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1.
J Biol Chem ; 295(36): 12755-12771, 2020 09 04.
Artículo en Inglés | MEDLINE | ID: mdl-32719005

RESUMEN

Collagen VI is a ubiquitous heterotrimeric protein of the extracellular matrix (ECM) that plays an essential role in the proper maintenance of skeletal muscle. Mutations in collagen VI lead to a spectrum of congenital myopathies, from the mild Bethlem myopathy to the severe Ullrich congenital muscular dystrophy. Collagen VI contains only a short triple helix and consists primarily of von Willebrand factor type A (VWA) domains, protein-protein interaction modules found in a range of ECM proteins. Disease-causing mutations occur commonly in the VWA domains, and the second VWA domain of the α3 chain, the N2 domain, harbors several such mutations. Here, we investigate structure-function relationships of the N2 mutations to shed light on their possible myopathy mechanisms. We determined the X-ray crystal structure of N2, combined with monitoring secretion efficiency in cell culture of selected N2 single-domain mutants, finding that mutations located within the central core of the domain severely affect secretion efficiency. In longer α3 chain constructs, spanning N6-N3, small-angle X-ray scattering demonstrates that the tandem VWA array has a modular architecture and samples multiple conformations in solution. Single-particle EM confirmed the presence of multiple conformations. Structural adaptability appears intrinsic to the VWA domain region of collagen VI α3 and has implications for binding interactions and modulating stiffness within the ECM.


Asunto(s)
Colágeno Tipo VI/química , Enfermedades Musculares , Mutación , Colágeno Tipo VI/genética , Cristalografía por Rayos X , Humanos , Dominios Proteicos
2.
J Biol Chem ; 292(29): 12208-12219, 2017 07 21.
Artículo en Inglés | MEDLINE | ID: mdl-28578314

RESUMEN

Toxoplasma gondii is an obligate, intracellular eukaryotic apicomplexan protozoan parasite that can cause fetal damage and abortion in both animals and humans. Sphingolipids are essential and ubiquitous components of eukaryotic membranes that are both synthesized and scavenged by the Apicomplexa. Here we report the identification, isolation, and analyses of the Toxoplasma serine palmitoyltransferase, an enzyme catalyzing the first and rate-limiting step in sphingolipid biosynthesis: the condensation of serine and palmitoyl-CoA. In all eukaryotes analyzed to date, serine palmitoyltransferase is a highly conserved heterodimeric enzyme complex. However, biochemical and structural analyses demonstrated the apicomplexan orthologue to be a functional, homodimeric serine palmitoyltransferase localized to the endoplasmic reticulum. Furthermore, phylogenetic studies indicated that it was evolutionarily related to the prokaryotic serine palmitoyltransferase, identified in the Sphingomonadaceae as a soluble homodimeric enzyme. Therefore this enzyme, conserved throughout the Apicomplexa, is likely to have been obtained via lateral gene transfer from a prokaryote.


Asunto(s)
Retículo Endoplásmico/enzimología , Modelos Moleculares , Filogenia , Proteínas Protozoarias/metabolismo , Serina C-Palmitoiltransferasa/metabolismo , Toxoplasma/enzimología , Secuencia de Aminoácidos , Dominio Catalítico , Biología Computacional , Secuencia Conservada , Dimerización , Eliminación de Gen , Duplicación de Gen , Transferencia de Gen Horizontal , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Transporte de Proteínas , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , Proteínas Protozoarias/aislamiento & purificación , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Serina C-Palmitoiltransferasa/química , Serina C-Palmitoiltransferasa/genética , Serina C-Palmitoiltransferasa/aislamiento & purificación , Homología Estructural de Proteína
3.
Anal Biochem ; 556: 23-34, 2018 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-29908863

