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1.
J Virol ; 97(6): e0040023, 2023 06 29.
Artículo en Inglés | MEDLINE | ID: mdl-37289084

RESUMEN

Induction of type I interferon (IFN) gene expression is among the first lines of cellular defense a virus encounters during primary infection. We previously identified the tegument protein M35 of murine cytomegalovirus (MCMV) as an essential antagonist of this antiviral system, showing that M35 interferes with type I IFN induction downstream of pattern-recognition receptor (PRR) activation. Here, we report structural and mechanistic details of M35's function. Determination of M35's crystal structure combined with reverse genetics revealed that homodimerization is a key feature for M35's immunomodulatory activity. In electrophoretic mobility shift assays (EMSAs), purified M35 protein specifically bound to the regulatory DNA element that governs transcription of the first type I IFN gene induced in nonimmune cells, Ifnb1. DNA-binding sites of M35 overlapped with the recognition elements of interferon regulatory factor 3 (IRF3), a key transcription factor activated by PRR signaling. Chromatin immunoprecipitation (ChIP) showed reduced binding of IRF3 to the host Ifnb1 promoter in the presence of M35. We furthermore defined the IRF3-dependent and the type I IFN signaling-responsive genes in murine fibroblasts by RNA sequencing of metabolically labeled transcripts (SLAM-seq) and assessed M35's global effect on gene expression. Stable expression of M35 broadly influenced the transcriptome in untreated cells and specifically downregulated basal expression of IRF3-dependent genes. During MCMV infection, M35 impaired expression of IRF3-responsive genes aside of Ifnb1. Our results suggest that M35-DNA binding directly antagonizes gene induction mediated by IRF3 and impairs the antiviral response more broadly than formerly recognized. IMPORTANCE Replication of the ubiquitous human cytomegalovirus (HCMV) in healthy individuals mostly goes unnoticed but can impair fetal development or cause life-threatening symptoms in immunosuppressed or -deficient patients. Like other herpesviruses, CMV extensively manipulates its hosts and establishes lifelong latent infections. Murine CMV (MCMV) presents an important model system as it allows the study of CMV infection in the host organism. We previously showed that during entry into host cells, MCMV virions release the evolutionary conserved protein M35 protein to immediately dampen the antiviral type I interferon (IFN) response induced by pathogen detection. Here, we show that M35 dimers bind to regulatory DNA elements and interfere with recruitment of interferon regulatory factor 3 (IRF3), a key cellular factor for antiviral gene expression. Thereby, M35 interferes with expression of type I IFNs and other IRF3-dependent genes, reflecting the importance for herpesviruses to avoid IRF3-mediated gene induction.


Asunto(s)
Infecciones por Citomegalovirus , Elementos de Facilitación Genéticos , Factor 3 Regulador del Interferón , Interferón Tipo I , Proteínas de la Matriz Viral , Animales , Humanos , Ratones , Infecciones por Citomegalovirus/genética , ADN/metabolismo , Factor 3 Regulador del Interferón/metabolismo , Interferón Tipo I/metabolismo , Interferón beta/genética , Interferón beta/metabolismo , Muromegalovirus/genética , Muromegalovirus/metabolismo , Proteínas de la Matriz Viral/metabolismo
2.
Cell Microbiol ; 23(5): e13313, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33491325

RESUMEN

ProA is a secreted zinc metalloprotease of Legionella pneumophila causing lung damage in animal models of Legionnaires' disease. Here we demonstrate that ProA promotes infection of human lung tissue explants (HLTEs) and dissect the contribution to cell type specific replication and extracellular virulence mechanisms. For the first time, we reveal that co-incubation of HLTEs with purified ProA causes a significant increase of the alveolar septal thickness. This destruction of connective tissue fibres was further substantiated by collagen IV degradation assays. The moderate attenuation of a proA-negative mutant in A549 epithelial cells and THP-1 macrophages suggests that effects of ProA in tissue mainly result from extracellular activity. Correspondingly, ProA contributes to dissemination and serum resistance of the pathogen, which further expands the versatile substrate spectrum of this thermolysin-like protease. The crystal structure of ProA at 1.48 Å resolution showed high congruence to pseudolysin of Pseudomonas aeruginosa, but revealed deviations in flexible loops, the substrate binding pocket S1 ' and the repertoire of cofactors, by which ProA can be distinguished from respective homologues. In sum, this work specified virulence features of ProA at different organisational levels by zooming in from histopathological effects in human lung tissue to atomic details of the protease substrate determination.


