RESUMEN
Protein-based therapeutics are highly successful in clinic and currently enjoy unprecedented recognition of their potential. More than 100 genuine and similar number of modified therapeutic proteins are approved for clinical use in the European Union and the USA with 2010 sales of US$108 bln; monoclonal antibodies (mAbs) accounted for almost half (48%) of the sales. Based on their pharmacological activity, they can be divided into five groups: (a) replacing a protein that is deficient or abnormal; (b) augmenting an existing pathway; (c) providing a novel function or activity; (d) interfering with a molecule or organism; and (e) delivering other compounds or proteins, such as a radionuclide, cytotoxic drug, or effector proteins. Therapeutic proteins can also be grouped based on their molecular types that include antibody-based drugs, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, engineered protein scaffolds, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytics. They can also be classified based on their molecular mechanism of activity as (a) binding non-covalently to target, e.g., mAbs; (b) affecting covalent bonds, e.g., enzymes; and (c) exerting activity without specific interactions, e.g., serum albumin. Most protein therapeutics currently on the market are recombinant and hundreds of them are in clinical trials for therapy of cancers, immune disorders, infections, and other diseases. New engineered proteins, including bispecific mAbs and multispecific fusion proteins, mAbs conjugated with small molecule drugs, and proteins with optimized pharmacokinetics, are currently under development. However, in the last several decades, there are no conceptually new methodological developments comparable, e.g., to genetic engineering leading to the development of recombinant therapeutic proteins. It appears that a paradigm change in methodologies and understanding of mechanisms is needed to overcome major challenges, including resistance to therapy, access to targets, complexity of biological systems, and individual variations.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Fragmentos Fc de Inmunoglobulinas/uso terapéutico , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión/uso terapéutico , Anticuerpos Monoclonales/efectos adversos , Anticuerpos Monoclonales/clasificación , Anticuerpos Monoclonales/economía , Antitrombina III/uso terapéutico , Unión Europea , Humanos , Enfermedades del Sistema Inmune/tratamiento farmacológico , Terapia Molecular Dirigida/economía , Terapia Molecular Dirigida/métodos , Neoplasias/tratamiento farmacológico , Ingeniería de Proteínas/economía , Proteínas Recombinantes de Fusión/clasificación , Estados UnidosRESUMEN
Phospholipases D are the major dermonecrotic component of Loxosceles venom and catalyze the hydrolysis of phospholipids, resulting in the formation of lipid mediators such as ceramide-1-phosphate and lysophosphatidic acid which can induce pathological and biological responses. Phospholipases D can be classified into two classes depending on their catalytic efficiency and the presence of an additional disulfide bridge. In this work, both wild-type and H12A-mutant forms of the class II phospholipase D from L. intermedia venom were crystallized. Wild-type and H12A-mutant crystals were grown under very similar conditions using PEG 200 as a precipitant and belonged to space group P12(1)1, with unit-cell parameters a = 50.1, b = 49.5, c = 56.5â Å, ß = 105.9°. Wild-type and H12A-mutant crystals diffracted to maximum resolutions of 1.95 and 1.60â Å, respectively.
Asunto(s)
Fosfolipasa D/química , Fosfolipasa D/clasificación , Venenos de Araña/enzimología , Arañas/enzimología , Secuencia de Aminoácidos , Animales , Cristalización , Cristalografía por Rayos X/métodos , Difusión , Disulfuros/química , Escherichia coli/genética , Histidina/química , Calor , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Peso Molecular , Mutación , Fosfolipasa D/genética , Fosfolipasa D/aislamiento & purificación , Hidrolasas Diéster Fosfóricas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/clasificación , Proteínas Recombinantes de Fusión/aislamiento & purificación , Homología de Secuencia de Aminoácido , Transformación Bacteriana , Difracción de Rayos XRESUMEN
The correct localization of integral membrane proteins to subcellular compartments is important for their functions. Synaptotagmin contains a single transmembrane domain that functions as a type I signal-anchor sequence in its N terminus and two calcium-binding domains (C(2)A and C(2)B) in its C terminus. Here, we demonstrate that the localization of an Arabidopsis synaptotagmin homolog, SYT1, to the plasma membrane (PM) is modulated by tandem C2 domains. An analysis of the roots of a transformant-expressing green fluorescent protein-tagged SYT1 driven by native SYT1 promoter suggested that SYT1 is synthesized in the endoplasmic reticulum, and then delivered to the PM via the exocytotic pathway. We transiently expressed a series of truncated proteins in protoplasts, and determined that tandem C(2)A-C(2)B domains were necessary for the localization of SYT1 to the PM. The PM localization of SYT1 was greatly reduced following mutation of the calcium-binding motifs of the C(2)B domain, based on sequence comparisons with other homologs, such as endomembrane-localized SYT5. The localization of SYT1 to the PM may have been required for the functional divergence that occurred in the molecular evolution of plant synaptotagmins.