RESUMEN

Biophysical screening techniques, such as surface plasmon resonance, enable detailed kinetic analysis of ligands binding to solubilised G-protein coupled receptors. The activity of a receptor solubilised out of the membrane is crucially dependent on the environment in which it is suspended. Finding the right conditions is challenging due to the number of variables to investigate in order to determine the optimum solubilisation buffer for any given receptor. In this study we used surface plasmon resonance technology to screen a variety of solubilisation conditions including buffers and detergents for two model receptors: CXCR4 and CCR5. We tested 950 different combinations of solubilisation conditions for both receptors. The activity of both receptors was monitored by using conformation dependent monoclonal antibodies and the binding of small molecule ligands. Despite both receptors belonging to the chemokine receptor family they show some differences in their preference for solubilisation conditions that provide the highest level of binding for both the conformation dependent antibodies and small molecules. The study described here is focused not only on finding the best solubilisation conditions for each receptor, but also on factors that determine the sensitivity of the assay for each receptor. We also suggest how these data about different buffers and detergents can be used as a guide for selecting solubilisation conditions for other membrane proteins.


Asunto(s)
Anticuerpos Monoclonales/química , Receptores CCR5/análisis , Receptores CXCR4/análisis , Resonancia por Plasmón de Superficie/métodos , Humanos , Solubilidad
4.
Mol Microbiol ; 102(3): 365-385, 2016 11.
Artículo en Inglés | MEDLINE | ID: mdl-27426054

RESUMEN

Thymidine kinase (TK) is a key enzyme in the pyrimidine salvage pathway which catalyzes the transfer of the γ-phosphate of ATP to 2'-deoxythymidine (dThd) forming thymidine monophosphate (dTMP). Unlike other type II TKs, the Trypanosoma brucei enzyme (TbTK) is a tandem protein with two TK homolog domains of which only the C-terminal one is active. In this study, we establish that TbTK is essential for parasite viability and cell cycle progression, independently of extracellular pyrimidine concentrations. We show that expression of TbTK is cell cycle regulated and that depletion of TbTK leads to strongly diminished dTTP pools and DNA damage indicating intracellular dThd to be an essential intermediate metabolite for the synthesis of thymine-derived nucleotides. In addition, we report the X-ray structure of the catalytically active domain of TbTK in complex with dThd and dTMP at resolutions up to 2.2 Å. In spite of the high conservation of the active site residues, the structures reveal a widened active site cavity near the nucleobase moiety compared to the human enzyme. Our findings strongly support TbTK as a crucial enzyme in dTTP homeostasis and identify structural differences within the active site that could be exploited in the process of rational drug design.


Asunto(s)
Timidina Quinasa/metabolismo , Trypanosoma brucei brucei/citología , Trypanosoma brucei brucei/enzimología , Puntos de Control del Ciclo Celular/fisiología , Nucleósido-Fosfato Quinasa/metabolismo , Relación Estructura-Actividad , Timidina/metabolismo , Timidina Quinasa/química , Timidina Monofosfato/metabolismo , Nucleótidos de Timina/metabolismo , Trypanosoma brucei brucei/metabolismo
5.
Adv Exp Med Biol ; 922: 1-11, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27553231

RESUMEN

The production of recombinant integral membrane proteins for structural and functional studies remains technically challenging due to their relatively low levels of expression. To address this problem, screening strategies have been developed to identify the optimal membrane sequence and expression host for protein production. A common approach is to genetically fuse the membrane protein to a fluorescent reporter, typically Green Fluorescent Protein (GFP) enabling expression levels, localization and detergent solubilisation to be assessed. Initially developed for screening the heterologous expression of bacterial membrane proteins in Escherichia coli, the method has been extended to eukaryotic hosts, including insect and mammalian cells. Overall, GFP-based expression screening has made a major impact on the number of membrane protein structures that have been determined in the last few years.


