RESUMEN
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
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
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
Ion channels play fundamental roles in both excitable and non-excitable tissues and therefore constitute attractive drug targets for myriad neurological, cardiovascular and metabolic diseases as well as for cancer and immunomodulation. However, achieving selectivity for specific ion channel subtypes with small-molecule drugs has been challenging, and there currently is a growing trend to target ion channels with biologics. One approach is to improve the pharmacokinetics of existing or novel venom-derived peptides. In parallel, after initial studies with polyclonal antibodies demonstrated the technical feasibility of inhibiting channel function with antibodies, multiple preclinical programmes are now using the full spectrum of available technologies to generate conventional monoclonal and engineered antibodies or nanobodies against extracellular loops of ion channels. After a summary of the current state of ion channel drug discovery, this Review discusses recent developments using the purinergic receptor channel P2X purinoceptor 7 (P2X7), the voltage-gated potassium channel KV1.3 and the voltage-gated sodium channel NaV1.7 as examples of targeting ion channels with biologics.
Asunto(s)
Anticuerpos Bloqueadores/farmacología , Canales Iónicos/efectos de los fármacos , Ponzoñas/farmacología , Animales , Anticuerpos Bloqueadores/uso terapéutico , Descubrimiento de Drogas , Humanos , Canales Iónicos/inmunología , Péptidos/farmacología , Péptidos/uso terapéutico , Ponzoñas/uso terapéuticoRESUMEN
It is now well established that antibodies have numerous potential benefits when developed as therapeutics. Here, we evaluate the technical challenges of raising antibodies to membrane-spanning proteins together with enabling technologies that may facilitate the discovery of antibody therapeutics to ion channels. Additionally, we discuss the potential targeting opportunities in the anti-ion channel antibody landscape, along with a number of case studies where functional antibodies that target ion channels have been reported. Antibodies currently in development and progressing towards the clinic are highlighted.
Asunto(s)
Anticuerpos , Desarrollo de Medicamentos/métodos , Descubrimiento de Drogas/métodos , Canales Iónicos/antagonistas & inhibidores , Animales , Anticuerpos/química , Anticuerpos/farmacología , HumanosRESUMEN
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
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
Current methods for producing immunoglobulin G (IgG) antibodies in engineered cells often require refolding steps or secretion across one or more biological membranes. Here, we describe a robust expression platform for biosynthesis of full-length IgG antibodies in the Escherichia coli cytoplasm. Synthetic heavy and light chains, both lacking canonical export signals, are expressed in specially engineered E. coli strains that permit formation of stable disulfide bonds within the cytoplasm. IgGs with clinically relevant antigen- and effector-binding activities are readily produced in the E. coli cytoplasm by grafting antigen-specific variable heavy and light domains into a cytoplasmically stable framework and remodelling the fragment crystallizable domain with amino-acid substitutions that promote binding to Fcγ receptors. The resulting cytoplasmic IgGsnamed 'cyclonals'effectively bypass the potentially rate-limiting steps of membrane translocation and glycosylation.
Asunto(s)
Formación de Anticuerpos/genética , Citoplasma/metabolismo , Escherichia coli/genética , Inmunoglobulina G/biosíntesis , Organismos Modificados Genéticamente/genética , Anticuerpos , Bacteriófagos/genética , Western Blotting , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Glicosilación , Plásmidos/genética , Transporte de Proteínas , Resonancia por Plasmón de SuperficieRESUMEN
Yeast glycan biosynthetic pathways are commonly studied through metabolic incorporation of an exogenous radiolabeled compound into a target glycan. In Saccharomyces cerevisiae glycosylphosphatidylinositol (GPI) biosynthesis, [(3) H]inositol has been widely used to identify intermediates that accumulate in conditional GPI synthesis mutants. However, this approach also labels non-GPI lipid species that overwhelm detection of early GPI intermediates during chromatography. In this study, we show that despite lacking the ability to metabolize N-acetylglucosamine (GlcNAc), S. cerevisiae is capable of importing low levels of extracellular GlcNAc via almost all members of the hexose transporter family. Furthermore, expression of a heterologous GlcNAc kinase gene permits efficient incorporation of exogenous [(14) C]GlcNAc into nascent GPI structures in vivo, dramatically lowering the background signal from non-GPI lipids. Utilizing this new method with several conditional GPI biosynthesis mutants, we observed and characterized novel accumulating lipids that were not previously visible using [(3) H]inositol labeling. Chemical and enzymatic treatments of these lipids indicated that each is a GPI intermediate likely having one to three mannoses and lacking ethanolamine phosphate (Etn-P) side-branches. Our data support a model of yeast GPI synthesis that bifurcates after the addition of the first mannose and that includes a novel branch that produces GPI species lacking Etn-P side-branches.