Asunto(s)
Arabidopsis/citología , Arabidopsis/metabolismo , Membrana Celular/metabolismo , Sinaptotagmina I/química , Sinaptotagmina I/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Retículo Endoplásmico/metabolismo , Eliminación de Gen , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Hojas de la Planta/citología , Hojas de la Planta/metabolismo , Raíces de Plantas/citología , Raíces de Plantas/metabolismo , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/clasificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Sinaptotagmina I/clasificación , Sinaptotagmina I/genéticaRESUMEN
A straightforward method using mild enzymatic digestions combined with MALDI mass spectrometry (MS) was used to enhance determination of the multiple phosphorylation sites of a set of recombinant nucleotide-binding proteins in Escherichia coli, including kinases and cystathionine beta-synthase (CBS) domain containing proteins. The protein kinases reveal abundant phosphorylations in the kinase domains and relatively low phosphogluconoylation (258 Da) at the N-terminal His-tag. In contrast, the CBS domain-containing proteins possess a highly conserved phosphorylation in vivo at Ser-2 of the His-tag. Multistage MS/MS and selected reaction monitoring established that the CBS domain proteins also contain a combined modification of gluconoylation (178 Da) and phosphorylation (80 Da) at two different sites, instead of an isobaric phosphogluconoylation (258 Da) event at the N-terminus. Functional analysis of 20 recombinant proteins as identified by mass spectrometry has shown the phosphorylation at the N-terminal His-tag is relevant to nucleotide binding and phosphotransfer reaction catalyzed by a serine protein kinase.
Asunto(s)
Escherichia coli/química , Fragmentos de Péptidos/química , Proteínas Recombinantes de Fusión/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Secuencia de Aminoácidos , Proteínas Portadoras/química , Proteínas Portadoras/clasificación , Proteínas Portadoras/metabolismo , Escherichia coli/metabolismo , Histidina/química , Datos de Secuencia Molecular , Oligopéptidos/química , Fragmentos de Péptidos/metabolismo , Fosforilación , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/clasificación , Proteínas Recombinantes de Fusión/metabolismo , Espectrometría de Masas en Tándem , Tripsina/metabolismoRESUMEN
BACKGROUND: CNTO 530is a biopharmaceutical consisting of a novel peptide that mimics the actions of erythropoietin, fused to the Fc fragment of human IgG4. Pharmacokinetic and pharmacodynamic studies showed that CNTO 530 produced sustained increases in red blood cell parameters in rats and rabbits and that the serum half life of CNTO 530 was 2 days in rabbits and 3 days in rats. METHODS: For the evaluation of embryofetal development, CNTO 530 was injected at loading doses of 0, 0.9/1, 6, or 60 mg/kg subcutaneously (SC) on gestation day (GD)7 followed by maintenance doses of 0, 0.3, 2, or 20 mg/kg SC every 3 days through GD16 in rats and every 2 days through GD19 in rabbits (GD0 was the day of mating). Rats were Caesarean sectioned on GD21, rabbits on GD29. RESULTS: Administration of CNTO 530 was associated with an increase in hematocrit at all dose levels and a decrease in maternal body weight gains. Fetuses exhibited reduced body weight and delayed ossification. Soft tissue changes were limited to cardiovascular alterations in the high-dose rabbits only. Rat and rabbit fetuses were exposed to CNTO 530 in all dose groups. CONCLUSIONS: These studies show that the embryo/fetal development effects observed following CNTO 530 treatment during organogenesis are qualitatively similar to those seen with other erythropoietin agonists and are likely a secondary consequence of increased hematocrit in the dams. Unlike other erythropoietin receptor agonists, CNTO 530 was able to cross the placental barrier, which was considered likely the result of FcRn-mediated transcytosis.