Asunto(s)
Genes Reporteros , Proteínas Luminiscentes/análisis , Proteínas de la Membrana/análisis , Animales , Células Cultivadas , Escherichia coli/metabolismo , Células Eucariotas/metabolismo , Expresión Génica , Proteínas Fluorescentes Verdes/análisis , Células HEK293/metabolismo , Humanos , Insectos/citología , Proteínas Luminiscentes/genética , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Levaduras/metabolismo
6.
BMC Biotechnol ; 14: 92, 2014 Nov 14.
Artículo en Inglés | MEDLINE | ID: mdl-25394427

RESUMEN

BACKGROUND: Neurotrophic factors influence survival, differentiation, proliferation and death of neuronal cells within the central nervous system. Human ciliary neurotrophic factor (hCNTF) has neuroprotective properties and is also known to influence energy balance. Consequently, hCNTF has potential therapeutic applications in neurodegenerative, obesity and diabetes related disorders. Clinical and biological applications of hCNTF necessitate a recombinant expression system to produce large amounts of functional protein in soluble form. Earlier attempts to express hCNTF in Escherichia coli (E. coli) were limited by low amounts and the need to refold from inclusion bodies. RESULTS: In this report, we describe a strategy to effectively identify constructs and conditions for soluble expression of hCNTF in E. coli. Small-scale expression screening with soluble fusion tags identified many conditions that yielded soluble expression. Codon optimized 6-His-hCNTF construct showed soluble expression in all the conditions tested. Large-scale culture of the 6-His-hCNTF construct yielded high (10 - 20 fold) soluble expression (8 - 9 fold) as compared to earlier published reports. Functional activity of recombinant 6-His-hCNTF produced was confirmed by its binding to hCNTF receptor (hCNTFRα) with an EC50 = 36 nM. CONCLUSION: Our results highlight the combination of codon optimization and screening soluble fusion tags as a successful strategy for high yielding soluble expression of hCNTF in E. coli. Codon optimization of the hCNTF sequence seems to be sufficient for soluble expression of hCNTF. The combined approach of codon optimization and soluble fusion tag screen can be an effective strategy for soluble expression of pharmaceutical proteins in E. coli.


Asunto(s)
Factor Neurotrófico Ciliar/genética , Codón , Expresión Génica , Ingeniería de Proteínas/métodos , Factor Neurotrófico Ciliar/química , Factor Neurotrófico Ciliar/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Solubilidad
7.
J Biol Chem ; 287(52): 43246-61, 2012 Dec 21.
Artículo en Inglés | MEDLINE | ID: mdl-23132860

RESUMEN

Inside-out activation of integrins is mediated via the binding of talin and kindlin to integrin ß-subunit cytoplasmic tails. The kindlin FERM domain is interrupted by a pleckstrin homology (PH) domain within its F2 subdomain. Here, we present data confirming the importance of the kindlin-1 PH domain for integrin activation and its x-ray crystal structure at a resolution of 2.1 Å revealing a C-terminal second α-helix integral to the domain but found only in the kindlin protein family. An isoform-specific salt bridge occludes the canonical phosphoinositide binding site, but molecular dynamics simulations display transient switching to an alternative open conformer. Molecular docking reveals that the opening of the pocket would enable potential ligands to bind within it. Although lipid overlay assays suggested the PH domain binds inositol monophosphates, surface plasmon resonance demonstrated weak affinities for inositol 3,4,5-triphosphate (Ins(3,4,5)P(3); K(D) ∼100 µM) and no monophosphate binding. Removing the salt bridge by site-directed mutagenesis increases the PH domain affinity for Ins(3,4,5)P(3) as measured by surface plasmon resonance and enables it to bind PtdIns(3,5)P(2) on a dot-blot. Structural comparison with other PH domains suggests that the phosphate binding pocket in the kindlin-1 PH domain is more occluded than in kindlins-2 and -3 due to its salt bridge. In addition, the apparent affinity for Ins(3,4,5)P(3) is affected by the presence of PO(4) ions in the buffer. We suggest the physiological ligand of the kindlin-1 PH domain is most likely not an inositol phosphate but another phosphorylated species.


