RESUMEN
Encapsulation of enzymes allows to preserve their biological activities in various environmental conditions, such as exposure to elevated temperature or to proteases. This is particularly relevant for in vivo applications, where proteases represent a severe obstacle to maintaining the activity of enzymes. Polyelectrolyte multilayer capsules are suitable for enzyme encapsulation, where CaCO3 particles and temperature-dependent capsule formation are the best templates and the most adequate method, respectively. In this work, these two areas are combined and, ALP (alkaline phosphatase), which is a robust and therapeutically relevant enzyme, is encapsulated into thermally shrunk polyelectrolyte multilayer (PDADMAC/PSS)4 capsules templated on calcium carbonate particles (original average diameter: ≈3.5 µm). The activity of the encapsulated enzyme and the optimal temperature range for encapsulation are investigated. The enzymatic activity is almost four times higher upon encapsulation when the temperature range for encapsulation is situated just above the glass transition temperature (40 °C), while its optimal conditions are dictated, on the one hand, by the enzyme activity (better at lower temperatures) and, on the other hand, by the size and mechanical properties of capsules (better at higher temperatures).
Asunto(s)
Fosfatasa Alcalina/metabolismo , Carbonato de Calcio/química , Polielectrolitos/química , Temperatura , Cápsulas , Microscopía de Fuerza Atómica , Tamaño de la Partícula , Polietilenos/química , Compuestos de Amonio Cuaternario/químicaRESUMEN
Composite bioceramic and hydrogel-based containers harboring alkaline phosphatase are generated through encapsulation of this enzyme by its immobilization into CaCO3-based bioceramic materials in combination with a hydrogel assembly technique and subsequent gelification. A refined way of synthesis and modification allows preparing the enzyme delivery system with functionalized protection layers. The particles are characterized by electron microscopy, Fourier transform infrared (FTIR) spectroscopy, and enzyme activity measurements. Loading efficiency and loading capacity are investigated depending on particle size, time of enzyme loading, and various container compositions and enzyme concentrations. Our results reveal that the size of particles influences their morphology and this, in turn, affects the activity of the encapsulated enzymes. Various functionalizations of the surfaces, including protection by the hydrogel layer, formation of hollow silver alginate, or calcium alginate encapsulation, decrease the enzymatic activity. The presence of a good therapeutic effect on osteoblastic cells coupled with a relatively high loading capacity, biocompatibility, and ease of fabrication suggests that the developed carriers are promising candidates for efficient drug delivery, especially in the field of bone reconstruction.
RESUMEN
We investigated melting transitions in native biological membranes containing their membrane proteins. The membranes originated from E. coli, B. subtilis, lung surfactant and nerve tissue from the spinal cord of several mammals. For some preparations, we studied the pressure, pH and ionic strength dependence of the transition. For porcine spine, we compared the transition of the native membrane to that of the extracted lipids. All preparations displayed melting transitions of 10-20° below physiological or growth temperature, independent of the organism of origin and the respective cell type. We found that the position of the transitions in E. coli membranes depends on the growth temperature. We discuss these findings in the context of the thermodynamic theory of membrane fluctuations close to transition that predicts largely altered elastic constants, an increase in fluctuation lifetime and in membrane permeability. We also discuss how to distinguish lipid melting from protein unfolding transitions. Since the feature of a transition slightly below physiological temperature is conserved even when growth conditions change, we conclude that the transitions are likely to be of major biological importance for the survival and the function of the cell.
Asunto(s)
Membrana Celular/química , Membrana Celular/metabolismo , Temperatura de Transición , Animales , Bacterias , Fenómenos Fisiológicos Bacterianos , Membrana Celular/fisiología , Lípidos/análisis , Mamíferos/fisiología , Concentración Osmolar , Transición de Fase , Temperatura , TermodinámicaRESUMEN
Human guanylate kinase (hGMPK) is the only known enzyme responsible for cellular GDP production, making it essential for cellular viability and proliferation. Moreover, hGMPK has been assigned a critical role in metabolic activation of antiviral and antineoplastic nucleoside-analog prodrugs. Given that hGMPK is indispensable for producing the nucleotide building blocks of DNA, RNA, and cGMP and that cancer cells possess elevated GTP levels, it is surprising that a detailed structural and functional characterization of hGMPK is lacking. Here, we present the first high-resolution structure of hGMPK in the apo form, determined with NMR spectroscopy. The structure revealed that hGMPK consists of three distinct regions designated as the LID, GMP-binding (GMP-BD), and CORE domains and is in an open configuration that is nucleotide binding-competent. We also demonstrate that nonsynonymous single-nucleotide variants (nsSNVs) of the hGMPK CORE domain distant from the nucleotide-binding site of this domain modulate enzymatic activity without significantly affecting hGMPK's structure. Finally, we show that knocking down the hGMPK gene in lung adenocarcinoma cell lines decreases cellular viability, proliferation, and clonogenic potential while not altering the proliferation of immortalized, noncancerous human peripheral airway cells. Taken together, our results provide an important step toward establishing hGMPK as a potential biomolecular target, from both an orthosteric (ligand-binding sites) and allosteric (location of CORE domain-located nsSNVs) standpoint.