Asunto(s)
Embrión de Mamíferos/efectos de los fármacos , Desarrollo Embrionario/efectos de los fármacos , Desarrollo Fetal/efectos de los fármacos , Receptores de Eritropoyetina/agonistas , Proteínas Recombinantes de Fusión/toxicidad , Teratógenos/toxicidad , Anomalías Inducidas por Medicamentos/etiología , Animales , Peso Corporal/efectos de los fármacos , Huesos/efectos de los fármacos , Huesos/embriología , Embrión de Mamíferos/embriología , Femenino , Peso Fetal/efectos de los fármacos , Inyecciones Subcutáneas , Osteogénesis/efectos de los fármacos , Policitemia/inducido químicamente , Embarazo , Conejos , Ratas , Proteínas Recombinantes de Fusión/clasificación , Teratógenos/clasificaciónRESUMEN
Translation initiation is down-regulated in eukaryotes by phosphorylation of the alpha-subunit of eIF2 (eukaryotic initiation factor 2), which inhibits its guanine nucleotide exchange factor, eIF2B. The N-terminal S1 domain of phosphorylated eIF2alpha interacts with a subcomplex of eIF2B formed by the three regulatory subunits alpha/GCN3, beta/GCD7, and delta/GCD2, blocking the GDP-GTP exchange activity of the catalytic epsilon-subunit of eIF2B. These regulatory subunits have related sequences and have sequences in common with many archaeal proteins, some of which are involved in methionine salvage and CO(2) fixation. Our sequence analyses however predicted that members of one phylogenetically distinct and coherent group of these archaeal proteins [designated aIF2Bs (archaeal initiation factor 2Bs)] are functional homologs of the alpha, beta, and delta subunits of eIF2B. Three of these proteins, from different archaea, have been shown to bind in vitro to the alpha-subunit of the archaeal aIF2 from the cognate archaeon. In one case, the aIF2B protein was shown further to bind to the S1 domain of the alpha-subunit of yeast eIF2 in vitro and to interact with eIF2Balpha/GCN3 in vivo in yeast. The aIF2B-eIF2alpha interaction was however independent of eIF2alpha phosphorylation. Mass spectrometry has identified several proteins that co-purify with aIF2B from Thermococcus kodakaraensis, and these include aIF2alpha, a sugar-phosphate nucleotidyltransferase with sequence similarity to eIF2Bvarepsilon, and several large-subunit (50S) ribosomal proteins. Based on this evidence that aIF2B has functions in common with eIF2B, the crystal structure established for an aIF2B was used to construct a model of the eIF2B regulatory subcomplex. In this model, the evolutionarily conserved regions and sites of regulatory mutations in the three eIF2B subunits in yeast are juxtaposed in one continuous binding surface for phosphorylated eIF2alpha.
Asunto(s)
Archaea/química , Proteínas Arqueales/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Factor 2 Procariótico de Iniciación/metabolismo , Secuencia de Aminoácidos , Archaea/metabolismo , Proteínas Arqueales/química , Proteínas Arqueales/clasificación , Proteínas Arqueales/genética , Sitios de Unión , Factor 2 Eucariótico de Iniciación/química , Factor 2 Eucariótico de Iniciación/genética , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Filogenia , Factor 2 Procariótico de Iniciación/química , Factor 2 Procariótico de Iniciación/clasificación , Factor 2 Procariótico de Iniciación/genética , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Recombinantes de Fusión/clasificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Subunidades Ribosómicas Grandes de Archaea/genética , Subunidades Ribosómicas Grandes de Archaea/metabolismo , Alineación de SecuenciaRESUMEN
C-terminal binding protein (CtBP) binds to adenovirus early region 1A (AdE1A) through a highly conserved PXDLS motif close to the C terminus. We now have demonstrated that CtBP1 also interacts directly with the transcriptional activation domain (conserved region 3 [CR3]) of adenovirus type 5 E1A (Ad5E1A) and requires the integrity of the entire CR3 region for optimal binding. The interaction appears to be at least partially mediated through a sequence ((161)RRNTGDP(167)) very similar to a recently characterized novel CtBP binding motif in ZNF217 as well as other regions of CR3. Using reporter assays, we further demonstrated that CtBP1 represses Ad5E1A CR3-dependent transcriptional activation. Ad5E1A also appears to be recruited to the E-cadherin promoter through its interaction with CtBP. Significantly, Ad5E1A, CtBP1, and ZNF217 form a stable complex which requires CR3 and the PLDLS motif. It has been shown that Ad513SE1A, containing the CR3 region, is able to overcome the transcriptional repressor activity of a ZNF217 polypeptide fragment in a GAL4 reporter assay through recruitment of CtBP1. These results suggest a hitherto-unsuspected complexity in the association of Ad5E1A with CtBP, with the interaction resulting in transcriptional activation by recruitment of CR3-bound factors to CtBP1-containing complexes.