Asunto(s)
Proteínas Bacterianas/metabolismo , Colágeno Tipo IV/metabolismo , Legionella pneumophila/enzimología , Legionella pneumophila/patogenicidad , Pulmón/microbiología , Metaloendopeptidasas/metabolismo , Alveolos Pulmonares/patología , Factores de Virulencia/metabolismo , Células A549 , Proteínas Bacterianas/química , Actividad Bactericida de la Sangre , Humanos , Legionella pneumophila/crecimiento & desarrollo , Pulmón/patología , Metaloendopeptidasas/química , Proteolisis , Alveolos Pulmonares/metabolismo , Células THP-1 , Virulencia , Factores de Virulencia/química
3.
Proc Natl Acad Sci U S A ; 116(41): 20644-20654, 2019 10 08.
Artículo en Inglés | MEDLINE | ID: mdl-31548418

RESUMEN

cis-Aconitate decarboxylase (CAD, also known as ACOD1 or Irg1) converts cis-aconitate to itaconate and plays central roles in linking innate immunity with metabolism and in the biotechnological production of itaconic acid by Aspergillus terreus We have elucidated the crystal structures of human and murine CADs and compared their enzymological properties to CAD from A. terreus Recombinant CAD is fully active in vitro without a cofactor. Murine CAD has the highest catalytic activity, whereas Aspergillus CAD is best adapted to a more acidic pH. CAD is not homologous to any known decarboxylase and appears to have evolved from prokaryotic enzymes that bind negatively charged substrates. CADs are homodimers, the active center is located in the interface between 2 distinct subdomains, and structural modeling revealed conservation in zebrafish and Aspergillus We identified 8 active-site residues critical for CAD function and rare naturally occurring human mutations in the active site that abolished CAD activity, as well as a variant (Asn152Ser) that increased CAD activity and is common (allele frequency 20%) in African ethnicity. These results open the way for 1) assessing the potential impact of human CAD variants on disease risk at the population level, 2) developing therapeutic interventions to modify CAD activity, and 3) improving CAD efficiency for biotechnological production of itaconic acid.


Asunto(s)
Carboxiliasas/química , Carboxiliasas/genética , Mutación , Succinatos/metabolismo , Células A549 , Secuencia de Aminoácidos , Animales , Carboxiliasas/metabolismo , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , Evolución Molecular , Humanos , Ratones , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Conformación Proteica , Homología de Secuencia
4.
Methods ; 95: 3-12, 2016 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-26658354

RESUMEN

Stable mammalian production cell lines in suspension culture enable the reproducible expression of target genes in any desired scale using bioreactor technology. Targeted integration methods have been developed to cut down timelines for the generation of stable producer cell lines. The powerful Flp recombinase mediated cassette exchange (RMCE) technique allows fast integration of target genes in preselected and optimized high expression loci in so called master cell lines. Up to now, these cells only enable the expression from a single locus. Here, we describe the set-up required for the generation of multiple tagged master cell lines on the example of a binary RMCE expression system in the glycosylation mutant CHO Lec3.2.8.1 cell line. We show how this technology is used for the expression of proteins from multiple loci by generating a binary RMCE expression system. The tools and strategy for the construction of binary master cell lines with different combinations of expression level are described in detail. The binary production cell lines show independent expression of the individual exchange loci of the producer cell lines. The expression level for the model protein tdTomato is the cumulative expression for the chosen combination of the expression loci of the master cell line. This binary RMCE expression system can be further developed to a multi RMCE expression system for co-expression of protein complex subunits with predetermined expression ratio of each individual exchange locus.


Asunto(s)
Clonación Molecular/métodos , Sitios Genéticos , Vectores Genéticos/química , Genoma , Transgenes , Animales , Células CHO , Línea Celular , Cricetulus , ADN Nucleotidiltransferasas/genética , ADN Nucleotidiltransferasas/metabolismo , Efecto Fundador , Expresión Génica , Vectores Genéticos/metabolismo , Genómica/métodos , Plásmidos/química , Plásmidos/metabolismo , Recombinación Genética , Transformación Genética
5.
Proc Natl Acad Sci U S A ; 109(19): 7257-62, 2012 May 08.
Artículo en Inglés | MEDLINE | ID: mdl-22509006

