RESUMEN
Quorum sensing is a cell-cell communication process that bacteria use to transition between individual and social lifestyles. In vibrios, homologous small RNAs called the Qrr sRNAs function at the center of quorum-sensing pathways. The Qrr sRNAs regulate multiple mRNA targets including those encoding the quorum-sensing regulatory components luxR, luxO, luxM, and aphA. We show that a representative Qrr, Qrr3, uses four distinct mechanisms to control its particular targets: the Qrr3 sRNA represses luxR through catalytic degradation, represses luxM through coupled degradation, represses luxO through sequestration, and activates aphA by revealing the ribosome binding site while the sRNA itself is degraded. Qrr3 forms different base-pairing interactions with each mRNA target, and the particular pairing strategy determines which regulatory mechanism occurs. Combined mathematical modeling and experiments show that the specific Qrr regulatory mechanism employed governs the potency, dynamics, and competition of target mRNA regulation, which in turn, defines the overall quorum-sensing response.
Asunto(s)
Percepción de Quorum , ARN Bacteriano/metabolismo , ARN Pequeño no Traducido/metabolismo , Vibrio/metabolismo , Secuencia de Bases , Escherichia coli/genética , Secuencias Invertidas Repetidas , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN Bacteriano/química , ARN Bacteriano/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Pequeño no Traducido/química , ARN Pequeño no Traducido/genética , Vibrio/genéticaRESUMEN
Recombinant insulin is a life-saving therapeutic for millions of patients affected by diabetes mellitus. Standard mutagenesis has led to insulin variants with improved control of blood glucose; for instance, the fast-acting insulin lispro contains two point mutations that suppress dimer formation and expedite absorption. However, insulins undergo irreversible denaturation, a process accelerated for the insulin monomer. Here we replace ProB29 of insulin lispro with 4R-fluoroproline, 4S-fluoroproline, and 4,4-difluoroproline. All three fluorinated lispro variants reduce blood glucose in diabetic mice, exhibit similar secondary structure as measured by CD, and rapidly dissociate from the zinc- and resorcinol-bound hexamer upon dilution. Notably, however, we find that 4S-fluorination of ProB29 delays the formation of undesired insulin fibrils that can accumulate at the injection site in vivo and can complicate insulin production and storage. These results demonstrate how subtle molecular changes achieved through non-canonical amino acid mutagenesis can improve the stability of protein therapeutics.
Asunto(s)
Halogenación , Insulina Lispro , Animales , Ratones , Humanos , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/genética , Glucemia/metabolismo , Hipoglucemiantes/farmacología , Hipoglucemiantes/química , MasculinoRESUMEN
Pancreatic ductal progenitor cells have been proposed to contribute to adult tissue maintenance and regeneration after injury, but the identity of such ductal cells remains elusive. Here, from adult mice, we identify a near homogenous population of ductal progenitor-like clusters, with an average of 8 cells per cluster. They are a rare subpopulation, about 0.1% of the total pancreatic cells, and can be sorted using a fluorescence-activated cell sorter with the CD133highCD71lowFSCmid-high phenotype. They exhibit properties in self-renewal and tri-lineage differentiation (including endocrine-like cells) in a unique 3-dimensional colony assay system. An in vitro lineage tracing experiment, using a novel HprtDsRed/+ mouse model, demonstrates that a single cell from a cluster clonally gives rise to a colony. Droplet RNAseq analysis demonstrates that these ductal clusters express embryonic multipotent progenitor cell markers Sox9, Pdx1, and Nkx6-1, and genes involved in actin cytoskeleton regulation, inflammation responses, organ development, and cancer. Surprisingly, these ductal clusters resist prolonged trypsin digestion in vitro, preferentially survive in vivo after a severe acinar cell injury and become proliferative within 14 days post-injury. Thus, the ductal clusters are the fundamental units of progenitor-like cells in the adult murine pancreas with implications in diabetes treatment and tumorigenicity.