Asunto(s)
Proteínas E1A de Adenovirus/química , Proteínas E1A de Adenovirus/metabolismo , Proteínas E1A de Adenovirus/genética , Oxidorreductasas de Alcohol/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Anticuerpos Monoclonales/metabolismo , Sitios de Unión , Carcinoma de Células Pequeñas/metabolismo , Línea Celular Tumoral , Secuencia Conservada , Proteínas de Unión al ADN/metabolismo , Genes Reporteros , Glutatión Transferasa/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Células HCT116 , Células HT29 , Células HeLa , Humanos , Luciferasas/análisis , Neoplasias Pulmonares/metabolismo , Datos de Secuencia Molecular , Mutación , Osteosarcoma/metabolismo , Plásmidos , Unión Proteica , Estructura Terciaria de Proteína , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/clasificación , Proteínas Recombinantes de Fusión/metabolismo , Factores de Tiempo , TransfecciónRESUMEN
Phosphoinositide 3-kinase (PI3K) and its product phosphatidylinositol(3,4,5)-trisphosphate (PIP3) control cell growth, migration, and other processes by recruiting proteins with pleckstrin homology (PH) domains and possibly other domains to the plasma membrane (PM). However, previous experimental and structural work with PH domains left conflicting evidence about which ones are PIP3 regulated. Here we used live-cell confocal imaging of 130 YFP-conjugated mouse PH domains and found that 20% translocated to the PM in response to receptor-generated PIP3 production. We developed a recursive-learning algorithm to predict PIP3 regulation of 1200 PH domains from different eukaryotes and validated that it accurately predicts PIP3 regulation. Strikingly, this algorithm showed that PIP3 regulation is specified by amino acids across the PH domain, not just the PIP3-binding pocket, and must have evolved several times independently from PIP3-insensitive ancestral PH domains. Finally, our algorithm and live-cell experiments provide a functional survey of PH domains in different species, showing that PI3K regulation increased from approximately two C. elegans and four Drosophila to 40 vertebrate proteins.
Asunto(s)
Algoritmos , Proteínas de Caenorhabditis elegans/metabolismo , Microscopía Confocal/métodos , Modelos Teóricos , Fosfatos de Fosfatidilinositol/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Células 3T3 NIH , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositol 4,5-Difosfato , Filogenia , Unión Proteica , Conformación Proteica , Proteoma/análisis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/clasificación , Proteínas Recombinantes de Fusión/genética , Alineación de SecuenciaRESUMEN
Malonyl coenzyme A (CoA)-acyl carrier protein (ACP) transacylase (MCAT) is an essential enzyme in fatty acid and mycolic acid biosynthesis of Mycobacterium tuberculosis. fabd2 is a novel gene coding MCAT in M. tuberculosis besides another known fabd. In our study, fabd2 was inserted into a bacterial expression vector pET28a resulting in a 6x Histidine-tag fabd2 fusion gene construction. The protein was purified by nickel affinity chromatography and the characterizations of FabD2 have been investigated. The molecular weight of FabD2 was estimated to be 26 kDa by MALDI-TOF. Consistent with the biosynthesis specialty of reported MCATs, FabD2 resulted in a typical activity of bacterial MCATs, which catalyzes the transacylation of malonate from malonyl-CoA to activated holo-ACP. Some physical and chemical differences between FabD2 and FabD also have been found. FabD2 shows dissimilarity with FabD in secondary structure in different pH buffer and MCAT genes RT-PCR results reveal different transcript condition with each other. Furthermore, FabD2 shows low similarity in protein sequence when alignment with other MCATs.
Asunto(s)
S-Maloniltransferasa de la Proteína Transportadora de Grupos Acilo/aislamiento & purificación , S-Maloniltransferasa de la Proteína Transportadora de Grupos Acilo/metabolismo , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Ácido Graso Sintasas/química , Mycobacterium tuberculosis/enzimología , S-Maloniltransferasa de la Proteína Transportadora de Grupos Acilo/clasificación , S-Maloniltransferasa de la Proteína Transportadora de Grupos Acilo/genética , Proteínas Bacterianas/clasificación , Proteínas Bacterianas/genética , Cationes Bivalentes/química , Ácido Graso Sintasas/aislamiento & purificación , Ácido Graso Sintasas/metabolismo , Concentración de Iones de Hidrógeno , Peso Molecular , Mycobacterium tuberculosis/genética , Filogenia , Estructura Secundaria de Proteína , Proteínas Recombinantes de Fusión/clasificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
ClC-4 and ClC-5 are mammalian ClC isoforms with unique ion conduction and gating properties. Macroscopic current recordings in heterologous expression systems revealed very small currents at negative potentials, whereas a substantially larger instantaneous current amplitude and a subsequent activation were observed upon depolarization. Neither the functional basis nor the physiological impact of these channel features are currently understood. Here, we used whole-cell recordings to study pore properties of human ClC-4 channels heterologously expressed in tsA201 or HEK293 cells. Variance analysis demonstrated that the prominent rectification of the instantaneous macroscopic current amplitude is due to a voltage-dependent unitary current conductance. The single channel amplitudes are very small, i.e., 0.10 +/- 0.02 pA at +140 mV for external Cl(-) and internal I(-). Conductivity and permeability sequences were determined for various external and internal anions, and both values increase for anions with lower dehydration energies. ClC-4 exhibits pore properties that are distinct from other ClC isoforms. These differences can be explained by assuming differences in the size of the pore narrowing and the electrostatic potentials within the ion conduction pathways.