Asunto(s)
Proteínas Portadoras/química , Simulación de Dinámica Molecular , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Cristalografía por Rayos X , Ratones , Mutagénesis , Fosfatos/química , Fosfatos/metabolismo , Fosfatos de Fosfatidilinositol/química , Fosfatos de Fosfatidilinositol/genética , Fosfatos de Fosfatidilinositol/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Relación Estructura-Actividad
8.
Acta Crystallogr D Biol Crystallogr ; 69(Pt 6): 1090-8, 2013 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-23695253

RESUMEN

Uridine at position 34 of bacterial transfer RNAs is commonly modified to uridine-5-oxyacetic acid (cmo(5)U) to increase the decoding capacity. The protein CmoA is involved in the formation of cmo(5)U and was annotated as an S-adenosyl-L-methionine-dependent (SAM-dependent) methyltransferase on the basis of its sequence homology to other SAM-containing enzymes. However, both the crystal structure of Escherichia coli CmoA at 1.73 Å resolution and mass spectrometry demonstrate that it contains a novel cofactor, S-adenosyl-S-carboxymethyl-L-homocysteine (SCM-SAH), in which the donor methyl group is substituted by a carboxymethyl group. The carboxyl moiety forms a salt-bridge interaction with Arg199 that is conserved in a large group of CmoA-related proteins but is not conserved in other SAM-containing enzymes. This raises the possibility that a number of enzymes that have previously been annotated as SAM-dependent are in fact SCM-SAH-dependent. Indeed, inspection of electron density for one such enzyme with known X-ray structure, PDB entry 1im8, suggests that the active site contains SCM-SAH and not SAM.


Asunto(s)
Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Transferasas del Grupo 1-Carbono/química , ARN de Transferencia/metabolismo , S-Adenosilhomocisteína/metabolismo , Cristalografía por Rayos X , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Transferasas del Grupo 1-Carbono/metabolismo , Espectrometría de Masas en Tándem
9.
Blood ; 117(25): 6928-38, 2011 Jun 23.
Artículo en Inglés | MEDLINE | ID: mdl-21364188

RESUMEN

Congenital dyserythropoietic anemia type 1 (CDA-1), a rare inborn anemia characterized by abnormal chromatin ultrastructure in erythroblasts, is caused by abnormalities in codanin-1, a highly conserved protein of unknown function. We have produced 3 monoclonal antibodies to codanin-1 that demonstrate its distribution in both nucleus and cytoplasm by immunofluorescence and allow quantitative measurements of patient and normal material by Western blot. A detailed analysis of chromatin structure in CDA-1 erythroblasts shows no abnormalities in overall histone composition, and the genome-wide epigenetic landscape of several histone modifications is maintained. However, immunofluorescence analysis of intermediate erythroblasts from patients with CDA-1 reveals abnormal accumulation of HP1α in the Golgi apparatus. A link between mutant codanin-1 and the aberrant localization of HP1α is supported by the finding that codanin-1 can be coimmunoprecipitated by anti-HP1α antibodies. Furthermore, we show colocalization of codanin-1 with Sec23B, the protein defective in CDA-2 suggesting that the CDAs might be linked at the molecular level. Mice containing a gene-trapped Cdan1 locus demonstrate its widespread expression during development. Cdan1(gt/gt) homozygotes die in utero before the onset of primitive erythropoiesis, suggesting that Cdan1 has other critical roles during embryogenesis.


Asunto(s)
Anemia Diseritropoyética Congénita/genética , Anemia Diseritropoyética Congénita/patología , Proteínas Cromosómicas no Histona/análisis , Eritroblastos/patología , Glicoproteínas/genética , Mutación , Animales , Proteínas Portadoras/genética , Línea Celular Tumoral , Células Cultivadas , Cromatina/patología , Homólogo de la Proteína Chromobox 5 , Eritroblastos/metabolismo , Femenino , Glicoproteínas/análisis , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Nucleares , Proteínas de Transporte Vesicular/análisis
10.
Methods ; 55(1): 29-37, 2011 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-21856427

RESUMEN

A suite of protein fusion vectors is presented that has been designed so that nine separate fusion vectors can be constructed from one PCR product using InFusion™ cloning. These vectors in combination with a small scale Escherichia coli expression screen can be used to assess in parallel the effect of fusion tags on solubility. The vectors were tested with 20 target proteins and the results suggest that the vectors are useful both as a rescue strategy if the N-terminal hexa-histidine tagged construct does not express and also as part of a primary expression experiment.