Asunto(s)
Células Acinares , Conductos Pancreáticos , Ratones , Animales , Páncreas , Células Madre , Diferenciación CelularRESUMEN
Composite materials built in part from living organisms have the potential to exhibit useful autonomous, adaptive, and self-healing behavior. The physicochemical, biological, and mechanical properties of such materials can be engineered through the genetic manipulation of their living components. Successful development of living materials will require not only new methods for design and preparation but also new analytical tools that are capable of real-time noninvasive mapping of chemical compositions. Here, we establish a strategy based on stimulated Raman scattering microscopy to monitor phosphatase-catalyzed mineralization of engineered bacterial films in situ. Real-time label-free imaging elucidates the mineralization process, quantifies both the organic and inorganic components of the material as functions of time, and reveals spatial heterogeneity at multiple scales. In addition, we correlate the mechanical performance of films with the extent of mineralization. This work introduces a promising strategy for quantitatively analyzing living materials, which should contribute to the accelerated development of such materials in the future.
Asunto(s)
Microscopía Óptica no Lineal , Microscopía Óptica no Lineal/métodos , Espectrometría Raman/métodos , Factores de Tiempo , Monoéster Fosfórico Hidrolasas/metabolismoRESUMEN
The incorporation of living cells into materials promises both significant challenges and new possibilities. Although recent years have seen important advances in this field, there is still much to be learned about engineering interfaces between cells and materials. Here, we present a new class of 3D-printable materials, based on poly(N-hydroxymethylacrylamide) (PNHMAA), in which the spore-forming bacterium Bacillus subtilis is effectively cross-linked into the surrounding polymeric scaffold. After dehydration and subsequent re-swelling in nutrient-rich media, embedded cells and spores become metabolically active and are capable of heterologous protein production and secretion. Strikingly, the leak-free scaffold allows protein production while preventing escape of embedded cells. The successful construction of complex three-dimensional structures by stereolithographic printing of living PNHMAA composite materials suggests utility in a broad range of applications.
Asunto(s)
Bacillus subtilis , Impresión Tridimensional , Proteínas RecombinantesRESUMEN
Though most bacteria in nature are nutritionally limited and grow slowly, our understanding of core processes like transcription comes largely from studies in model organisms doubling rapidly. We previously identified a small protein of unknown function, SutA, in a screen of proteins synthesized in Pseudomonas aeruginosa during dormancy. SutA binds RNA polymerase (RNAP), causing widespread changes in gene expression, including upregulation of the ribosomal RNA genes. Here, using biochemical and structural methods, we examine how SutA interacts with RNAP and the functional consequences of these interactions. We show that SutA comprises a central α-helix with unstructured N- and C-terminal tails, and binds to the ß1 domain of RNAP. It activates transcription from the rrn promoter by both the housekeeping sigma factor holoenzyme (Eσ70 ) and the stress sigma factor holoenzyme (EσS ) in vitro, but has a greater impact on EσS . In both cases, SutA appears to affect intermediates in the open complex formation and its N-terminal tail is required for activation. The small magnitudes of in vitro effects are consistent with a role in maintaining activity required for homeostasis during dormancy. Our results add SutA to a growing list of transcription regulators that use their intrinsically disordered regions to remodel transcription complexes.
Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Pseudomonas aeruginosa/crecimiento & desarrollo , Transcripción Genética , ARN Polimerasas Dirigidas por ADN/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Regiones Promotoras Genéticas , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , Factor sigma/genética , Factor sigma/metabolismo , Activación TranscripcionalRESUMEN
Microbial quiescence and slow growth are ubiquitous physiological states, but their study is complicated by low levels of metabolic activity. To address this issue, we used a time-selective proteome-labeling method [bioorthogonal noncanonical amino acid tagging (BONCAT)] to identify proteins synthesized preferentially, but at extremely low rates, under anaerobic survival conditions by the opportunistic pathogen Pseudomonas aeruginosa. One of these proteins is a transcriptional regulator that has no homology to any characterized protein domains and is posttranscriptionally up-regulated during survival and slow growth. This small, acidic protein associates with RNA polymerase, and chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing suggests that the protein associates with genomic DNA through this interaction. ChIP signal is found both in promoter regions and throughout the coding sequences of many genes and is particularly enriched at ribosomal protein genes and in the promoter regions of rRNA genes. Deletion of the gene encoding this protein affects expression of these and many other genes and impacts biofilm formation, secondary metabolite production, and fitness in fluctuating conditions. On the basis of these observations, we have designated the protein SutA (survival under transitions A).