RESUMEN

Given worldwide increases in the incidence of obesity and type 2 diabetes, new strategies for preventing and treating metabolic diseases are needed. The nuclear receptor PPARγ (peroxisome proliferator-activated receptor gamma) plays a central role in lipid and glucose metabolism; however, current PPARγ-targeting drugs are characterized by undesirable side effects. Natural products from edible biomaterial provide a structurally diverse resource to alleviate complex disorders via tailored nutritional intervention. We identified a family of natural products, the amorfrutins, from edible parts of two legumes, Glycyrrhiza foetida and Amorpha fruticosa, as structurally new and powerful antidiabetics with unprecedented effects for a dietary molecule. Amorfrutins bind to and activate PPARγ, which results in selective gene expression and physiological profiles markedly different from activation by current synthetic PPARγ drugs. In diet-induced obese and db/db mice, amorfrutin treatment strongly improves insulin resistance and other metabolic and inflammatory parameters without concomitant increase of fat storage or other unwanted side effects such as hepatoxicity. These results show that selective PPARγ-activation by diet-derived ligands may constitute a promising approach to combat metabolic disease.


Asunto(s)
Productos Biológicos/farmacología , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Fabaceae/química , Hipoglucemiantes/farmacología , Salicilatos/farmacología , Células 3T3-L1 , Animales , Productos Biológicos/química , Productos Biológicos/metabolismo , Western Blotting , Células CHO , Cricetinae , Cricetulus , Cristalografía por Rayos X , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/etiología , Dieta Alta en Grasa/efectos adversos , Suplementos Dietéticos , Expresión Génica/efectos de los fármacos , Glycyrrhiza/química , Humanos , Hipoglucemiantes/química , Hipoglucemiantes/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Estructura Molecular , Obesidad/complicaciones , Obesidad/tratamiento farmacológico , Obesidad/etiología , PPAR gamma/genética , PPAR gamma/metabolismo , Unión Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Salicilatos/química , Salicilatos/metabolismo
6.
Front Immunol ; 15: 1427457, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-39156902

RESUMEN

Aconitate decarboxylase-1 (ACOD1) is expressed by activated macrophages and generates itaconate that exerts anti-microbial and immunoregulatory effects. ACOD1-itaconate is essential for macrophage-mediated control of the intracellular pathogen Coxiella (C.) burnetii, which causes Q fever. Two isomers of itaconate, mesaconate and citraconate, have overlapping yet distinct activity on macrophage metabolism and inflammatory gene expression. Here, we found that all three isomers inhibited the growth of C. burnetii in axenic culture in ACCM-2 medium. However, only itaconate reduced C. burnetii replication efficiently in Acod1-/- macrophages. In contrast, addition of citraconate strongly increased C. burnetii replication in Acod1+/- macrophages, whereas mesaconate weakly enhanced bacterial burden in Acod1-/- macrophages. Analysis of intracellular isomers showed that exogenous citraconate and mesaconate inhibited the generation of itaconate by infected Acod1+/- macrophages. Uptake of added isomers into Acod1-/- macrophages was increased after infection for itaconate and mesaconate, but not for citraconate. Mesaconate, but not citraconate, competed with itaconate for uptake into macrophages. Taken together, inhibition of itaconate generation by macrophages and interference with the uptake of extracellular itaconate could be identified as potential mechanisms behind the divergent effects of citraconate and mesaconate on C. burnetii replication in macrophages or in axenic culture.


Asunto(s)
Cultivo Axénico , Carboxiliasas , Coxiella burnetii , Macrófagos , Succinatos , Coxiella burnetii/efectos de los fármacos , Coxiella burnetii/crecimiento & desarrollo , Succinatos/farmacología , Animales , Macrófagos/microbiología , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/efectos de los fármacos , Ratones , Carboxiliasas/metabolismo , Ratones Noqueados , Fiebre Q/inmunología , Fiebre Q/microbiología , Ratones Endogámicos C57BL , Hidroliasas
7.
BMC Biotechnol ; 13: 52, 2013 Jun 26.
Artículo en Inglés | MEDLINE | ID: mdl-23802841

