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1.
PLoS Comput Biol ; 16(12): e1007578, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-33270624

RESUMEN

Phosphorylation of specific substrates by protein kinases is a key control mechanism for vital cell-fate decisions and other cellular processes. However, discovering specific kinase-substrate relationships is time-consuming and often rather serendipitous. Computational predictions alleviate these challenges, but the current approaches suffer from limitations like restricted kinome coverage and inaccuracy. They also typically utilise only local features without reflecting broader interaction context. To address these limitations, we have developed an alternative predictive model. It uses statistical relational learning on top of phosphorylation networks interpreted as knowledge graphs, a simple yet robust model for representing networked knowledge. Compared to a representative selection of six existing systems, our model has the highest kinome coverage and produces biologically valid high-confidence predictions not possible with the other tools. Specifically, we have experimentally validated predictions of previously unknown phosphorylations by the LATS1, AKT1, PKA and MST2 kinases in human. Thus, our tool is useful for focusing phosphoproteomic experiments, and facilitates the discovery of new phosphorylation reactions. Our model can be accessed publicly via an easy-to-use web interface (LinkPhinder).


Asunto(s)
Proteínas Quinasas/metabolismo , Simulación por Computador , Humanos , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal , Especificidad por Sustrato
2.
Proteomics ; 20(19-20): e2000062, 2020 10.
Artículo en Inglés | MEDLINE | ID: mdl-32864787

RESUMEN

Expression of the macrophage immunometabolism regulator gene (MACIR) is associated with severity of autoimmune disease pathology and with the regulation of macrophage biology through unknown mechanisms. The encoded 206 amino acid protein lacks homology to any characterized protein sequence and is a disordered protein according to structure prediction algorithms. To identify interactions of MACIR with proteins from all subcellular compartments, a membrane solubilization buffer is employed, that together with a high affinity EF hand based pull down method, increases the resolution of quantitative mass spectrometry analysis with significant enrichment of interactions from membrane bound nuclear and mitochondrial compartments compared to samples prepared with radioimmunoprecipitation assay buffer. A total of 63 significant interacting proteins are identified and interaction with the nuclear transport receptor TNPO1 and the trafficking proteins UNC119 homolog A and B are validated by immunoprecipitation. Mutational analysis in two candidate nuclear localization signal motifs in the MACIR amino acid sequence shows the interaction with TNPO1 is likely via a non-classical proline/tyrosine-nuclear localization signal motif (aa98-117). It is shown that employing a highly specific and high affinity pull down method that performs efficiently in this glycerol and detergent rich buffer is a powerful approach for the analysis of uncharacterized protein interactomes.


Asunto(s)
Macrófagos , Proteínas de la Membrana , Proteómica , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Humanos , Inmunoprecipitación , beta Carioferinas
3.
Biochemistry ; 57(30): 4383-4390, 2018 07 31.
Artículo en Inglés | MEDLINE | ID: mdl-29956929

RESUMEN

The process of isolating recombinant G protein-coupled receptors from membrane preparations is challenging because the process requires solubilization in detergent micelles and multistep affinity chromatography protocols. Solubilization buffers contain high concentrations of salts, detergents, and glycerol that create stringent conditions necessary to stabilize the receptor but in which affinity chromatography resins perform poorly, and these resins also require the addition of eluting agents that complicate downstream assays. To simplify this process we have developed a high affinity fragment complementation molecular switch as a highly specific system for receptor capture in solubilization buffer with a calcium chelation-based elution step releasing functional protein in a simple buffer. Here we describe in detail the design, methodology, interpretation, and limitations of this novel affinity chromatography system in the isolation and purification of the cannabinoid G protein-coupled receptor CB2, in comparison with commercially available systems. This powerful tool may be applied to any recombinant membrane bound protein and can be further optimized to enhance the yield and purity of the most challenging protein targets for study.