Asunto(s)
Proteínas Bacterianas/metabolismo , Pseudomonas aeruginosa/metabolismo , Anaerobiosis , Biopelículas , ARN Polimerasas Dirigidas por ADN/metabolismo , Unión Proteica , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crecimiento & desarrollo , Procesamiento Postranscripcional del ARN , Ribosomas/genética , Transcripción Genética , Regulación hacia ArribaRESUMEN
Networks assembled by reversible association of telechelic polymers constitute a common class of soft materials. Various mechanisms of chain migration in associative networks have been proposed; yet there remains little quantitative experimental data to discriminate among them. Proposed mechanisms for chain migration include multichain aggregate diffusion as well as single-chain mechanisms such as "walking" and "hopping", wherein diffusion is achieved by either partial ("walking") or complete ("hopping") disengagement of the associated chain segments. Here, we provide evidence that hopping can dominate the effective diffusion of chains in associative networks due to a strong entropic penalty for bridge formation imposed by local network structure; chains become conformationally restricted upon association with two or more spatially separated binding sites. This restriction decreases the effective binding strength of chains with multiple associative domains, thereby increasing the probability that a chain will hop. For telechelic chains this manifests as binding asymmetry, wherein the first association is effectively stronger than the second. We derive a simple thermodynamic model that predicts the fraction of chains that are free to hop as a function of tunable molecular and network properties. A large set of self-diffusivity measurements on a series of model associative polymers finds good agreement with this model.
Asunto(s)
Polímeros/química , Difusión , EntropíaRESUMEN
Protein synthesis is a dynamic process that tunes the cellular proteome in response to internal and external demands. Metabolic labeling approaches identify the general proteomic response but cannot visualize specific newly synthesized proteins within cells. Here we describe a technique that couples noncanonical amino acid tagging or puromycylation with the proximity ligation assay to visualize specific newly synthesized proteins and monitor their origin, redistribution and turnover in situ.
Asunto(s)
Fibroblastos/metabolismo , Proteínas/química , Proteínas/metabolismo , Animales , Anticuerpos , Células Cultivadas , Regulación de la Expresión Génica/fisiología , Hipocampo/citología , Ratones , Neuronas/metabolismo , Ratas , Coloración y EtiquetadoRESUMEN
Proteomic plasticity undergirds stress responses in plants, and understanding such responses requires accurate measurement of the extent to which proteins levels are adjusted to counter external stimuli. Here, we adapt bioorthogonal noncanonical amino acid tagging (BONCAT) to interrogate protein synthesis in vegetative Arabidopsis (Arabidopsis thaliana) seedlings. BONCAT relies on the translational incorporation of a noncanonical amino acid probe into cellular proteins. In this study, the probe is the Met surrogate azidohomoalanine (Aha), which carries a reactive azide moiety in its amino acid side chain. The azide handle in Aha can be selectively conjugated to dyes and functionalized beads to enable visualization and enrichment of newly synthesized proteins. We show that BONCAT is sensitive enough to detect Arabidopsis proteins synthesized within a 30-min interval defined by an Aha pulse and that the method can be used to detect proteins made under conditions of light stress, osmotic shock, salt stress, heat stress, and recovery from heat stress. We further establish that BONCAT can be coupled to tandem liquid chromatography-mass spectrometry to identify and quantify proteins synthesized during heat stress and recovery from heat stress. Our results are consistent with a model in which, upon the onset of heat stress, translation is rapidly reprogrammed to enhance the synthesis of stress mitigators and is again altered during recovery. All experiments were carried out with commercially available reagents, highlighting the accessibility of the BONCAT method to researchers interested in stress responses as well as translational and posttranslational regulation in plants.