RESUMEN

BACKGROUND: The demand of monospecific high affinity binding reagents, particularly monoclonal antibodies, has been steadily increasing over the last years. Enhanced throughput of antibody generation has been addressed by optimizing in vitro selection using phage display which moved the major bottleneck to the production and purification of recombinant antibodies in an end-user friendly format. Single chain (sc)Fv antibody fragments require additional tags for detection and are not as suitable as immunoglobulins (Ig)G in many immunoassays. In contrast, the bivalent scFv-Fc antibody format shares many properties with IgG and has a very high application compatibility. RESULTS: In this study transient expression of scFv-Fc antibodies in human embryonic kidney (HEK) 293 cells was optimized. Production levels of 10-20 mg/L scFv-Fc antibody were achieved in adherent HEK293T cells. Employment of HEK293-6E suspension cells expressing a truncated variant of the Epstein Barr virus (EBV) nuclear antigen (EBNA) 1 in combination with production under serum free conditions increased the volumetric yield up to 10-fold to more than 140 mg/L scFv-Fc antibody. After vector optimization and process optimization the yield of an scFv-Fc antibody and a cytotoxic antibody-RNase fusion protein further increased 3-4-fold to more than 450 mg/L. Finally, an entirely new mammalian expression vector was constructed for single step in frame cloning of scFv genes from antibody phage display libraries. Transient expression of more than 20 different scFv-Fc antibodies resulted in volumetric yields of up to 600 mg/L and 400 mg/L in average. CONCLUSION: Transient production of recombinant scFv-Fc antibodies in HEK293-6E in combination with optimized vectors and fed batch shake flasks cultivation is efficient and robust, and integrates well into a high-throughput recombinant antibody generation pipeline.


Asunto(s)
Fragmentos Fc de Inmunoglobulinas/biosíntesis , Anticuerpos de Cadena Única/biosíntesis , Clonación Molecular , Vectores Genéticos , Células HEK293 , Humanos , Biblioteca de Péptidos , Proteínas Recombinantes de Fusión/biosíntesis , Ribonucleasas/biosíntesis
8.
BMC Biol ; 10: 62, 2012 Jul 19.
Artículo en Inglés | MEDLINE | ID: mdl-22809326

RESUMEN

BACKGROUND: The family of lysosome-associated membrane proteins (LAMP) comprises the multifunctional, ubiquitous LAMP-1 and LAMP-2, and the cell type-specific proteins DC-LAMP (LAMP-3), BAD-LAMP (UNC-46, C20orf103) and macrosialin (CD68). LAMPs have been implicated in a multitude of cellular processes, including phagocytosis, autophagy, lipid transport and aging. LAMP-2 isoform A acts as a receptor in chaperone-mediated autophagy. LAMP-2 deficiency causes the fatal Danon disease. The abundant proteins LAMP-1 and LAMP-2 are major constituents of the glycoconjugate coat present on the inside of the lysosomal membrane, the 'lysosomal glycocalyx'. The LAMP family is characterized by a conserved domain of 150 to 200 amino acids with two disulfide bonds. RESULTS: The crystal structure of the conserved domain of human DC-LAMP was solved. It is the first high-resolution structure of a heavily glycosylated lysosomal membrane protein. The structure represents a novel ß-prism fold formed by two ß-sheets bent by ß-bulges and connected by a disulfide bond. Flexible loops and a hydrophobic pocket represent possible sites of molecular interaction. Computational models of the glycosylated luminal regions of LAMP-1 and LAMP-2 indicate that the proteins adopt a compact conformation in close proximity to the lysosomal membrane. The models correspond to the thickness of the lysosomal glycoprotein coat of only 5 to 12 nm, according to electron microscopy. CONCLUSION: The conserved luminal domain of lysosome-associated membrane proteins forms a previously unknown ß-prism fold. Insights into the structure of the lysosomal glycoprotein coat were obtained by computational models of the LAMP-1 and LAMP-2 luminal regions.


Asunto(s)
Secuencia Conservada , Glicocálix/metabolismo , Proteína 3 de la Membrana Asociada a Lisosoma/química , Proteína 3 de la Membrana Asociada a Lisosoma/metabolismo , Lisosomas/metabolismo , Secuencia de Aminoácidos , Cristalografía por Rayos X , Glicosilación , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Membranas Intracelulares/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína
9.
Nat Genet ; 30(4): 385-93, 2002 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-11912495

RESUMEN

Proteome analysis is a fundamental step in systematic functional genomics. Here we have resolved 8,767 proteins from the mouse brain proteome by large-gel two-dimensional electrophoresis. We detected 1,324 polymorphic proteins from the European collaborative interspecific backcross. Of these, we mapped 665 proteins genetically and identified 466 proteins by mass spectrometry. Qualitatively polymorphic proteins, to 96%, reflect changes in conformation and/or mass. Quantitatively polymorphic proteins show a high frequency (73%) of allele-specific transmission in codominant heterozygotes. Variations in protein isoforms and protein quantity often mapped to chromosomal positions different from that of the structural gene, indicating that single proteins may act as polygenic traits. Genetic analysis of proteomes may detect the types of polymorphism that are most relevant in disease-association studies.