Asunto(s)
Acetilglucosamina/metabolismo , Etanolaminas/metabolismo , Glicosilfosfatidilinositoles/metabolismo , Manosa/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Saccharomyces cerevisiae/metabolismo , Tritio/metabolismo , Etanolaminas/química , Inositol/metabolismo , Manosa/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Phosphoglycerate mutases (PGM) interconvert 2- and 3-phosphoglycerate in the glycolytic and gluconeogenic pathways. A putative cofactor-independent phosphoglycerate mutase gene (iPGM) was identified in the genome sequence of the Wolbachia endosymbiont from the filarial nematode, Brugia malayi (wBm). Since iPGM has no sequence or structural similarity to the cofactor-dependent phosphoglycerate mutase (dPGM) found in mammals, it may represent an attractive Wolbachia drug target. In the present study, wBm-iPGM cloned and expressed in Escherichia coli was mostly insoluble and inactive. However, the protein was successfully produced in the yeast Kluyveromyces lactis and the purified recombinant wBm-iPGM showed typical PGM activity. Our results provide a foundation for further development of wBm-iPGM as a promising new drug target for novel anti-filarial therapies that selectively target the endosymbiont.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Brugia Malayi/microbiología , Fosfoglicerato Mutasa/genética , Fosfoglicerato Mutasa/metabolismo , Wolbachia/enzimología , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/aislamiento & purificación , Clonación Molecular , Escherichia coli/genética , Expresión Génica , Kluyveromyces/genética , Datos de Secuencia Molecular , NAD/metabolismo , Fosfoglicerato Mutasa/aislamiento & purificación , Alineación de Secuencia , Wolbachia/genéticaRESUMEN
As the primary microtubule-organizing centre of the mammalian cell, the centrosome plays many important roles during cell growth and organization. This is evident across a broad range of cell types and processes, such as the proliferation, differentiation and polarity of neural cells. Additionally, given its localization and function, there are likely to be many more processes that rely on the centrosome that have not yet been characterized. Currently, little is known about centrosomal dynamics during mammalian development. In this study, we have analyzed Nedd1 protein expression to characterize the localization of the centrosome during some aspects of mouse embryogenesis. Using a Nedd1 antibody we have demonstrated the colocalization of Nedd1 with centrosomal markers. We found strong expression of Nedd1, and therefore the centrosome, in highly proliferating cells during neural development. Additionally, Nedd1 was found to have high expression in the cytoplasm of a subset of cells in the dorsal root ganglia. We have also shown a distinct, polarized centrosomal localization of Nedd1 in the developing lens, retina and other polarized tissues. This study reveals the localization of Nedd1 and the centrosome during important processes in mouse embryogenesis and provides a basis for further study into its role in development.
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Sistema Nervioso Central/metabolismo , Centrosoma/metabolismo , Ojo/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Animales , Línea Celular , Línea Celular Tumoral , Polaridad Celular , Sistema Nervioso Central/embriología , Cilios/metabolismo , Desarrollo Embrionario , Ojo/embriología , Ganglios Espinales/embriología , Ganglios Espinales/metabolismo , Humanos , Ratones , Especificidad de ÓrganosRESUMEN
Mytilus californianus foot protein three (Mcfp-3) was successfully expressed in the yeast, Kluyveromyces lactis. The first nine amino acids (YPYDVPDYA) from the human-influenza-virus hemagglutinin (HA) protein were fused to the amino terminus of Mcfp-3 (HA-Mcfp-3) to facilitate identification and purification. HA-Mcfp-3 was purified to a concentration of 1mg/L using HA affinity chromatography. The recovered polypeptide was resolved by SDS-PAGE and migrated primarily at 36 kDa, an increase of approximately 29 kDa over the calculated molecular weight of a HA-Mcfp-3 monomer. Significantly, release of Mcfp-3 by enterokinase treatment coincided with the formation of high molecular weight complexes. It is noteworthy that the complexes mimicked the previously reported insolubility of Mcfps found in vivo to denaturing and reducing conditions. These data demonstrate the successful expression of Mcfp-3 in K. lactis and show an intrinsic ability of Mcfp-3 to self-assemble into stable, higher molecular weight forms.