Asunto(s)
Alanina/análogos & derivados , Proteínas de Arabidopsis/biosíntesis , Arabidopsis/metabolismo , Azidas/química , Colorantes/química , Biosíntesis de Proteínas , Alanina/química , Arabidopsis/efectos de los fármacos , Arabidopsis/efectos de la radiación , Cromatografía Liquida , Sequías , Calor , Immunoblotting , Luz , Reproducibilidad de los Resultados , Plantones/efectos de los fármacos , Plantones/metabolismo , Plantones/efectos de la radiación , Cloruro de Sodio/farmacología , Coloración y Etiquetado/métodos , Estrés Fisiológico , Espectrometría de Masas en Tándem , Factores de TiempoRESUMEN
Proteomic analysis of rare cells in heterogeneous environments presents difficult challenges. Systematic methods are needed to enrich, identify, and quantify proteins expressed in specific cells in complex biological systems including multicellular plants and animals. Here, we have engineered a Caenorhabditis elegans phenylalanyl-tRNA synthetase capable of tagging proteins with the reactive noncanonical amino acid p-azido-L-phenylalanine. We achieved spatiotemporal selectivity in the labeling of C. elegans proteins by controlling expression of the mutant synthetase using cell-selective (body wall muscles, intestinal epithelial cells, neurons, and pharyngeal muscle) or state-selective (heat-shock) promoters in several transgenic lines. Tagged proteins are distinguished from the rest of the protein pool through bioorthogonal conjugation of the azide side chain to probes that permit visualization and isolation of labeled proteins. By coupling our methodology with stable-isotope labeling of amino acids in cell culture (SILAC), we successfully profiled proteins expressed in pharyngeal muscle cells, and in the process, identified proteins not previously known to be expressed in these cells. Our results show that tagging proteins with spatiotemporal selectivity can be achieved in C. elegans and illustrate a convenient and effective approach for unbiased discovery of proteins expressed in targeted subsets of cells.
Asunto(s)
Proteínas de Caenorhabditis elegans/biosíntesis , Caenorhabditis elegans/citología , Caenorhabditis elegans/metabolismo , Regulación de la Expresión Génica/fisiología , Proteoma/biosíntesis , Proteómica/métodos , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Marcaje Isotópico/métodos , Mutación , Proteoma/genéticaRESUMEN
Programmable colloidal assembly enables the creation of mesoscale materials in a bottom-up manner. Although DNA oligonucleotides have been used extensively as the programmable units in this paradigm, proteins, which exhibit more diverse modes of association and function, have not been widely used to direct colloidal assembly. Here we use protein-protein interactions to drive controlled aggregation of polystyrene microparticles, either through reversible coiled-coil interactions or through intermolecular isopeptide linkages. The sizes of the resulting aggregates are tunable and can be controlled by the concentration of immobilized surface proteins. Moreover, particles coated with different protein pairs undergo orthogonal assembly. We demonstrate that aggregates formed by association of coiled-coil proteins, in contrast to those linked by isopeptide bonds, are dispersed by treatment with chemical denaturants or soluble competing proteins. Finally, we show that protein-protein interactions can be used to assemble complex core-shell aggregates. This work illustrates a versatile strategy for engineering colloidal systems for use in materials science and biotechnology.
Asunto(s)
Proteínas Bacterianas/metabolismo , Coloides/metabolismo , Proteínas Inmovilizadas/metabolismo , Poliestirenos/metabolismo , Mapas de Interacción de Proteínas , Streptococcus pyogenes/metabolismo , Proteínas Bacterianas/química , Coloides/química , Dimerización , Proteínas Inmovilizadas/química , Modelos Moleculares , Tamaño de la Partícula , Poliestirenos/química , Streptococcus pyogenes/químicaRESUMEN
Daily injections of insulin provide lifesaving benefits to millions of diabetics. But currently available prandial insulins are suboptimal: The onset of action is delayed by slow dissociation of the insulin hexamer in the subcutaneous space, and insulin forms amyloid fibrils upon storage in solution. Here we show, through the use of noncanonical amino acid mutagenesis, that replacement of the proline residue at position 28 of the insulin B-chain (ProB28) by (4S)-hydroxyproline (Hzp) yields an active form of insulin that dissociates more rapidly, and fibrillates more slowly, than the wild-type protein. Crystal structures of dimeric and hexameric insulin preparations suggest that a hydrogen bond between the hydroxyl group of Hzp and a backbone amide carbonyl positioned across the dimer interface may be responsible for the altered behavior. The effects of hydroxylation are stereospecific; replacement of ProB28 by (4R)-hydroxyproline (Hyp) causes little change in the rates of fibrillation and hexamer disassociation. These results demonstrate a new approach that fuses the concepts of medicinal chemistry and protein design, and paves the way to further engineering of insulin and other therapeutic proteins.