Asunto(s)
Encéfalo/fisiología , Polimorfismo Genético , Alelos , Animales , Encéfalo/metabolismo , Mapeo Cromosómico , Clonación Molecular , Cruzamientos Genéticos , ADN/metabolismo , Electroforesis en Gel Bidimensional , Femenino , Ligamiento Genético , Marcadores Genéticos , Genotipo , Heterocigoto , Espectrometría de Masas , Ratones , Modelos Genéticos , Hibridación de Ácido Nucleico , Fenotipo , Reacción en Cadena de la Polimerasa , Conformación Proteica , Isoformas de Proteínas
10.
Sci Rep ; 13(1): 10360, 2023 06 26.
Artículo en Inglés | MEDLINE | ID: mdl-37365251

RESUMEN

cis-Aconitate decarboxylase (ACOD1, IRG1) converts cis-aconitate to the immunomodulatory and antibacterial metabolite itaconate. Although the active site residues of human and mouse ACOD1 are identical, the mouse enzyme is about fivefold more active. Aiming to identify the cause of this difference, we mutated positions near the active site in human ACOD1 to the corresponding residues of mouse ACOD1 and measured resulting activities in vitro and in transfected cells. Interestingly, Homo sapiens is the only species with methionine instead of isoleucine at residue 154 and introduction of isoleucine at this position increased the activity of human ACOD1 1.5-fold in transfected cells and 3.5-fold in vitro. Enzyme activity of gorilla ACOD1, which is almost identical to the human enzyme but has isoleucine at residue 154, was similar to the mouse enzyme in vitro. Met154 in human ACOD1 forms a sulfur-π bond to Phe381, which is positioned to impede access of the substrate to the active site. It appears that the ACOD1 sequence has changed at position 154 during human evolution, resulting in a pronounced decrease in activity. This change might have offered a selective advantage in diseases such as cancer.


Asunto(s)
Aminoácidos , Carboxiliasas , Isoleucina , Animales , Humanos , Ratones , Dominio Catalítico , Carboxiliasas/química
11.
J Struct Biol ; 175(2): 159-70, 2011 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-21382497

RESUMEN

Escherichia coli (E. coli) remains the most commonly used host for recombinant protein expression. It is well known that a variety of experimental factors influence the protein production level as well as the solubility profile of over-expressed proteins. This becomes increasingly important for optimizing production of protein complexes using co-expression strategies. In this study, we focus on the effect of the choice of the expression vector system: by standardizing experimental factors including bacterial strain, cultivation temperature and growth medium composition, we compare the effectiveness of expression technologies used by the partners of the Structural Proteomics in Europe 2 (SPINE2-complexes) consortium. Four different protein complexes, including three binary and one ternary complex, all known to be produced in the soluble form in E. coli, are used as the benchmark targets. The respective genes were cloned by each partner into their preferred set of vectors. The resulting constructs were then used for comparative co-expression analysis done in parallel and under identical conditions at a single site. Our data show that multiple strategies can be applied for the expression of protein complexes in high yield. While there is no 'silver bullet' approach that was infallible even for this small test set, our observations are useful as a guideline to delineate co-expression strategies for particular protein complexes.


Asunto(s)
Clonación Molecular/métodos , Escherichia coli/genética , Vectores Genéticos/normas , Complejos Multiproteicos/biosíntesis , Proteínas Recombinantes/biosíntesis , Academias e Institutos , Factor de Unión a CCAAT/biosíntesis , Factor de Unión a CCAAT/genética , Proteínas de Ciclo Celular/biosíntesis , Proteínas de Ciclo Celular/genética , Europa (Continente) , Geminina , Cooperación Internacional , Israel , Complejos Multiproteicos/química , Complejos Multiproteicos/aislamiento & purificación , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Factores de Transcripción TFII/biosíntesis , Factores de Transcripción TFII/genética
12.
J Clin Immunol ; 31(6): 998-1009, 2011 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-21901394

RESUMEN

BACKGROUND: To clarify the impact of T cell responses towards enteric antigens for chronic intestinal inflammation, we determined T helper 1 reactivity towards conserved Escherichia coli proteins in patients with Crohn's disease (CD) and healthy individuals and patients with ankylosing spondylitis (AS), who also often show microscopic inflammatory lesions within the gut or even develop overt inflammatory bowel disease. METHODS: We determined the frequency of IFNγ+CD40L+ cells/CD4+ T cells after stimulation of whole blood with pools of E. coli proteins. RESULTS: The E. coli-specific Th1 response was significantly reduced in CD patients and to a lower extent also in AS patients. CONCLUSIONS: E. coli is a target for polyclonal Th1 responses in healthy individuals. The impairment of these responses in CD and AS patients might be due to recruitment of enterobacteria-specific Th1 cells to the gut or might reflect inadequate priming of adaptive immune response.


Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Enfermedad de Crohn/inmunología , Proteínas de Escherichia coli/inmunología , Intestinos/patología , Espondilitis Anquilosante/inmunología , Células TH1/metabolismo , Inmunidad Adaptativa , Adolescente , Adulto , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/patología , Ligando de CD40/metabolismo , Movimiento Celular , Niño , Preescolar , Enfermedad de Crohn/fisiopatología , Femenino , Humanos , Terapia de Inmunosupresión , Lactante , Inflamación , Interferón gamma/metabolismo , Masculino , Espondilitis Anquilosante/fisiopatología , Células TH1/inmunología , Células TH1/patología
13.
Nucleic Acids Res ; 35(6): e43, 2007.
Artículo en Inglés | MEDLINE | ID: mdl-17311810

RESUMEN

A vector system is presented that allows generation of E. coli co-expression clones by a standardized, robust cloning procedure. The number of co-expressed proteins is not limited. Five 'pQLink' vectors for expression of His-tag and GST-tag fusion proteins as well as untagged proteins and for cloning by restriction enzymes or Gateway cloning were generated. The vectors allow proteins to be expressed individually; to achieve co-expression, two pQLink plasmids are combined by ligation-independent cloning. pQLink co-expression plasmids can accept an unrestricted number of genes. As an example, the co-expression of a heterotetrameric human transport protein particle (TRAPP) complex from a single plasmid, its isolation and analysis of its stoichiometry are shown. pQLink clones can be used directly for pull-down experiments if the proteins are expressed with different tags. We demonstrate pull-down experiments of human valosin-containing protein (VCP) with fragments of the autocrine motility factor receptor (AMFR). The cloning method avoids PCR or gel isolation of restriction fragments, and a single resistance marker and origin of replication are used, allowing over-expression of rare tRNAs from a second plasmid. It is expected that applications are not restricted to bacteria, but could include co-expression in other hosts such as Bacluovirus/insect cells.


Asunto(s)
Clonación Molecular/métodos , Vectores Genéticos/química , Proteínas Recombinantes/biosíntesis , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Escherichia coli/genética , Expresión Génica , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Proteínas de la Membrana/metabolismo , Subunidades de Proteína/genética , Subunidades de Proteína/aislamiento & purificación , Subunidades de Proteína/metabolismo , Receptores del Factor Autocrino de Motilidad , Receptores de Citocinas/genética , Receptores de Citocinas/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteína que Contiene Valosina , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/aislamiento & purificación , Proteínas de Transporte Vesicular/metabolismo
14.
PLoS One ; 14(4): e0215062, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30990863

RESUMEN

Interferon α (IFNα) counteracts viral infections by activating various IFNα-stimulated genes (ISGs). These genes encode proteins that block viral transport into the host cell and inhibit viral replication, gene transcription and translation. Due to the existence of 14 different, highly homologous isoforms of mouse IFNα, an IFNα knockout mouse has not yet been established by genetic knockout strategies. An scFv intrabody for holding back IFNα isoforms in the endoplasmic reticulum (ER) and thus counteracting IFNα secretion is reported. The intrabody was constructed from the variable domains of the anti-mouse IFNα rat monoclonal antibody 4EA1 recognizing the 5 isoforms IFNα1, IFNα2, IFNα4, IFNα5, IFNα6. A soluble form of the intrabody had a KD of 39 nM to IFNα4. It could be demonstrated that the anti-IFNα intrabody inhibits clearly recombinant IFNα4 secretion by HEK293T cells. In addition, the secretion of IFNα4 was effectively inhibited in stably transfected intrabody expressing RAW 264.7 macrophages and dendritic D1 cells. Colocalization of the intrabody with IFNα4 and the ER marker calnexin in HEK293T cells indicated complex formation of intrabody and IFNα4 inside the ER. Intracellular binding of intrabody and antigen was confirmed by co-immunoprecipitation. Complexes of endogenous IFNα and intrabody could be visualized in the ER of Poly (I:C) stimulated RAW 264.7 macrophages and D1 dendritic cells. Infection of macrophages and dendritic cells with the vesicular stomatitis virus VSV-AV2 is attenuated by IFNα and IFNß. The intrabody increased virus proliferation in RAW 264.7 macrophages and D1 dendritic cells under IFNß-neutralizing conditions. To analyze if all IFNα isoforms are recognized by the intrabody was not in the focus of this study. Provided that binding of the intrabody to all isoforms was confirmed, the establishment of transgenic intrabody mice would be promising for studying the function of IFNα during viral infection and autoimmune diseases.