Asunto(s)
Permeabilidad de la Membrana Celular/fisiología , Canales de Cloruro/clasificación , Canales de Cloruro/fisiología , Cloro/fisiología , Riñón/embriología , Potenciales de la Membrana/fisiología , Aniones/farmacocinética , Línea Celular , Conductividad Eléctrica , Humanos , Activación del Canal Iónico/fisiología , Riñón/fisiología , Porosidad , Proteínas Recombinantes de Fusión/clasificación , Proteínas Recombinantes de Fusión/fisiología , Especificidad de la EspecieRESUMEN
Skeletal-type E-C coupling is thought to require a direct interaction between RyR1 and the alpha(1S)-DHPR. Most available evidence suggests that the cytoplasmic II-III loop of the dihydropyridine receptor (DHPR) is the primary source of the orthograde signal. However, identification of the region(s) of RyR1 involved in bidirectional signaling with the alpha(1S)-DHPR remains elusive. To identify these regions we have designed a series of chimeric RyR cDNAs in which different segments of RyR1 were inserted into the corresponding region of RyR3 and expressed in dyspedic 1B5 myotubes. RyR3 provides a preferable background than RyR2 for defining domains essential for E-C coupling because it possesses less sequence homology to RyR1 than the RyR2 backbone used in previous studies. Our data show that two regions of RyR1 (chimera Ch-10 aa 1681-2641 and Ch-9 aa 2642-3770), were independently able to restore skeletal-type E-C coupling to RyR3. These two regions were further mapped and the critical RyR1 residues were 1924-2446 (Ch-21) and 2644-3223 (Ch-19). These results both support and refine the previous hypothesis that multiple domains of RyR1 combine to functionally interact with the DHPR during E-C coupling.
Asunto(s)
Calcio/metabolismo , Contracción Muscular/fisiología , Fibras Musculares Esqueléticas/química , Fibras Musculares Esqueléticas/fisiología , Canal Liberador de Calcio Receptor de Rianodina/química , Canal Liberador de Calcio Receptor de Rianodina/fisiología , Potenciales de Acción/fisiología , Animales , Cafeína/farmacología , Humanos , Contracción Muscular/efectos de los fármacos , Fibras Musculares Esqueléticas/efectos de los fármacos , Músculo Esquelético/química , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/fisiología , Isoformas de Proteínas , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/clasificación , Proteínas Recombinantes de Fusión/deficiencia , Proteínas Recombinantes de Fusión/fisiología , Canal Liberador de Calcio Receptor de Rianodina/clasificación , Canal Liberador de Calcio Receptor de Rianodina/deficienciaRESUMEN
The specificity of biological regulatory mechanisms relies on selective interactions between different proteins in different cell types and in response to different extracellular signals. We describe a bimolecular fluorescence complementation (BiFC) approach for the simultaneous visualization of multiple protein interactions in the same cell. This approach is based on complementation between fragments of fluorescent proteins with different spectral characteristics. We have identified 12 bimolecular fluorescent complexes that correspond to 7 different spectral classes. Bimolecular complex formation between fragments of different fluorescent proteins did not differentially affect the dimerization efficiency of the bZIP domains of Fos and Jun or the subcellular sites of interactions between these domains. Multicolor BiFC enables visualization of interactions between different proteins in the same cell and comparison of the efficiencies of complex formation with alternative interaction partners.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Colorantes Fluorescentes , Microscopía Fluorescente/métodos , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico , Células COS , Chlorocebus aethiops , Color , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/clasificación , Factores de Unión a la G-Box , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/clasificación , Proteínas Recombinantes de Fusión/metabolismo , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Factores de Transcripción/análisis , Factores de Transcripción/clasificación , Dedos de ZincRESUMEN
The production of hybrid enzymes with novel properties and the research for new methods for enzyme immobilization in bioreactors are of major interest in biotechnology. We report here the second part of a study concerning the improvement of the properties of the endoxylanase XYN3A4 from the anaerobic fungi Neocallimastix frontalis. The effects of gene fusion and immobilization on metal-chelate matrix are also compared for the reference enzymes XYN3, XYN3A, XYN4 used for the construction of the fusion protein XYN3A4. The influence of the metal ion in the immobilization process was first investigated and best immobilization yields were obtained with the Cu(II) ion whereas best coupling efficiencies were reached with the Ni(II) ion. It was also observed that XYN3, XYN3A and XYN34 had a lower rate of hydrolysis when immobilized on Ni(II)-IDA and more difficulties to accomodate small substrates than the soluble enzymes. Nevertheless, a major difference was noted during the hydrolysis of birchwood xylan and it appears that the reaction using the immobilized XYN3A4 chimeric enzyme leads to the accumulation of a specific product.