Asunto(s)
Guanilato-Quinasas/metabolismo , Regulación Alostérica , Animales , Línea Celular Tumoral , Cristalografía por Rayos X , Guanilato-Quinasas/química , Guanilato-Quinasas/genética , Humanos , Cinética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones , Mutagénesis Sitio-Dirigida , Resonancia Magnética Nuclear Biomolecular , Estructura Terciaria de Proteína , Interferencia de ARN , ARN Interferente Pequeño , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
5-Aminoimidazole-4-carboxamide 1-ß-d-ribofuranoside (AICAR, or acadesine) is a precursor of the monophosphate derivative 5-amino-4-imidazole carboxamide ribonucleoside 5'-phosphate (ZMP), an intermediate in de novo purine biosynthesis. AICAR proved to have promising anti-proliferative properties, although the molecular basis of its toxicity is poorly understood. To exert cytotoxicity, AICAR needs to be metabolized, but the AICAR-derived toxic metabolite was not identified. Here, we show that ZMP is the major toxic derivative of AICAR in yeast and establish that its metabolization to succinyl-ZMP, ZDP, or ZTP (di- and triphosphate derivatives of AICAR) strongly reduced its toxicity. Affinity chromatography identified 74 ZMP-binding proteins, including 41 that were found neither as AMP nor as AICAR or succinyl-ZMP binders. Overexpression of karyopherin-ß Kap123, one of the ZMP-specific binders, partially rescued AICAR toxicity. Quantitative proteomic analyses revealed 57 proteins significantly less abundant on nuclei-enriched fractions from AICAR-fed cells, this effect being compensated by overexpression of KAP123 for 15 of them. These results reveal nuclear protein trafficking as a function affected by AICAR.
Asunto(s)
Aminoimidazol Carboxamida/análogos & derivados , Núcleo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Proteómica , Ribonucleótidos , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Aminoimidazol Carboxamida/farmacocinética , Aminoimidazol Carboxamida/farmacología , Núcleo Celular/química , Núcleo Celular/genética , Cromatografía de Afinidad , Ribonucleótidos/farmacocinética , Ribonucleótidos/farmacología , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Human guanylate kinase (hGMPK) is a critical enzyme that, in addition to phosphorylating its physiological substrate (d)GMP, catalyzes the second phosphorylation step in the conversion of anti-viral and anti-cancer nucleoside analogs to their corresponding active nucleoside analog triphosphates. Until now, a high-resolution structure of hGMPK is unavailable and thus, we studied free hGMPK by NMR and assigned the chemical shift resonances of backbone and side chain 1H, 13C, and 15N nuclei as a first step towards the enzyme's structural and mechanistic analysis with atomic resolution.
Asunto(s)
Guanilato-Quinasas/química , Resonancia Magnética Nuclear Biomolecular , HumanosRESUMEN
Immunogenicity is one of the most common complications occurring during therapy making use of protein drugs of nonhuman origin. A notable example of such a case is bacterial l-asparaginases (L-ASNases) used for the treatment of acute lymphoblastic leukemia (ALL). The replacement of the bacterial enzymes by human ones is thought to set the basis for a major improvement of antileukemic therapy. Recently, we solved the crystal structure of a human enzyme possessing L-ASNase activity, designated hASNase-3. This enzyme is expressed as an inactive precursor protein and post-translationally undergoes intramolecular processing leading to the generation of two subunits which remain noncovalently, yet tightly associated and constitute the catalytically active form of the enzyme. We discovered that this intramolecular processing can be drastically and selectively accelerated by the free amino acid glycine. In the present study, we report on the molecular engineering of hASNase-3 aiming at the improvement of its catalytic properties. We created a fluorescence-activated cell sorting (FACS)-based high-throughput screening system for the characterization of rationally designed mutant libraries, capitalizing on the finding that free glycine promotes autoproteolytic cleavage, which activates the mutant proteins expressed in an E. coli strain devoid of aspartate biosynthesis. Successive screening rounds led to the isolation of catalytically improved variants showing up to 6-fold better catalytic efficiency as compared to the wild-type enzyme. Our work establishes a powerful strategy for further exploitation of the human asparaginase sequence space to facilitate the identification of in vitro-evolved enzyme species that will lay the basis for improved ALL therapy.