Asunto(s)
Células Dendríticas/inmunología , Retículo Endoplásmico/inmunología , Interferón-alfa/antagonistas & inhibidores , Macrófagos/inmunología , Anticuerpos de Cadena Única/farmacología , Replicación Viral/efectos de los fármacos , Animales , Células Cultivadas , Células Dendríticas/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Células HEK293 , Humanos , Interferón-alfa/efectos de los fármacos , Interferón-alfa/inmunología , Interferón-alfa/metabolismo , Macrófagos/efectos de los fármacos , Ratones , Células RAW 264.7
15.
Proteomics ; 8(4): 612-25, 2008 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-18210369

RESUMEN

The sequence infrastructure that has arisen through large-scale genomic projects dedicated to protein analysis, has provided a wealth of information and brought together scientists and institutions from all over the world. As a consequence, the development of novel technologies and methodologies in proteomics research is helping to unravel the biochemical and physiological mechanisms of complex multivariate diseases at both a functional and molecular level. In the late sixties, when X-ray crystallography had just been established, the idea of determining protein structure on an almost universal basis was akin to an impossible dream or a miracle. Yet only forty years after, automated protein structure determination platforms have been established. The widespread use of robotics in protein crystallography has had a huge impact at every stage of the pipeline from protein cloning, over-expression, purification, crystallization, data collection, structure solution, refinement, validation and data management- all of which have become more or less automated with minimal human intervention necessary. Here, recent advances in protein crystal structure analysis in the context of structural genomics will be discussed. In addition, this review aims to give an overview of recent developments in high throughput instrumentation, and technologies and strategies to accelerate protein structure/function analysis.


Asunto(s)
Cristalografía por Rayos X/métodos , Proteínas/química , Proteómica/instrumentación , Automatización , Cristalización , Procesamiento Automatizado de Datos , Histidina/química , Estructura Cuaternaria de Proteína , Proteínas/aislamiento & purificación , Sincrotrones
16.
J Clin Invest ; 115(5): 1352-60, 2005 May.
Artículo en Inglés | MEDLINE | ID: mdl-15841210

RESUMEN

MS is a chronic inflammatory and demyelinating disease of the CNS with as yet unknown etiology. A hallmark of this disease is the occurrence of oligoclonal IgG antibodies in the cerebrospinal fluid (CSF). To assess the specificity of these antibodies, we screened protein expression arrays containing 37,000 tagged proteins. The 2 most frequent MS-specific reactivities were further mapped to identify the underlying high-affinity epitopes. In both cases, we identified peptide sequences derived from EBV proteins expressed in latently infected cells. Immunoreactivities to these EBV proteins, BRRF2 and EBNA-1, were significantly higher in the serum and CSF of MS patients than in those of control donors. Oligoclonal CSF IgG from MS patients specifically bound both EBV proteins. Also, CD8(+) T cell responses to latent EBV proteins were higher in MS patients than in controls. In summary, these findings demonstrate an increased immune response to EBV in MS patients, which suggests that the virus plays an important role in the pathogenesis of disease.


Asunto(s)
Herpesvirus Humano 4/inmunología , Esclerosis Múltiple/inmunología , Proteínas Virales/inmunología , Anticuerpos/genética , Anticuerpos/inmunología , Humanos , Inmunoglobulina G/líquido cefalorraquídeo , Inmunoglobulina G/inmunología , Esclerosis Múltiple/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Análisis por Matrices de Proteínas , Linfocitos T/inmunología
17.
FEBS J ; 275(18): 4627-40, 2008 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-18699778