Asunto(s)
Quelantes , Metales/química , Neocallimastix/enzimología , Xilosidasas/biosíntesis , Xilosidasas/química , Catálisis , Endo-1,4-beta Xilanasas , Activación Enzimática , Enzimas Inmovilizadas/química , Escherichia coli/enzimología , Escherichia coli/genética , Regulación Enzimológica de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Neocallimastix/genética , Níquel/química , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/clasificación , Proteínas Recombinantes de Fusión/genética , Especificidad por Sustrato , Transfección/métodos , Xilosidasas/clasificación , Xilosidasas/genéticaRESUMEN
In response to the rapidly growing field of proteomics, the use of recombinant proteins has increased greatly in recent years. Recombinant hybrids containing a polypeptide fusion partner, termed affinity tag, to facilitate the purification of the target polypeptides are widely used. Many different proteins, domains, or peptides can be fused with the target protein. The advantages of using fusion proteins to facilitate purification and detection of recombinant proteins are well-recognized. Nevertheless, it is difficult to choose the right purification system for a specific protein of interest. This review gives an overview of the most frequently used and interesting systems: Arg-tag, calmodulin-binding peptide, cellulose-binding domain, DsbA, c-myc-tag, glutathione S-transferase, FLAG-tag, HAT-tag, His-tag, maltose-binding protein, NusA, S-tag, SBP-tag, Strep-tag, and thioredoxin.
Asunto(s)
Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes/aislamiento & purificación , Secuencia de Aminoácidos , Sitios de Unión , Calmodulina/química , Proteínas Portadoras/química , Celulosa/química , Glutatión Transferasa/química , Histidina/química , Industrias , Proteínas de Unión a Maltosa , Oligopéptidos/química , Fragmentos de Péptidos/síntesis química , Péptidos/química , Unión Proteica , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión/clasificación , Proteínas Recombinantes de Fusión/genética , Ribonucleasas/síntesis químicaRESUMEN
Group B streptococci (GBS) are a major cause of pneumonia, sepsis, and meningitis in newborns and infants. GBS initiate infection of the lung by colonizing mucosal surfaces of the respiratory tract; adherence of the bacteria to host cells is presumed to be the initial step in and prerequisite for successful colonization (G. S. Tamura, J. M. Kuypers, S. Smith, H. Raff, and C. E. Rubens, Infect. Immun. 62:2450-2458, 1994). We have performed a genome-wide screen to identify novel genes of GBS that mediate adherence to fibronectin. A shotgun phage display library was constructed from chromosomal DNA of a serotype Ia GBS strain and affinity selected on immobilized fibronectin. DNA sequence analysis of different clones identified 19 genes with homology to known bacterial adhesin genes, virulence genes, genes involved in transport or metabolic processes, and genes with yet-unknown function. One of the isolated phagemid clones showed significant homology to the gene (scpB) for the GBS C5a peptidase, a surface-associated serine protease that specifically cleaves the complement component C5a, a chemotaxin for polymorphonuclear leukocytes. In this work we have demonstrated that affinity-purified recombinant ScpB and a peptide ScpB fragment (ScpB-PDF), similar to the peptide identified in the phagemid, bound fibronectin in a concentration-dependent manner. Adherence assays to fibronectin were performed, comparing an isogenic scpB mutant to the wild-type strain. Approximately 50% less binding was observed with the mutant than with the wild-type strain. The mutant phenotype could be fully restored by in trans complementation of the mutant with the cloned wild-type scpB gene, providing further evidence for the role of ScpB in fibronectin adherence. Our results suggest that C5a peptidase is a bifunctional protein, which enzymatically cleaves C5a and mediates adherence to fibronectin. Since binding of fibronectin has been implicated in attachment and invasion of eukaryotic cells by streptococci, our results may imply a second important role for this surface protein in the pathogenesis of GBS infections.
