/metabolismo , beta Carioferinas/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Células HeLa , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Proteína Relacionada con la Hormona Paratiroidea , Fragmentos de Péptidos/química , Hormonas Peptídicas/genética , Fosforilación , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Secuencias Repetitivas de Aminoácido , Transducción de Señal , Electricidad Estática , Propiedades de Superficie , beta Carioferinas/químicaRESUMEN
The primary function of telomerase is to maintain preexisting telomere tracts. In the ciliate Euplotes crassus, however, telomerase RNP structure and substrate recognition are altered during macronuclear development to facilitate de novo telomere addition. We found that E. crassus harbors three TERT genes encoding the telomerase catalytic subunit that not only vary in their nucleotide and predicted protein sequences, but also in their expression profiles. Expression of EcTERT-1 and -3 correlates with the requirement for telomere maintenance, while that of EcTERT-2 correlates with de novo telomere synthesis. All three genes appear to require ribosomal frameshifting for expression of catalytically active protein. The transcriptionally active form of EcTERT-2 exists only transiently in mated cells and is absent from the vegetative macronucleus. Thus, telomerase expression in Euplotes is controlled by unique regulatory mechanisms that culminate in a developmental switch to a different catalytic subunit with properties suited to de novo telomere addition.