RESUMEN

Iba2 is a homolog of ionized calcium-binding adapter molecule 1 (Iba1), a 17-kDa protein that binds and cross-links filamentous actin (F-actin) and localizes to membrane ruffles and phagocytic cups. Here, we present the crystal structure of human Iba2 and its homodimerization properties, F-actin cross-linking activity, cellular localization and recruitment upon bacterial invasion in comparison with Iba1. The Iba2 structure comprises two central EF-hand motifs lacking bound Ca2+. Iba2 crystallized as a homodimer stabilized by a disulfide bridge and zinc ions. Analytical ultracentrifugation revealed a different mode of dimerization under reducing conditions that was independent of Ca2+. Furthermore, no binding of Ca2+ up to 0.1 mM was detected by equilibrium dialysis. Correspondingly, Iba EF-hand motifs lack residues essential for strong Ca2+ coordination. Sedimentation experiments and microscopy detected pronounced, indistinguishable F-actin binding and cross-linking activity of Iba1 and Iba2 with induction of F-actin bundles. Fluorescent Iba fusion proteins were expressed in HeLa cells and co-localized with F-actin. Iba1 was recruited into cellular projections to a larger extent than Iba2. Additionally, we studied Iba recruitment in a Shigella invasion model that induces cytoskeletal rearrangements. Both proteins were recruited into the bacterial invasion zone and Iba1 was again concentrated slightly higher in the cellular extensions.


Asunto(s)
Proteínas de Unión al Calcio/química , Proteínas de Unión al ADN/química , Proteínas de Microfilamentos/química , Actinas/metabolismo , Secuencia de Aminoácidos , Calcio/metabolismo , Proteínas de Unión al Calcio/análisis , Proteínas de Unión al Calcio/metabolismo , Cristalografía por Rayos X , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/metabolismo , Dimerización , Células HeLa , Humanos , Proteínas de Microfilamentos/análisis , Proteínas de Microfilamentos/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Alineación de Secuencia , Shigella/patogenicidad
18.
Methods Mol Biol ; 426: 163-73, 2008.
Artículo en Inglés | MEDLINE | ID: mdl-18542862

RESUMEN

The systematic structural analysis of many target proteins involves generating expression clones in high throughput. This requires robust laboratory procedures and benefits from laboratory automation and data management systems. This chapter gives an overview of the Protein Structure Factory, a structural genomics project focusing on human proteins, and presents the authors' method for cloning bacterial expression clones with the restriction enzymes BamHI and NotI and compatible enzymes. PCR amplification, product purification and digestion and vector ligation were adapted to the 96-well microtiter plate format.


Asunto(s)
Clonación Molecular/métodos , Vectores Genéticos/metabolismo , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , ADN Complementario/genética , Desoxirribonucleasa BamHI/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Vectores Genéticos/genética , Humanos , Reacción en Cadena de la Polimerasa/métodos , Proteínas Recombinantes/genética
19.
PLoS One ; 13(7): e0201605, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30063768

RESUMEN

The identification of inhibitors of eukaryotic protein biosynthesis, which are targeting single translation factors, is highly demanded. Here we report on a small molecule inhibitor, gephyronic acid, isolated from the myxobacterium Archangium gephyra that inhibits growth of transformed mammalian cell lines in the nM range. In direct comparison, primary human fibroblasts were shown to be less sensitive to toxic effects of gephyronic acid than cancer-derived cells. Gephyronic acid is targeting the protein translation system. Experiments with IRES dual luciferase reporter assays identified it as an inhibitor of the translation initiation. DARTs approaches, co-localization studies and pull-down assays indicate that the binding partner could be the eukaryotic initiation factor 2 subunit alpha (eIF2α). Gephyronic acid seems to have a different mode of action than the structurally related polyketides tedanolide, myriaporone, and pederin and is a valuable tool for investigating the eukaryotic translation system. Because cancer derived cells were found to be especially sensitive, gephyronic acid could potentially find use as a drug candidate.


Asunto(s)
Factor 2 Eucariótico de Iniciación/antagonistas & inhibidores , Myxococcales/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , Ácidos Grasos Monoinsaturados/farmacología , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Técnicas Microbiológicas , Myxococcales/genética , Myxococcales/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Transducción de Señal/efectos de los fármacos
20.
Curr Opin Chem Biol ; 10(1): 4-10, 2006 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-16376134

RESUMEN

Technological innovations and novel applications have greatly advanced the field of protein microarrays. Over the past two years, different types of protein microarrays have been used for serum profiling, protein abundance determinations, and identification of proteins that bind DNA or small compounds. However, considerable development is still required to ensure common quality standards and to establish large content repertoires. Here, we summarize applications available to date and discuss recent technological achievements and efforts on standardization.


Asunto(s)
Análisis por Matrices de Proteínas/métodos , Animales , Anticuerpos/química , Anticuerpos/inmunología , Reacciones Antígeno-Anticuerpo , Humanos , Inmovilización , Análisis por Matrices de Proteínas/normas , Sensibilidad y Especificidad
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