Asunto(s)
Quelantes del Calcio/química , Cromatografía de Afinidad/métodos , Receptor Cannabinoide CB2/aislamiento & purificación , Animales , Anticuerpos Monoclonales/química , Tampones (Química) , Calcio/química , Escherichia coli/genética , Humanos , Liposomas/química , Ratones , Modelos Moleculares , Conejos , Receptor Cannabinoide CB2/genética , Receptor Cannabinoide CB2/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Solubilidad
4.
Methods Mol Biol ; 2095: 385-396, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31858480

RESUMEN

Purification of proteins for the biophysical analysis of protein interactions occurring in human cells can benefit from methods that facilitate the capture of small amounts of natively processed protein obtained using transient mammalian expression systems. We have used a novel calcium-dependent fragment complementation-based affinity method to effectively purify full length glycogen synthase kinase 3 (GSK3) α and ß isoforms to study their interaction with amyloid ß peptide (Aß42). Using these proteins, purified from 1 mg of total cell lysate, we measured an apparent KD of ≤100 pM between GSK3α/ß and immobilized Aß42 with surface plasmon resonance technology. This approach can be used to retrieve useful quantities of protein for biophysical experiments with small scale mammalian cell culture.


Asunto(s)
Péptidos beta-Amiloides/metabolismo , Calcio/metabolismo , Motivos EF Hand , Glucógeno Sintasa Quinasas/aislamiento & purificación , Calcio/química , Expresión Génica , Glucógeno Sintasa Quinasa 3/aislamiento & purificación , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta/aislamiento & purificación , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Glucógeno Sintasa Quinasas/química , Glucógeno Sintasa Quinasas/genética , Glucógeno Sintasa Quinasas/metabolismo , Células HEK293 , Humanos , Isoformas de Proteínas/genética , Isoformas de Proteínas/aislamiento & purificación , Isoformas de Proteínas/metabolismo , Resonancia por Plasmón de Superficie
5.
ACS Chem Neurosci ; 7(2): 161-70, 2016 Feb 17.
Artículo en Inglés | MEDLINE | ID: mdl-26618561

RESUMEN

Amyloid ß peptide (Aß42) assemblies are considered central to the development of Alzheimer's disease, but the mechanism of this toxicity remains unresolved. We screened protein microarrays with on-pathway oligomeric Aß42 to identify candidate proteins interacting with toxic Aß42 species. Samples prepared from Alexa546-Aß42 and Aß42 monomers at 1:5 molar ratio were incubated with the array during a time window of the amyloid fibril formation reaction during which the maximum number of transient oligomers exist in the reaction flux. A specific interaction was detected between Aß42 and glycogen synthase kinase 3α (GSK3α), a kinase previously implicated in the disease pathology. This interaction was validated with anti-GSK3α immunoprecipitation assays in neuronal cell lysates. Confocal microscopy studies further identified colocalization of Aß42 and GSK3α in neurites of mature primary mouse neurons. A high binding affinity (KD = 1 nM) was measured between Alexa488-Aß42 and GSK3α in solution using thermophoresis. An even lower apparent KD was estimated between GSK3α and dextran-immobilized Aß42 in surface plasmon resonance experiments. Parallel experiments with GSK3ß also identified colocalization and high affinity binding to this isoform. GSK3α-mediated hyperphosphorylation of the protein tau was found to be stimulated by Aß42 in in vitro phosphorylation assays and identified a functional relationship between the proteins. We uncover a direct and functional molecular link between Aß42 and GSK3α, which opens an important avenue toward understanding the mechanism of Aß42-mediated neuronal toxicity in Alzheimer's disease.


Asunto(s)
Péptidos beta-Amiloides/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Neuronas/metabolismo , Fragmentos de Péptidos/metabolismo , Proteínas tau/metabolismo , Péptidos beta-Amiloides/farmacología , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Células Cultivadas , Corteza Cerebral/citología , Relación Dosis-Respuesta a Droga , Embrión de Mamíferos , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3/farmacología , Glucógeno Sintasa Quinasa 3 beta/genética , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Humanos , Inmunoprecipitación , Ratones , Microscopía Confocal , Mutación/genética , Neuronas/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Resonancia por Plasmón de Superficie , Factores de Tiempo , Transfección , Proteínas tau/genética
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