Asunto(s)
Hidroxiprolina/química , Insulina/química , Amiloide/química , Cristalografía por Rayos X , Dimerización , Hidroxilación , Modelos Biológicos , Modelos Moleculares , Proinsulina/químicaRESUMEN
Coiled-coil domains can direct the assembly of protein block copolymers into physically cross-linked, viscoelastic hydrogels. Here, we describe the use of fluorescence recovery after photobleaching (FRAP) to probe chain mobility in reversible hydrogels assembled from engineered proteins bearing terminal coiled-coil domains. We show that chain mobility can be related to the underlying dynamics of the coiled-coil domains by application of a three-state "hopping" model of chain migration. We further show that genetic programming allows the effective mobility of network chains to be varied 500-fold through modest changes in protein sequence. Destabilization of the coiled-coil domains by site-directed mutagenesis increases the effective diffusivity of probe chains. Conversely, probe mobility is reduced by expanding the hydrophobic surface area of the coiled-coil domains through introduction of the bulky leucine surrogate homoisoleucine. Predictions from the three-state model imply asymmetric sequential binding of the terminal domains. Brownian Dynamics simulations suggest that binding asymmetry is a general feature of reversible gels, arising from a loss in entropy as chains transition to a conformationally restricted bridged state.
Asunto(s)
Hidrogeles/química , Proteínas/química , Fluorescencia , Interacciones Hidrofóbicas e Hidrofílicas , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Ingeniería de Proteínas , Proteínas/genética , Propiedades de SuperficieRESUMEN
Protein-based hydrogels have emerged as promising alternatives to synthetic hydrogels for biomedical applications, owing to the precise control of structure and function enabled by protein engineering. Nevertheless, strategies for assembling 3D molecular networks that carry the biological information encoded in full-length proteins remain underdeveloped. Here we present a robust protein gelation strategy based on a pair of genetically encoded reactive partners, SpyTag and SpyCatcher, that spontaneously form covalent isopeptide linkages under physiological conditions. The resulting "network of Spies" may be designed to include cell-adhesion ligands, matrix metalloproteinase-1 cleavage sites, and full-length globular proteins [mCherry and leukemia inhibitory factor (LIF)]. The LIF network was used to encapsulate mouse embryonic stem cells; the encapsulated cells remained pluripotent in the absence of added LIF. These results illustrate a versatile strategy for the creation of information-rich biomaterials.
Asunto(s)
Hidrogeles/farmacología , Factor Inhibidor de Leucemia/farmacología , Ingeniería de Proteínas/métodos , Células 3T3 , Secuencia de Aminoácidos , Animales , Células Inmovilizadas/citología , Células Inmovilizadas/efectos de los fármacos , Células Inmovilizadas/metabolismo , Células Madre Embrionarias/citología , Células Madre Embrionarias/efectos de los fármacos , Células Madre Embrionarias/metabolismo , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Proteínas Luminiscentes/metabolismo , Ratones , Datos de Secuencia Molecular , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/efectos de los fármacos , Células Madre Pluripotentes/metabolismo , Proteína Fluorescente RojaRESUMEN
Pathogenic microbes have evolved complex secretion systems to deliver virulence factors into host cells. Identification of these factors is critical for understanding the infection process. We report a powerful and versatile approach to the selective labeling and identification of secreted pathogen proteins. Selective labeling of microbial proteins is accomplished via translational incorporation of azidonorleucine (Anl), a methionine surrogate that requires a mutant form of the methionyl-tRNA synthetase for activation. Secreted pathogen proteins containing Anl can be tagged by azide-alkyne cycloaddition and enriched by affinity purification. Application of the method to analysis of the type III secretion system of the human pathogen Yersinia enterocolitica enabled efficient identification of secreted proteins, identification of distinct secretion profiles for intracellular and extracellular bacteria, and determination of the order of substrate injection into host cells. This approach should be widely useful for the identification of virulence factors in microbial pathogens and the development of potential new targets for antimicrobial therapy.
Asunto(s)
Aminoácidos/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Técnicas Microbiológicas , Antibacterianos/química , Células HeLa , Humanos , Espectrometría de Masas , Metionina-ARNt Ligasa/química , Microscopía Confocal , Microscopía Fluorescente , Modelos Biológicos , Mutación , Norleucina/química , Proteoma , Proteómica/métodos , Factores de Tiempo , Factores de Virulencia , Yersinia enterocolitica/metabolismoRESUMEN
Reliable methods to determine the subcellular localization of bacterial proteins are needed for the study of prokaryotic cell biology. We describe here a simple and general technique for imaging of bacterial proteins in situ by fluorescence microscopy. The method uses the eukaryotic enzyme N-myristoyltransferase to modify the N-terminus of the protein of interest with an azido fatty acid. Subsequent strain-promoted azide-alkyne cycloaddition allows conjugation of dyes and imaging of tagged proteins by confocal fluorescence microscopy. We demonstrate the method by labeling the chemotaxis proteins Tar and CheA and the cell division proteins FtsZ and FtsA in Escherichia coli. We observe distinct spatial patterns for each of these proteins in both fixed and live cells. The method should prove broadly useful for protein imaging in bacteria.