Asunto(s)
Asparaginasa/aislamiento & purificación , Mutación , Asparaginasa/genética , Asparaginasa/metabolismo , Asparaginasa/uso terapéutico , Fluorescencia , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológicoRESUMEN
Choline kinase beta (CKß) is one of the CK isozymes involved in the biosynthesis of phosphatidylcholine. CKß is important for normal mitochondrial function and muscle development as the lack of the ckß gene in human and mice results in the development of muscular dystrophy. In contrast, CKα is implicated in tumorigenesis and has been extensively studied as an anticancer target. Phosphorylation of human CKα was found to regulate the enzyme's activity and its subcellular location. This study provides evidence for CKß phosphorylation by protein kinase A (PKA). In vitro phosphorylation of CKß by PKA was first detected by phosphoprotein staining, as well as by in-gel kinase assays. The phosphorylating kinase was identified as PKA by Western blotting. CKß phosphorylation by MCF-7 cell lysate was inhibited by a PKA-specific inhibitor peptide, and the intracellular phosphorylation of CKß was shown to be regulated by the level of cyclic adenosine monophosphate (cAMP), a PKA activator. Phosphorylation sites were located on CKß residues serine-39 and serine-40 as determined by mass spectrometry and site-directed mutagenesis. Phosphorylation increased the catalytic efficiencies for the substrates choline and ATP about 2-fold, without affecting ethanolamine phosphorylation, and the S39D/S40D CKß phosphorylation mimic behaved kinetically very similar. Remarkably, phosphorylation drastically increased the sensitivity of CKß to hemicholinium-3 (HC-3) inhibition by about 30-fold. These findings suggest that CKß, in concert with CKα, and depending on its phosphorylation status, might play a critical role as a druggable target in carcinogenesis.
Asunto(s)
Colina Quinasa/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Línea Celular Tumoral , Colina Quinasa/antagonistas & inhibidores , Humanos , FosforilaciónRESUMEN
A new method of fabrication of calcium carbonate microparticles of ellipsoidal, rhomboidal, and spherical geometries is reported by adjusting the relative concentration ratios of the initial salt solutions and/or the ethylene glycol content in the reaction medium. Morphology, porosity, crystallinity, and loading capacity of synthesized CaCO3 templates were characterized in detail. Particles harboring dextran or the enzyme guanylate kinase were obtained through encapsulation of these macromolecules using the layer-by-layer assembly technique to deposit positively and negatively charged polymers on these differently shaped CaCO3 templates and were characterized by confocal laser scanning fluorescence microscopy, fluorometric techniques, and enzyme activity measurements. The enzymatic activity, an important application of such porous particles and containers, has been analyzed in comparison with the loading capacity and geometry. Our results reveal that the particles' shape influences morphology of particles and that, as a result, affects the activity of the encapsulated enzymes, in addition to the earlier reported influence on cellular uptake. These particles are promising candidates for efficient drug delivery due to their relatively high loading capacity, biocompatibility, and easy fabrication and handling.
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Carbonato de Calcio/química , Sistemas de Liberación de Medicamentos , Anisotropía , Tamaño de la Partícula , Polímeros/químicaRESUMEN
Bio-catalysis is the outcome of a subtle interplay between internal motions in enzymes and chemical kinetics. Small-angle X-ray scattering (SAXS) investigation of an enzyme's internal motions during catalysis offers an integral view of the protein's structural plasticity, dynamics, and function, which is useful for understanding allosteric effects and developing novel medicines. Guanylate kinase (GMPK) is an essential enzyme involved in the guanine nucleotide metabolism of unicellular and multicellular organisms. It is also required for the intracellular activation of numerous antiviral and anticancer purine nucleoside analog prodrugs. Catalytically active recombinant human GMPK (hGMPK) was purified for the first time and changes in the size and shape of open/closed hGMPK were tracked by SAXS. The binding of substrates (GMP + AMPPNP or Ap5G or GMP + ADP) resulted in the compaction of size and shape of hGMPK. The structural changes between open and completely closed hGMPK conformation were confirmed by observing differences in the hGMPK secondary structures with circular dichroism spectroscopy.