Asunto(s)
Adhesinas Bacterianas/metabolismo , Endopeptidasas/metabolismo , Fibronectinas/metabolismo , Glicoproteínas de Membrana/metabolismo , Serina Endopeptidasas/metabolismo , Streptococcus agalactiae/metabolismo , Adhesinas Bacterianas/clasificación , Adhesinas Bacterianas/genética , Endopeptidasas/clasificación , Endopeptidasas/genética , Prueba de Complementación Genética , Genoma Bacteriano , Glicoproteínas de Membrana/clasificación , Glicoproteínas de Membrana/genética , Mutagénesis , Biblioteca de Péptidos , Péptidos/metabolismo , Proteínas Recombinantes de Fusión/clasificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Serina Endopeptidasas/clasificación , Serina Endopeptidasas/genética , Streptococcus agalactiae/genéticaRESUMEN
The genome of Saccharomyces cerevisiae encodes three close homologues of the Escherichia coli 2'-O-rRNA methyltransferase FtsJ/RrmJ, designated Trm7p, Spb1p and Mrm2p. We present evidence that Trm7p methylates the 2'-O-ribose of nucleotides at positions 32 and 34 of the tRNA anticodon loop, both in vivo and in vitro. In a trm7Delta strain, which is viable but grows slowly, translation is impaired, thus indicating that these tRNA modifications could be important for translation efficiency. We discuss the emergence of a family of three 2'-O-RNA methyltransferases in Eukaryota and one in Prokaryota from a common ancestor. We propose that each eukaryotic enzyme is located in a different cell compartment, in which it would methylate a different RNA that can adopt a very similar secondary structure.
Asunto(s)
Anticodón/metabolismo , ARN de Hongos/metabolismo , ARN de Transferencia/metabolismo , Ribosa/biosíntesis , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , ARNt Metiltransferasas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Catálisis , Proteínas de Ciclo Celular/química , Escherichia coli/enzimología , Células Eucariotas , Humanos , Metiltransferasas/química , Datos de Secuencia Molecular , Mutagénesis , Conformación de Ácido Nucleico , Biosíntesis de Proteínas , Estructura Terciaria de Proteína , ARN Mensajero , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/clasificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Ribosa/análogos & derivados , S-Adenosilmetionina/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/clasificación , Proteínas de Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , ARNt Metiltransferasas/química , ARNt Metiltransferasas/clasificación , ARNt Metiltransferasas/genéticaRESUMEN
We report here the molecular cloning and expression of a hemolytic sphingomyelinase from an aquatic bacterium, Pseudomonas sp. strain TK4. The sphingomyelinase gene was found to consist of 1,548 nucleotides encoding 516 amino acid residues. The recombinant 57.7-kDa enzyme hydrolyzed sphingomyelin but not phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, phosphatidic acid, or phosphatidylethanolamine, indicating that the enzyme is a sphingomyelin-specific sphingomyelinase C. The hydrolysis of sphingomyelin by the enzyme was found to be most efficient at pH 8.0 and activated by Mn(2+). The enzyme shows quite a broad specificity, i.e., it hydrolyzed 4-nitrobenz-2-oxa-1,3-diazole (NBD)-sphingomyelin with short-chain fatty acids and NBD-sphingosylphosphorylcholine, the latter being completely resistant to hydrolysis by any sphingomyelinase reported so far. Significant sequence similarities were found in sphingomyelinases from Bacillus cereus, Staphylococcus aureus, Listeria ivanovii, and Leptospira interrogans, as well as a hypothetical protein encoded in Chromobacterium violaceum, although the first three lacked one-third of the sequence corresponding to that from the C terminus of the TK4 enzyme. Interestingly, the deletion mutant of strain TK4 lacking 186 amino acids at the C-terminal end hydrolyzed sphingomyelin, whereas it lost all hemolytic activity, indicating that the C-terminal region of the TK4 enzyme is indispensable for the hemolytic activity.