Asunto(s)
Aciltransferasas/metabolismo , Proteínas Bacterianas/análisis , Proteínas Bacterianas/química , Proteínas del Citoesqueleto/análisis , Proteínas del Citoesqueleto/química , Proteínas de Escherichia coli/análisis , Proteínas de Escherichia coli/química , Escherichia coli/química , Escherichia coli/citología , Microscopía Fluorescente , Estructura MolecularRESUMEN
Methods for cell-selective analysis of proteome dynamics will facilitate studies of biological processes in multicellular organisms. Here we describe a mutant murine methionyl-tRNA synthetase (designated L274GMmMetRS) that charges the noncanonical amino acid azidonorleucine (Anl) to elongator tRNA(Met) in hamster (CHO), monkey (COS7), and human (HeLa) cell lines. Proteins made in cells that express the synthetase can be labeled with Anl, tagged with dyes or affinity reagents, and enriched on affinity resin to facilitate identification by mass spectrometry. The method does not require expression of orthogonal tRNAs or depletion of canonical amino acids. Successful labeling of proteins with Anl in several mammalian cell lines demonstrates the utility of L274GMmMetRS as a tool for cell-selective analysis of mammalian protein synthesis.
Asunto(s)
Aminoacil-ARNt Sintetasas/metabolismo , Biosíntesis de Proteínas , Aminoácidos/análisis , Aminoácidos/química , Aminoacil-ARNt Sintetasas/genética , Animales , Cricetinae , Escherichia coli/metabolismo , Haplorrinos , Células HeLa , Humanos , Mamíferos , Metionina-ARNt Ligasa/metabolismo , Ratones , Proteoma/metabolismo , ARN de Transferencia de Metionina/metabolismoRESUMEN
Although biochemically patterned hydrogels are capable of recapitulating many critical aspects of the heterogeneous cellular niche, exercising spatial and temporal control of the presentation and removal of biomolecular signalling cues in such systems has proved difficult. Here, we demonstrate a synthetic strategy that exploits two bioorthogonal photochemistries to achieve reversible immobilization of bioactive full-length proteins with good spatial and temporal control within synthetic, cell-laden biomimetic scaffolds. A photodeprotection-oxime-ligation sequence permits user-defined quantities of proteins to be anchored within distinct subvolumes of a three-dimensional matrix, and an ortho-nitrobenzyl ester photoscission reaction facilitates subsequent protein removal. By using this approach to pattern the presentation of the extracellular matrix protein vitronectin, we accomplished reversible differentiation of human mesenchymal stem cells to osteoblasts in a spatially defined manner. Our protein-patterning approach should provide further avenues to probe and direct changes in cell physiology in response to dynamic biochemical signalling.
Asunto(s)
Materiales Biomiméticos/química , Diferenciación Celular , Hidrogeles/química , Proteínas Inmovilizadas/química , Células Madre Mesenquimatosas/metabolismo , Osteoblastos/metabolismo , Humanos , Células Madre Mesenquimatosas/citología , Osteoblastos/citología , Procesos Fotoquímicos , Andamios del Tejido/química , Vitronectina/químicaRESUMEN
An approach to proteomic analysis that combines bioorthogonal noncanonical amino acid tagging (BONCAT) and pulsed stable isotope labeling with amino acids in cell culture (pSILAC) provides accurate quantitative information about rates of cellular protein synthesis on time scales of minutes. The method is capable of quantifying 1400 proteins produced by HeLa cells during a 30 min interval, a time scale that is inaccessible to isotope labeling techniques alone. Potential artifacts in protein quantification can be reduced to insignificant levels by limiting the extent of noncanonical amino acid tagging. We find no evidence for artifacts in protein identification in experiments that combine the BONCAT and pSILAC methods.