Asunto(s)
Mytilus/anatomía & histología , Mytilus/química , Proteínas/metabolismo , Secuencia de Aminoácidos , Animales , Western Blotting , Cromatografía de Afinidad , Clonación Molecular , Codón , Electroforesis en Gel de Poliacrilamida , Enteropeptidasa/farmacología , Hemaglutininas Virales/metabolismo , Humanos , Gripe Humana , Kluyveromyces/genética , Peso Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Proteínas/química , Proteínas/genética , Proteínas/aislamiento & purificación , Tinción con Nitrato de Plata , Proteínas Virales de Fusión/químicaRESUMEN
The yeast Kluyveromyces lactis has been extensively used as a host for heterologous protein expression. A necessary step in the construction of a stable expression strain is the introduction of an integrative expression vector into K. lactis cells, followed by selection of transformed strains using either medium containing antibiotic (e.g., G418) or nitrogen-free medium containing acetamide. In this study, we show that selection using acetamide yields K. lactis transformant populations nearly completely comprised of strains bearing multiple tandem insertions of the expression vector pKLAC1 at the LAC4 chromosomal locus, whereas an average of 16% of G418-selected transformants are multiply integrated. Additionally, the average copy number within transformant populations doubled when acetamide was used for selection compared to G418. Finally, we demonstrate that the high frequency of multicopy integration associated with using acetamide selection can be exploited to rapidly construct expression strains that simultaneously produce multiple heterologous proteins or multisubunit proteins, such as Fab antibodies.
Asunto(s)
Acetamidas/farmacología , Vectores Genéticos/genética , Kluyveromyces/genética , Transformación Genética/efectos de los fármacos , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Electroforesis en Gel de Campo Pulsado , Electroforesis en Gel de Poliacrilamida , Dosificación de Gen , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Proteínas de Unión a Maltosa , Modelos Genéticos , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Kluyveromyces lactis is both scientifically and biotechnologically one of the most important non-Saccharomyces yeasts. Its biotechnological significance builds on its history of safe use in the food industry and its well-known ability to produce enzymes like lactase and bovine chymosin on an industrial scale. In this article, we review the various strains, genetic techniques and molecular tools currently available for the use of K. lactis as a host for protein expression. Additionally, we present data illustrating the recent use of proteomics studies to identify cellular bottlenecks that impede heterologous protein expression.
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Kluyveromyces/genética , Kluyveromyces/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/genética , Ingeniería Genética/métodos , Microbiología Industrial , Proteínas Recombinantes/genéticaRESUMEN
The strong LAC4 promoter (P(LAC4)) from Kluyveromyces lactis has been extensively used to drive expression of heterologous proteins in this industrially important yeast. A drawback of this expression method is the serendipitous ability of P(LAC4) to promote gene expression in Escherichia coli. This can interfere with the process of assembling expression constructs in E. coli cells prior to their introduction into yeast cells, especially if the cloned gene encodes a protein that is detrimental to bacteria. In this study, we created a series of P(LAC4) variants by targeted mutagenesis of three DNA sequences (PBI, PBII, and PBIII) that resemble the E. coli Pribnow box element of bacterial promoters and that reside immediately upstream of two E. coli transcription initiation sites associated with P(LAC4). Mutation of PBI reduced the bacterial expression of a reporter protein (green fluorescent protein [GFP]) by approximately 87%, whereas mutation of PBII and PBIII had little effect on GFP expression. Deletion of all three sequences completely eliminated GFP expression. Additionally, each promoter variant expressed human serum albumin in K. lactis cells to levels comparable to wild-type P(LAC4). We created a novel integrative expression vector (pKLAC1) containing the P(LAC4) variant lacking PBI and used it to successfully clone and express the catalytic subunit of bovine enterokinase, a protease that has historically been problematic in E. coli cells. The pKLAC1 vector should aid in the cloning of other potentially toxic genes in E. coli prior to their expression in K. lactis.