Asunto(s)
Dominio Catalítico , Guanilato Ciclasa/química , Simulación de Dinámica Molecular , Secuencia de Aminoácidos , Humanos , Datos de Secuencia Molecular , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
A photo-electrochemical sensor for the specific detection of guanosine monophosphate (GMP) is demonstrated, based on three enzymes combined in a coupled reaction assay. The first reaction involves the adenosine triphosphate (ATP)-dependent conversion of GMP to guanosine diphosphate (GDP) by guanylate kinase, which warrants substrate specificity. The reaction products ADP and GDPare co-substrates for the enzymatic conversion of phosphoenolpyruvate to pyruvate in a second reaction mediated by pyruvate kinase. Pyruvate in turn is the co-substrate for lactate dehydrogenase that generates lactate via oxidation of nicotinamide adenine dinucleotide (reduced form) NADH to NAD(+). This third enzymatic reaction is electrochemically detected. For this purpose a CdS/ZnS quantum dot (QD) electrode is illuminated and the photocurrent response under fixed potential conditions is evaluated. The sequential enzyme reactions are first evaluated in solution. Subsequently, a sensor for GMP is constructed using polyelectrolytes for enzyme immobilization.
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Técnicas Biosensibles/instrumentación , Conductometría/instrumentación , Guanosina Monofosfato/análisis , L-Lactato Deshidrogenasa/química , Puntos Cuánticos , Espectrometría de Fluorescencia/instrumentación , Compuestos de Cadmio/química , Enzimas Inmovilizadas , Diseño de Equipo , Análisis de Falla de Equipo , Microelectrodos , Compuestos de Selenio/química , Compuestos de Zinc/químicaRESUMEN
Droplet-based microfluidic technologies are powerful tools for applications requiring high-throughput, for example, in biochemistry or material sciences. Several systems have been proposed for the high-throughput production of monodisperse emulsions by parallelizing multiple droplet makers. However, these systems have two main limitations: (1) they allow the use of only a single disperse phase; (2) they are based on multiple layer microfabrication techniques. We present here a pipette-and-play solution offering the possibility of manipulating simultaneously 10 different disperse phases on a single layer device. This system allows high-throughput emulsion production using aqueous flow rates of up to 26 ml/h (>110 000 drops/s) leading to emulsions with user-defined complex chemical composition. We demonstrate the multiplex capabilities of our system by measuring the kinetics of ß-galactosidase in droplets using nine different concentrations of a fluorogenic substrate.
RESUMEN
Colloidal particles play an important role in various areas of material and pharmaceutical sciences, biotechnology, and biomedicine. In this overview we describe micro- and nano-particles used for the preparation of polyelectrolyte multilayer capsules and as drug delivery vehicles. An essential feature of polyelectrolyte multilayer capsule preparations is the ability to adsorb polymeric layers onto colloidal particles or templates followed by dissolution of these templates. The choice of the template is determined by various physico-chemical conditions: solvent needed for dissolution, porosity, aggregation tendency, as well as release of materials from capsules. Historically, the first templates were based on melamine formaldehyde, later evolving towards more elaborate materials such as silica and calcium carbonate. Their advantages and disadvantages are discussed here in comparison to non-particulate templates such as red blood cells. Further steps in this area include development of anisotropic particles, which themselves can serve as delivery carriers. We provide insights into application of particles as drug delivery carriers in comparison to microcapsules templated on them.