Asunto(s)
Escherichia coli/genética , Expresión Génica , Ensayos Analíticos de Alto Rendimiento , Biología Molecular/métodos , Proteínas Recombinantes de Fusión/biosíntesis , Animales , Secuencia de Bases , Clonación Molecular , Escherichia coli/metabolismo , Vectores Genéticos/química , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Histidina/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Neisseria meningitidis , Oligopéptidos/metabolismo , Pseudomonas aeruginosa , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Solubilidad
11.
Acta Crystallogr Sect F Struct Biol Cryst Commun ; 68(Pt 12): 1427-33, 2012 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-23192019

RESUMEN

The structure of ribose 5-phosphate isomerase from the probiotic bacterium Lactobacillus salivarius UCC188 has been determined at 1.72 Šresolution. The structure was solved by molecular replacement, which identified the functional homodimer in the asymmetric unit. Despite only showing 57% sequence identity to its closest homologue, the structure adopted the typical α and ß D-ribose 5-phosphate isomerase fold. Comparison to other related structures revealed high homology in the active site, allowing a model of the substrate-bound protein to be proposed. The determination of the structure was expedited by the use of in situ crystallization-plate screening on beamline I04-1 at Diamond Light Source to identify well diffracting protein crystals prior to routine cryocrystallography.


Asunto(s)
Isomerasas Aldosa-Cetosa/química , Lactobacillus/enzimología , Isomerasas Aldosa-Cetosa/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Dimerización , Datos de Secuencia Molecular , Conformación Proteica , Pliegue de Proteína , Alineación de Secuencia
12.
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
13.
PLoS One ; 16(10): e0256070, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34653190

RESUMEN

Nontypeable Haemophilus influenzae (NTHi) is a significant pathogen in respiratory disease and otitis media. Important for NTHi survival, colonization and persistence in vivo is the Sap (sensitivity to antimicrobial peptides) ABC transporter system. Current models propose a direct role for Sap in heme and antimicrobial peptide (AMP) transport. Here, the crystal structure of SapA, the periplasmic component of Sap, in a closed, ligand bound conformation, is presented. Phylogenetic and cavity volume analysis predicts that the small, hydrophobic SapA central ligand binding cavity is most likely occupied by a hydrophobic di- or tri- peptide. The cavity is of insufficient volume to accommodate heme or folded AMPs. Crystal structures of SapA have identified surface interactions with heme and dsRNA. Heme binds SapA weakly (Kd 282 µM) through a surface exposed histidine, while the dsRNA is coordinated via residues which constitute part of a conserved motif (estimated Kd 4.4 µM). The RNA affinity falls within the range observed for characterized RNA/protein complexes. Overall, we describe in molecular-detail the interactions of SapA with heme and dsRNA and propose a role for SapA in the transport of di- or tri-peptides.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Proteínas Portadoras/metabolismo , Haemophilus influenzae/metabolismo , Hemo/metabolismo , ARN Bicatenario/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Antibacterianos/farmacología , Proteínas Portadoras/genética , Cristalografía por Rayos X , Farmacorresistencia Bacteriana Múltiple/genética , Infecciones por Haemophilus/microbiología , Infecciones por Haemophilus/patología , Haemophilus influenzae/efectos de los fármacos , Haemophilus influenzae/genética , Otitis Media/microbiología , Otitis Media/patología , Conformación Proteica , Transporte de Proteínas/fisiología , ARN Bicatenario/genética , Motivos de Unión al ARN/genética , Factores de Virulencia/metabolismo
14.
BMC Struct Biol ; 10: 13, 2010 May 28.
Artículo en Inglés | MEDLINE | ID: mdl-20509910