Asunto(s)
Escherichia coli/enzimología , Variación Genética , Vectores Genéticos , Kluyveromyces/enzimología , Lactasa/genética , Regiones Promotoras Genéticas/genética , Medios de Cultivo , Enteropeptidasa/genética , Enteropeptidasa/metabolismo , Escherichia coli/genética , Kluyveromyces/clasificación , Kluyveromyces/genética , Lactasa/metabolismo , Datos de Secuencia Molecular , Mutagénesis , Análisis de Secuencia de ADNRESUMEN
Endogenous proteins secreted from Kluyveromyces lactis were screened for their ability to bind to or to hydrolyze chitin. This analysis resulted in identification of a nucleus-encoded extracellular chitinase (KlCts1p) with a chitinolytic activity distinct from that of the plasmid-encoded killer toxin alpha-subunit. Sequence analysis of cloned KlCTS1 indicated that it encodes a 551-amino-acid chitinase having a secretion signal peptide, an amino-terminal family 18 chitinase catalytic domain, a serine-threonine-rich domain, and a carboxy-terminal type 2 chitin-binding domain. The association of purified KlCts1p with chitin is stable in the presence of high salt concentrations and pH 3 to 10 buffers; however, complete dissociation and release of fully active KlCts1p occur in 20 mM NaOH. Similarly, secreted human serum albumin harboring a carboxy-terminal fusion with the chitin-binding domain derived from KlCts1p also dissociates from chitin in 20 mM NaOH, demonstrating the domain's potential utility as an affinity tag for reversible chitin immobilization or purification of alkaliphilic or alkali-tolerant recombinant fusion proteins. Finally, haploid K. lactis cells harboring a cts1 null mutation are viable but exhibit a cell separation defect, suggesting that KlCts1p is required for normal cytokinesis, probably by facilitating the degradation of septum-localized chitin.
Asunto(s)
Núcleo Celular/enzimología , Quitinasas , Kluyveromyces/enzimología , Secuencia de Aminoácidos , Núcleo Celular/genética , Quitina/metabolismo , Quitinasas/química , Quitinasas/genética , Quitinasas/metabolismo , Clonación Molecular , Citocinesis , ADN de Hongos/análisis , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Eliminación de Gen , Kluyveromyces/genética , Datos de Secuencia Molecular , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Alineación de SecuenciaRESUMEN
Glycosylphosphatidylinositols (GPIs) are essential for viability in yeast and have key roles in cell wall construction. Assembly of Saccharomyces cerevisiae GPIs includes the addition of a fourth, side-branching mannose (Man) to the third Man of the core GPI glycan by the Smp3 mannosyltransferase. The SMP3 gene from the human pathogenic fungus Candida albicans has been cloned. CaSMP3 complements the inviable S. cerevisiae smp3 null mutant and, when expressed in an S. cerevisiae smp3/gpi13 double mutant, it permits in vivo conversion of the Man3-GPI precursor that accumulates in that mutant to a Man4-GPI. One allele of CaSMP3 was disrupted using the ura-blaster procedure, then the remaining allele was placed under the control of the glucose-repressible MAL2 promoter. Repression of CaSMP3 expression leads to accumulation of a GPI precursor glycolipid whose glycan headgroup contains three mannoses and bears a phosphodiester-linked substituent on its first Man. Under repressing conditions, cells exhibited morphological and cell wall defects and became inviable. CaSmp3p therefore adds a fourth, alpha1,2-linked Man to trimannosyl GPI precursors in C. albicans and is necessary for viability. Because addition of a fourth Man to GPIs is of less relative importance in mammals, Smp3p is a potential antifungal target.