Asunto(s)
Composición de Medicamentos , Sistemas de Liberación de Medicamentos , Modelos Químicos , Nanocápsulas/química , Nanopartículas/química , Polímeros/química , Cápsulas , Coloides , Composición de Medicamentos/tendencias , Microesferas , Nanotecnología/tendencias , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
The structural and functional characterization of human enzymes that are of potential medical and therapeutic interest is of prime significance for translational research. One of the most notable examples of a therapeutic enzyme is L-asparaginase, which has been established as an antileukemic protein drug for more than four decades. Up until now, only bacterial enzymes have been used in therapy despite a plethora of undesired side effects mainly attributed to the bacterial origins of these enzymes. Therefore, the replacement of the currently approved bacterial drugs by human homologs aiming at the elimination of adverse effects is of great importance. Recently, we structurally and biochemically characterized the enzyme human L-asparaginase 3 (hASNase3), which possesses L-asparaginase activity and belongs to the N-terminal nucleophile superfamily of enzymes. Inspired by the necessity for the development of a protein drug of human origin, in the present study, we focused on the characterization of another human L-asparaginase, termed hASNase1. This bacterial-type cytoplasmic L-asparaginase resides in the N-terminal subdomain of an overall 573-residue protein previously reported to function as a lysophospholipase. Our kinetic, mutagenesis, structural modeling, and fluorescence labeling data highlight allosteric features of hASNase1 that are similar to those of its Escherichia coli homolog, EcASNase1. Differential scanning fluorometry and urea denaturation experiments demonstrate the impact of particular mutations on the structural and functional integrity of the L-asparaginase domain and provide a direct comparison of sites critical for the conformational stability of the human and E. coli enzymes.
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Asparaginasa/química , Asparagina/química , Lisofosfolipasa/química , Modelos Moleculares , Regulación Alostérica/fisiología , Asparaginasa/genética , Asparaginasa/metabolismo , Asparagina/genética , Asparagina/metabolismo , Estabilidad de Enzimas , Escherichia coli/enzimología , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Lisofosfolipasa/genética , Lisofosfolipasa/metabolismo , Estructura Terciaria de Proteína , Homología Estructural de ProteínaRESUMEN
The limitations of the ganciclovir (GCV)/herpes simplex virus thymidine kinase (HSV1 TK: EC 2.7.1.21) system as a suicide gene therapy approach have been extensively studied over the years. In our study, we focused on improving the cytotoxic profile of the GCV/equine herpes virus-4 thymidine kinase (EHV4 TK: EC 2.7.1.21) system. Our approach involved the structure-guided mutagenesis of EHV4 TK in order to switch its ability to preferentially phosphorylate the natural substrate deoxythymidine (dT) to that of GCV. We performed steady-state kinetic analysis, genetic complementation in a thymidine kinase-deficient Escherichia coli strain, isothermal titration calorimetry, and analysis of GCV-induced cell killing through generation of HEK 293 stable cell-lines expressing EHV4 TK mutants and wild-type EHV4 TK. We found that the EHV4 TK S144H-GFP mutant preferentially phosphorylates GCV and confers increased GCV-induced cytotoxicity compared to wild-type EHV4 TK.
Asunto(s)
Ganciclovir/farmacología , Regulación Viral de la Expresión Génica/fisiología , Herpesvirus Équido 4/enzimología , Timidina Quinasa/metabolismo , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Proliferación Celular , Diseño de Fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Herpesvirus Équido 4/genética , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Conformación Proteica , Timidina Quinasa/genética , Proteínas Virales/genéticaRESUMEN
l-asparaginases hydrolyze l-asparagine to l-aspartic acid and ammonia. Enzymes of bacterial origin are used as therapeutic agents for the treatment of acute lymphoblastic leukemia. Recently, the structure of a human homolog, hASNase3, which possesses l-asparaginase activity, was solved setting the basis for the development of an anti-leukemic protein drug of human origin. Being an N-terminal hydrolase, hASNase3 undergoes intramolecular self-cleavage generating two protomers (subunits α and ß) which remain non-covalently associated and constitute the catalytically active form of the enzyme. However, recombinant expression of full-length hASNase3 in Escherichiacoli results in only partial processing towards the active enzyme. We developed a co-expression system for the two subunits that allowed production of the ß-subunit complexed to the α-subunit such that the N-terminal methionine is removed by endogenous methionine aminopeptidase to expose the catalytically essential threonine residue at the N-terminus of the ß-subunit. The enzyme produced by this co-expression strategy is fully active, thus obviating the necessity of self-activation by slow autoproteolytic cleavage.
RESUMEN
We report on the development of a sensitive real-time assay for monitoring the activity of L-asparaginase that hydrolyzes L-asparagine to L-aspartate and ammonia. In this method, L-aspartate is oxidized by L-aspartate oxidase to iminoaspartate and hydrogen peroxide (H2O2), and in the detection step horseradish peroxidase uses H2O2 to convert the colorless, nonfluorescent reagent Amplex Red to the red-colored and highly fluorescent product resorufin. The assay was validated in both the absorbance and the fluorescence modes. We show that, due to its high sensitivity and substrate selectivity, this assay can be used to measure enzymatic activity in human serum containing L-asparaginase.