RESUMEN

BACKGROUND: Zhx1 to 3 (zinc-fingers and homeoboxes) form a set of paralogous genes encoding multi-domain proteins. ZHX proteins consist of two zinc fingers followed by five homeodomains. ZHXs have biological roles in cell cycle control by acting as co-repressors of the transcriptional regulator Nuclear Factor Y. As part of a structural genomics project we have expressed single and multi-domain fragments of the different human ZHX genes for use in structure determination. RESULTS: A total of 30 single and multiple domain ZHX1-3 constructs selected from bioinformatics protocols were screened for soluble expression in E. coli using high throughput methodologies. Two homeodomains were crystallized leading to structures for ZHX1 HD4 and ZHX2 HD2. ZHX1 HD4, although closest matched to homeodomains from 'homez' and 'engrailed', showed structural differences, notably an additional C-terminal helix (helix V) which wrapped over helix I thereby making extensive contacts. Although ZHX2 HD2-3 was successfully expressed and purified, proteolysis occurred during crystallization yielding crystals of just HD2. The structure of ZHX2 HD2 showed an unusual open conformation with helix I undergoing 'domain-swapping' to form a homodimer. CONCLUSIONS: Although multiple-domain constructs of ZHX1 selected by bioinformatics studies could be expressed solubly, only single homeodomains yielded crystals. The crystal structure of ZHX1 HD4 showed additional hydrophobic interactions relative to many known homeodomains via extensive contacts formed by the novel C-terminal helix V with, in particular, helix I. Additionally, the replacement of some charged covariant residues (which are commonly observed to form salt bridges in non-homeotherms such as the Drosophila 'engrailed' homeodomain), by apolar residues further increases hydrophobic contacts within ZHX1 HD4, and potentially stability, relative to engrailed homeodomain. ZHX1 HD4 helix V points away from the normally observed DNA major groove binding site on homeodomains and thus would not obstruct the putative binding of nucleic acid. In contrast, for ZHX2 HD2 the observed altered conformation involving rearrangement of helix I, relative to the canonical homeodomain fold, disrupts the normal DNA binding site, although protein-protein binding is possible as observed in homodimer formation.


Asunto(s)
Proteínas de Homeodominio/química , Factores de Transcripción/química , Secuencia de Aminoácidos , Biología Computacional , Cristalografía por Rayos X , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
15.
Cell Biol Toxicol ; 26(1): 57-68, 2010 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-19655260

RESUMEN

Generating large amounts of recombinant protein in transgenic animals is often challenging and has a number of drawbacks compared to cell culture systems. The baculovirus expression vector system (BEVS) uses virus-infected insect cells to produce recombinant proteins to high levels, and these are usually processed in a similar way to the native protein. Interestingly, since the development of the BEVS, the virus most often used (Autographa californica multi-nucleopolyhedovirus; AcMNPV) has been little altered genetically from its wild-type parental virus. In this study, we modified the AcMNPV genome in an attempt to improve recombinant protein yield, by deleting genes that are non-essential in cell culture. We deleted the p26, p10 and p74 genes from the virus genome, replacing them with an antibiotic selection cassette, allowing us to isolate recombinants. We screened and identified recombinant viruses by restriction enzyme analysis, PCR and Western blot. Cell viability analysis showed that the deletions did not improve the viability of infected cells, compared to non-deletion viruses. However, expression studies showed that recombinant protein levels for the deletion viruses were significantly higher than the expression levels of non-deletion viruses. These results confirm that there is still great potential for improving the BEVS, further increasing recombinant protein expression yields and stability in insect cells.


Asunto(s)
Baculoviridae/genética , Eliminación de Gen , Genes Virales , Ingeniería Genética/métodos , Vectores Genéticos , Proteínas Recombinantes/biosíntesis , Animales , Línea Celular , Expresión Génica , Insectos , Regulación hacia Arriba
16.
Biotechnol Appl Biochem ; 56(3): 85-93, 2010 Jun 21.
Artículo en Inglés | MEDLINE | ID: mdl-20441568