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Candida albicans/crecimiento & desarrollo , Pared Celular/fisiología , Glicosilfosfatidilinositoles/metabolismo , Manosiltransferasas/antagonistas & inhibidores , Proteínas de Saccharomyces cerevisiae/fisiología , Candida albicans/genética , Candida albicans/metabolismo , Pared Celular/metabolismo , Glicosilfosfatidilinositoles/química , Manosa/metabolismo , Manosiltransferasas/genética , Manosiltransferasas/metabolismo , Unión Proteica , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Yeast and human glycosylphosphatidylinositol (GPI) precursors differ in the extent to which a fourth mannose is present as a side branch of the third core mannose. A fourth mannose addition to GPIs has scarcely been detected in studies of mammalian GPI synthesis but is an essential step in the Saccharomyces cerevisiae pathway. We report that human SMP3 encodes a functional homolog of the yeast Smp3 GPI fourth mannosyl-transferase. Expression of hSMP3 in yeast complements growth and biochemical defects of smp3 mutants and permits in vivo mannosylation of trimannosyl (Man(3))-GPIs. Immunolocalization shows that hSmp3p resides in the endoplasmic reticulum in human cells. Northern analysis of mRNA from human tissues and cell lines indicates that hSMP3 is expressed in most tissues, with the highest levels in brain and colon, but its mRNA is nearly absent from cultured human cell lines. Correspondingly, increasing expression of hSMP3 in cultured HeLa cells causes abundant formation of three putative tetramannosyl (Man(4))-GPIs. Our data indicate that hSmp3p functions as a mannosyltransferase that adds a fourth mannose to certain Man(3)-GPIs during biosynthesis of the human GPI precursor, and suggest it may do so in a tissue-specific manner.
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Glicosilfosfatidilinositoles/biosíntesis , Manosiltransferasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Glicosilfosfatidilinositoles/química , Humanos , Manosa , Manosiltransferasas/química , Manosiltransferasas/genética , Especificidad de Órganos , Unión Proteica , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Bcl-2 family proteins are key regulators of apoptosis. Both pro-apoptotic and anti-apoptotic members of this family are found in mammalian cells, but only the pro-apoptotic protein Debcl has been characterized in Drosophila: Here we report that Buffy, the second Drosophila Bcl-2-like protein, is a pro-survival protein. Ablation of Buffy by RNA interference leads to ectopic apoptosis, whereas overexpression of buffy results in the inhibition of developmental programmed cell death and gamma irradiation-induced apoptosis. Buffy interacts genetically and physically with Debcl to suppress Debcl-induced cell death. Genetic interactions suggest that Buffy acts downstream of Rpr, Grim and Hid, and upstream of the apical caspase Dronc. Furthermore, overexpression of buffy inhibits ectopic cell death in diap1 (th(5)) mutants. Taken together these data suggest that Buffy can act downstream of Rpr, Grim and Hid to block caspase-dependent cell death. Overexpression of Buffy in the embryo results in inhibition of the cell cycle, consistent with a G(1)/early-S phase arrest. Our data suggest that Buffy is functionally similar to the mammalian pro-survival Bcl-2 family of proteins.
Asunto(s)
Apoptosis/fisiología , Ciclo Celular/fisiología , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila/embriología , Proteínas Proto-Oncogénicas c-bcl-2/química , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , Apoptosis/genética , Apoptosis/efectos de la radiación , Supervivencia Celular/genética , Secuencia Conservada , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Rayos gamma/efectos adversos , Regulación del Desarrollo de la Expresión Génica , Datos de Secuencia Molecular , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-bcl-2/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Homología de Secuencia de Aminoácido , TransgenesRESUMEN
Caspase-2 is unique among mammalian caspases because it localizes to the nucleus in a prodomain-dependent manner. The caspase-2 prodomain also regulates caspase-2 activity via a caspase recruitment domain that mediates oligomerization of procaspase-2 molecules and their subsequent autoactivation. In this study we sought to map specific functional regions in the caspase-2 prodomain that regulate its nuclear transport and also its activation. Our data indicate that caspase-2 contains a classical nuclear localization signal (NLS) at the C terminus of the prodomain which is recognized by the importin alpha/beta heterodimer. The mutation of a conserved Lys residue in the NLS abolishes nuclear localization of caspase-2 and binding to the importin alpha/beta heterodimer. Although caspase-2 is imported into the nucleus, mutants lacking the NLS were still capable of inducing apoptosis upon overexpression in transfected cells. We define a region in the prodomain that regulates the ability of caspase-2 to form dot- and filament-like structures when ectopically expressed, which in turn promotes cell killing. Our data provides a mechanism for caspase-2 nuclear import and demonstrate that association of procaspase-2 into higher order structures, rather than its nuclear localization, is required for caspase-2 activation and its ability to induce apoptosis.