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Asparaginasa/metabolismo , Fluorometría , Oxazinas/química , Espectrometría de Fluorescencia , Pruebas de Enzimas , Humanos , Oxazinas/metabolismo , Oxidación-Reducción , Especificidad por SustratoRESUMEN
The present study focuses on the formation of microcapsules containing catalytically active L-asparaginase (L-ASNase), a protein drug of high value in antileukemic therapy. We make use of the layer-by-layer (LbL) technique to coat protein-loaded calcium carbonate (CaCO3) particles with two or three poly dextran/poly-L-arginine-based bilayers. To achieve high loading efficiency, the CaCO3 template was generated by coprecipitation with the enzyme. After assembly of the polymer shell, the CaCO3 core material was dissolved under mild conditions by dialysis against 20 mM EDTA. Biochemical stability of the encapsulated L-asparaginase was analyzed by treating the capsules with the proteases trypsin and thrombin, which are known to degrade and inactivate the enzyme during leukemia treatment, allowing us to test for resistance against proteolysis by physiologically relevant proteases through measurement of residual l-asparaginase activities. In addition, the thermal stability, the stability at the physiological temperature, and the long-term storage stability of the encapsulated enzyme were investigated. We show that encapsulation of l-asparaginase remarkably improves both proteolytic resistance and thermal inactivation at 37 °C, which could considerably prolong the enzyme's in vivo half-life during application in acute lymphoblastic leukemia (ALL). Importantly, the use of low EDTA concentrations for the dissolution of CaCO3 by dialysis could be a general approach in cases where the activity of sensitive biomacromolecules is inhibited, or even irreversibly damaged, when standard protocols for fabrication of such LbL microcapsules are used. Encapsulated and free enzyme showed similar efficacies in driving leukemic cells to apoptosis.
Asunto(s)
Asparaginasa/química , Portadores de Fármacos/química , Proteínas de Escherichia coli/química , Polímeros/química , Proteínas de Saccharomyces cerevisiae/química , Antineoplásicos/química , Antineoplásicos/farmacología , Asparaginasa/farmacología , Materiales Biocompatibles/química , Carbonato de Calcio/química , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular/efectos de los fármacos , Portadores de Fármacos/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Electrólitos/química , Estabilidad de Enzimas , Escherichia coli/enzimología , Proteínas de Escherichia coli/farmacología , Humanos , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/farmacologíaRESUMEN
Human asparaginase 3 (hASNase3), which belongs to the N-terminal nucleophile hydrolase superfamily, is synthesized as a single polypeptide that is devoid of asparaginase activity. Intramolecular autoproteolytic processing releases the amino group of Thr168, a moiety required for catalyzing asparagine hydrolysis. Recombinant hASNase3 purifies as the uncleaved, asparaginase-inactive form and undergoes self-cleavage to the active form at a very slow rate. Here, we show that the free amino acid glycine selectively acts to accelerate hASNase3 cleavage both in vitro and in human cells. Other small amino acids such as alanine, serine, or the substrate asparagine are not capable of promoting autoproteolysis. Crystal structures of hASNase3 in complex with glycine in the uncleaved and cleaved enzyme states reveal the mechanism of glycine-accelerated posttranslational processing and explain why no other amino acid can substitute for glycine.
Asunto(s)
Asparaginasa/metabolismo , Glicina/metabolismo , Asparaginasa/química , Asparaginasa/genética , Asparagina/metabolismo , Biocatálisis , Cristalografía por Rayos X , Células HEK293 , Humanos , Concentración de Iones de Hidrógeno , Hidrólisis , Simulación de Dinámica Molecular , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Especificidad por SustratoRESUMEN
We demonstrate the design and integration of droplet-based microfluidic devices with microoptical element arrays for enhanced detection of fluorescent signals. We show that the integration of microlenses and mirror surfaces in these devices results in an 8-fold increase in the fluorescence signal and in improved spatial resolution. Using an array of microlenses, massively parallel detection of droplets containing fluorescent dyes was achieved, leading to detection throughputs of about 2000 droplets per second and per lens, parallelized over 625 measurement points.