RESUMEN

Secretory and membrane-bound proteins are generally produced in lower amounts in insect cells compared with cytoplasmic and nuclear proteins. There may be many reasons for this, including degradation of recombinant proteins by proteases, competition for cellular resources between native and recombinant proteins, and physical blockage of the secretory pathways. In the present study, we describe the construction of a baculovirus in which chiA (chitinase) and cath (cathepsin) genes have been deleted and show improved recombinant protein expression using this vector. We confirmed the complete removal of both genes by PCR, restriction enzyme analysis and enzyme assays, and the modified virus DNA was shown to be stable in bacterial cells over multiple passages. A selection of recombinant genes were inserted into the double-deletion virus and their expression levels compared with recombinant viruses that had single or no gene deletions. In all instances, the double-deletion viruses showed greatly enhanced levels of protein production for both secreted and nuclear/cytoplasmic proteins. In summary, we have conclusively demonstrated the importance of this deletion vector for the high-level production of recombinant proteins.


Asunto(s)
Baculoviridae/genética , Proteínas de la Membrana/biosíntesis , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/biosíntesis , Animales , Baculoviridae/enzimología , Catepsinas/genética , Células Cultivadas , Quitinasas/genética , Eliminación de Gen , Expresión Génica , Humanos , Insectos/citología , Proteínas de la Membrana/genética
17.
Sci Rep ; 9(1): 6843, 2019 05 02.
Artículo en Inglés | MEDLINE | ID: mdl-31048732

RESUMEN

Outer membrane vesicle (OMV)- based vaccines have been used to provide strain-specific protection against capsular group B Neisseria meningitidis infections, but the full breadth of the immune response against the components of the OMV has not been established. Sera from adults vaccinated with an OMV vaccine were used to screen 91 outer membrane proteins (OMPs) incorporated in an antigen microarray panel. Antigen-specific IgG levels were quantified pre-vaccination, and after 12 and 18 weeks. These results were compared with IgG levels from mice vaccinated with the same OMV vaccine. The repertoires of highly responding antigens in humans and mice overlapped, but were not identical. The highest responding antigens to human IgG comprised four integral OMPs (PorA, PorB, OpcA and PilQ), a protein which promotes the stability of PorA and PorB (RmpM) and two lipoproteins (BamC and GNA1162). These observations will assist in evaluating the role of minor antigen components within OMVs in providing protection against meningococcal infection. In addition, the relative dominance of responses to integral OMPs in humans emphasizes the importance of this subclass and points to the value of maintaining conformational epitopes from integral membrane proteins in vaccine formulations.


Asunto(s)
Antígenos Bacterianos/inmunología , Vacunas Bacterianas/uso terapéutico , Neisseria meningitidis Serogrupo B/inmunología , Adolescente , Adulto , Animales , Vacunas Bacterianas/inmunología , Cromatografía en Gel , Femenino , Humanos , Inmunoglobulina G/inmunología , Masculino , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Porinas/inmunología , Porinas/metabolismo , Adulto Joven
18.
Mol Biochem Parasitol ; 231: 111187, 2019 07.
Artículo en Inglés | MEDLINE | ID: mdl-31103556

RESUMEN

Nucleoside diphosphate kinases (NDPKs) are crucial to keep the high triphosphate nucleotide levels in the biological process. The enzymatic mechanism has been extensively described; however, the structural characteristics and kinetic parameters have never been fully determined. In Schistosoma mansoni, NDPK (SmNDPK) is directly involved in the pyrimidine and purine salvage pathways, being essential for nucleotide metabolism. The SmNDPK enzymatic activity is the highest of the known purine metabolisms when compared to the mammalian NDPKs, suggesting the importance of this enzyme in the worm metabolism. Here, we report the recombinant expression of SmNDPK that resulted in 1.7 and 1.9 Å apo-form structure in different space-groups, as well as the 2.1 Å SmNDPK.ADP complex. The binding and kinetic assays reveal the ATP-dependence for enzyme activation. Moreover, in situ hybridization showed that SmNDPK transcripts are found in reproductive organs and in the esophagus gland of adult worms, which can be intrinsically related with the oviposition and digestive processes. These results will help us fully understand the crucial participation of this enzyme in Schistosoma mansoni and its importance for the pathology of the disease.