Asunto(s)
Expresión Génica , Proteínas Hemolisinas/genética , Manganeso/metabolismo , Pseudomonas/enzimología , Esfingomielina Fosfodiesterasa/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Bacteriano , Escherichia coli/metabolismo , Vectores Genéticos/metabolismo , Proteínas Hemolisinas/clasificación , Proteínas Hemolisinas/aislamiento & purificación , Proteínas Hemolisinas/metabolismo , Hidrólisis , Datos de Secuencia Molecular , Mutagénesis , Pseudomonas/genética , Proteínas Recombinantes de Fusión/clasificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Ovinos , Esfingomielina Fosfodiesterasa/clasificación , Esfingomielina Fosfodiesterasa/aislamiento & purificación , Esfingomielina Fosfodiesterasa/metabolismo , Esfingomielinas/metabolismo , Especificidad por SustratoRESUMEN
In Arabidopsis, monogalactosyldiacylglycerol (MGDG) is synthesized by a multigenic family of MGDG synthases consisting of two types of enzymes differing in their N-terminal portion: type A (atMGD1) and type B (atMGD2 and atMGD3). The present paper compares type B isoforms with the enzymes of type A that are known to sit in the inner membrane of plastid envelope. The occurrence of types A and B in 16:3 and 18:3 plants shows that both types are not specialized isoforms for the prokaryotic and eukaryotic glycerolipid biosynthetic pathways. Type A atMGD1 gene is abundantly expressed in green tissues and along plant development and encodes the most active enzyme. Its mature polypeptide is immunodetected in the envelope of chloroplasts from Arabidopsis leaves after cleavage of its transit peptide. atMGD1 is therefore likely devoted to the massive production of MGDG required to expand the inner envelope membrane and build up the thylakoids network. Transient expression of green fluorescent protein fusions in Arabidopsis leaves and in vitro import experiments show that type B precursors are targeted to plastids, owing to a different mechanism. Noncanonical addressing peptides, whose processing could not be assessed, are involved in the targeting of type B precursors, possibly to the outer envelope membrane where they might contribute to membrane expansion. Expression of type B enzymes was higher in nongreen tissues, i.e., in inflorescence (atMGD2) and roots (atMGD3), where they conceivably influence the eukaryotic structure prominence in MGDG. In addition, their expression of type B enzymes is enhanced under phosphate deprivation.
Asunto(s)
Galactosiltransferasas/genética , Glucolípidos/biosíntesis , Fotosíntesis/fisiología , Secuencia de Aminoácidos , Arabidopsis/enzimología , Arabidopsis/genética , Secuencia de Bases , Fraccionamiento Químico , Cloroplastos , ADN de Plantas , Diglicéridos/metabolismo , Escherichia coli , Células Eucariotas , Galactolípidos , Galactosiltransferasas/clasificación , Galactosiltransferasas/aislamiento & purificación , Galactosiltransferasas/metabolismo , Expresión Génica , Genes de Plantas , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Datos de Secuencia Molecular , Fosfatos/metabolismo , Filogenia , Células Procariotas , Proteínas Recombinantes de Fusión/clasificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Especificidad por Sustrato , Distribución TisularRESUMEN
P67, a new protein binding to a specific RNA probe, was purified from radish seedlings [Echeverria, M. and Lahmy, S. (1995) Nucleic Acids Res. 23, 4963-4970]. Amino acid sequence information obtained from P67 microsequencing allowed the isolation of genes encoding P67 in radish and Airabidopsis thaliana. Immunolocalisation experiments in transfected protoplasts demonstrated that this protein is addressed to the chloroplast. The RNA-binding activity of recombinant P67 was found to be similar to that of the native protein. A significant similarity with the maize protein CRP1 [Fisk, D.G., Walker, M.B. and Barkan, A. (1999) EMBO J. 18, 2621-2630] suggests that P67 belongs to the PPR family and could be involved in chloroplast RNA processing.
Asunto(s)
Cloroplastos/metabolismo , Proteínas de Plantas/metabolismo , Proteínas de Unión al ARN/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Arabidopsis , Secuencia de Bases , Brassica , Clonación Molecular , ADN de Plantas , Expresión Génica , Genes de Plantas , Datos de Secuencia Molecular , Proteínas de Plantas/clasificación , Proteínas de Plantas/genética , ARN Mensajero , Proteínas de Unión al ARN/clasificación , Proteínas de Unión al ARN/genética , Proteínas Recombinantes de Fusión/clasificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Fracciones SubcelularesRESUMEN
The tumour suppressor p53 protein integrates multiple signals regulating cell cycle progression and apoptosis. This regulation is mediated by several kinases that phosphorylate specific residues in the different functional domains of the p53 molecule. The human VRK1 protein is a new kinase related to a poxvirus kinase, and more distantly to the casein kinase 1 family. We have characterized the biochemical properties of human VRK1 from HeLa cells. VRK1 has a strong autophosphorylating activity in several Ser and Thr residues. VRK-1 phosphorylates acidic proteins, such as phosvitin and casein, and basic proteins such as histone 2b and myelin basic protein. Because some transcription factors are regulated by phosphorylation, we tested as substrates the N-transactivation domains of p53 and c-Jun fused to GST. Human c-Jun is not phosphorylated by VRK1. VRK1 phosphorylates murine p53 in threonine 18. This threonine is within the p53 hydrophobic loop (residues 13-23) required for the interaction of p53 with the cleft of its inhibitor mdm-2. The VRK1 C-terminus domain (residues 268-396) that contains a nuclear localization signal targets the protein to the nucleus, as determined by using fusion proteins with the green fluorescent protein. We conclude that VRK1 is an upstream regulator of p53 that belongs to a new signalling pathway.