Asunto(s)
Proteínas del Helminto/química , Proteínas del Helminto/metabolismo , Nucleósido-Difosfato Quinasa/química , Nucleósido-Difosfato Quinasa/metabolismo , Schistosoma mansoni/enzimología , Esquistosomiasis mansoni/parasitología , Adenosina Difosfato/química , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Dominio Catalítico , Esófago/química , Esófago/enzimología , Femenino , Tracto Gastrointestinal/química , Tracto Gastrointestinal/enzimología , Proteínas del Helminto/genética , Humanos , Cinética , Masculino , Modelos Moleculares , Nucleósido-Difosfato Quinasa/genética , Schistosoma mansoni/genética , Schistosoma mansoni/metabolismo , Alineación de Secuencia
19.
Mol Biochem Parasitol ; 229: 24-34, 2019 04.
Artículo en Inglés | MEDLINE | ID: mdl-30772423

RESUMEN

Schistosoma mansoni, the parasite responsible for schistosomiasis, lacks the "de novo" purine biosynthetic pathway and depends entirely on the purine salvage pathway for the supply of purines. Numerous reports of praziquantel resistance have been described, as well as stimulated efforts to develop new drugs against schistosomiasis. Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is a key enzyme of the purine salvage pathway. Here, we describe a crystallographic structure of the S. mansoni HPGRT-1 (SmHGPRT), complexed with IMP at a resolution of 2.8 Ǻ. Four substitutions were identified in the region of the active site between SmHGPRT-1 and human HGPRT. We also present data from RNA-Seq and WISH, suggesting that some isoforms of HGPRT might be involved in the process related to sexual maturation and reproduction in worms; furthermore, its enzymatic assays show that the isoform SmHGPRT-3 does not present the same catalytic efficiency as other isoforms. Finally, although other studies have previously suggested this enzyme as a potential antischistosomal chemotherapy target, the kinetics parameters reveal the impossibility to use SmHGPRT as an efficient chemotherapeutic target.


Asunto(s)
Proteínas del Helminto/química , Proteínas del Helminto/genética , Hipoxantina Fosforribosiltransferasa/química , Hipoxantina Fosforribosiltransferasa/genética , Isoenzimas/química , Isoenzimas/genética , Schistosoma mansoni/enzimología , Secuencia de Aminoácidos , Animales , Dominio Catalítico , Proteínas del Helminto/metabolismo , Hipoxantina Fosforribosiltransferasa/metabolismo , Isoenzimas/metabolismo , Cinética , Datos de Secuencia Molecular , Reproducción , Schistosoma mansoni/química , Schistosoma mansoni/genética , Schistosoma mansoni/fisiología , Alineación de Secuencia
20.
FEBS Lett ; 581(16): 2959-64, 2007 Jun 26.
Artículo en Inglés | MEDLINE | ID: mdl-17544401

RESUMEN

Dominant mutations in the ubiquitous enzyme glycyl-tRNA synthetase (GlyRS), including S581L, lead to motor nerve degeneration. We have determined crystal structures of wildtype and S581L-mutant human GlyRS. The S581L mutation is approximately 50A from the active site, and yet gives reduced aminoacylation activity. The overall structures of wildtype and S581L-GlyRS, including the active site, are very similar. However, residues 567-575 of the anticodon-binding domain shift position and in turn could indirectly affect glycine binding via the tRNA or alternatively inhibit conformational changes. Reduced enzyme activity may underlie neuronal degeneration, although a dominant-negative effect is more likely in this autosomal dominant disorder.


Asunto(s)
Miopatías Distales/enzimología , Glicina-ARNt Ligasa/química , Atrofia Muscular Espinal/enzimología , Sustitución de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Dimerización , Miopatías Distales/genética , Glicina-ARNt Ligasa/genética , Glicina-ARNt Ligasa/metabolismo , Humanos , Leucina/química , Modelos Moleculares , Atrofia Muscular Espinal/genética , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutación Missense , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Serina/química , Aminoacilación de ARN de Transferencia/genética
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