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The global incidence of invasive fungal infections (IFIs) has increased over the past few decades, mainly in immunocompromised patients, and is associated with high mortality and morbidity. Aspergillus fumigatus is one of the most common and deadliest IFI pathogens. Major hurdles to treating fungal infections remain the lack of rapid and definitive diagnosis, including the frequent need for invasive procedures to provide microbiological confirmation, and the lack of specificity of structural imaging methods. To develop an Aspergillus-specific positron emission tomography (PET) imaging agent, we focused on fungal-specific sugar metabolism. We radiolabeled cellobiose, a disaccharide known to be metabolized by Aspergillus species, and synthesized 2-deoxy-2-[18F]fluorocellobiose ([18F]FCB) by enzymatic conversion of 2-deoxy-2-[18F]fluoroglucose ([18F]FDG) with a radiochemical yield of 60 to 70%, a radiochemical purity of >98%, and 1.5 hours of synthesis time. Two hours after [18F]FCB injection in A. fumigatus pneumonia as well as A. fumigatus, bacterial, and sterile inflammation myositis mouse models, retained radioactivity was only seen in foci with live A. fumigatus infection. In vitro testing confirmed production of ß-glucosidase enzyme by A. fumigatus and not by bacteria, resulting in hydrolysis of [18F]FCB into glucose and [18F]FDG, the latter being retained by the live fungus. The parent molecule was otherwise promptly excreted through the kidneys, resulting in low background radioactivity and high target-to-nontarget ratios at A. fumigatus infectious sites. We conclude that [18F]FCB is a promising and clinically translatable Aspergillus-specific PET tracer.
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Aspergillus fumigatus , Celobiosa , Tomografía de Emisión de Positrones , Animales , Tomografía de Emisión de Positrones/métodos , Celobiosa/metabolismo , Aspergillus fumigatus/metabolismo , Ratones , Aspergilosis/diagnóstico por imagen , Fluorodesoxiglucosa F18/química , Aspergillus/metabolismo , Distribución Tisular , Radiofármacos/química , Radiofármacos/metabolismoRESUMEN
Very-long-chain polyunsaturated fatty acids (VLCPUFAs; C24-38) constitute a unique class of PUFA that have important biological roles, but the lack of a suitable dietary source has limited research in this field. We produced an n-3 C24-28-rich VLCPUFA-oil concentrated from fish oil to study its bioavailability and physiological functions in C57BL/6J mice. The serum and retinal C24:5 levels increased significantly compared to control after a single-dose gavage, and VLCPUFAs were incorporated into the liver, brain, and eyes after 8-week supplementation. Dietary VLCPUFAs resulted in favorable cardiometabolic changes, and improved electroretinography responses and visual performance. VLCPUFA supplementation changed the expression of genes involved in PPAR signaling pathways. Further in vitro studies demonstrated that the VLCPUFA-oil and chemically synthesized C24:5 are potent agonists for PPARs. The multiple potential beneficial effects of fish oil-derived VLCPUFAs on cardiometabolic risk and eye health in mice support future efforts to develop VLCPUFA-oil into a supplemental therapy.
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Protein palmitoylation, with more than 5000 substrates, is the most prevalent form of protein lipidation. Palmitoylated proteins participate in almost all areas of cellular physiology and have been linked to several human diseases. Twenty-three zDHHC enzymes catalyze protein palmitoylation with extensive overlap among the substrates of each zDHHC member. Currently, there is no global strategy to delineate the physiological substrates of individual zDHHC enzymes without perturbing the natural cellular pool. Here, we outline a general approach to accomplish this on the basis of synthetic orthogonal substrates that are only compatible with engineered zDHHC enzymes. We demonstrate the utility of this strategy by validating known substrates and use it to identify novel substrates of two human zDHHC enzymes. Finally, we employ this method to discover and explore conserved palmitoylation in a family of host restriction factors against pathogenic viruses, including SARS-CoV-2.
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Aciltransferasas , COVID-19 , Humanos , Aciltransferasas/metabolismo , Especificidad por Sustrato , SARS-CoV-2/metabolismo , Proteínas/metabolismo , LipoilaciónRESUMEN
The use of radiolabeled glucose for PET imaging resulted in the most commonly used tracer in the clinic, 2-deoxy-2-[18F]fluoroglucose (FDG). More recently, other radiolabeled sugars have been reported for various applications, including imaging tumors and infections. Therefore, in this study, we developed a series of fluorine-18-labeled L-rhamnose derivatives as potential PET tracers of various fungal and bacterial strains. Acetyl-protected triflate precursors of rhamnose were prepared and radiolabeled with fluorine-18 followed by hydrolysis to produce L-deoxy [18F]fluororhamnose. The overall radiochemical yield was 7-27% in a 90 min synthesis time with a radiochemical purity of 95%. In vivo biodistribution of the ligands using PET imaging showed that 2-deoxy-2-[18F]fluoro-L-rhamnose is stable for at least up to 60 min in mice and eliminated via renal clearance. The tracer also exhibited minimal tissue or skeletal uptake in healthy mice resulting in a low background signal.
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Radioisótopos de Flúor , Ramnosa , Ratones , Animales , Distribución Tisular , Línea Celular Tumoral , Tomografía de Emisión de Positrones/métodos , RadiofármacosRESUMEN
Huntington's disease is a neurodegenerative disorder caused by a CAG expansion in the first exon of the HTT gene, resulting in an extended polyglutamine (poly-Q) tract in huntingtin (httex1). The structural changes occurring to the poly-Q when increasing its length remain poorly understood due to its intrinsic flexibility and the strong compositional bias. The systematic application of site-specific isotopic labeling has enabled residue-specific NMR investigations of the poly-Q tract of pathogenic httex1 variants with 46 and 66 consecutive glutamines. Integrative data analysis reveals that the poly-Q tract adopts long α-helical conformations propagated and stabilized by glutamine side chain to backbone hydrogen bonds. We show that α-helical stability is a stronger signature in defining aggregation kinetics and the structure of the resulting fibrils than the number of glutamines. Our observations provide a structural perspective of the pathogenicity of expanded httex1 and pave the way to a deeper understanding of poly-Q-related diseases.
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Exones , Proteína Huntingtina/genética , Proteína Huntingtina/química , Espectroscopía de Resonancia Magnética , Conformación Proteica en Hélice alfaRESUMEN
Wild-type P53-induced phosphatase 1 (WIP1), also known as PPM1D or PP2Cδ, is a serine/threonine protein phosphatase induced by P53 after genotoxic stress. WIP1 inhibition has been proposed as a therapeutic strategy for P53 wild-type cancers in which it is overexpressed, but this approach would be ineffective in P53-negative cancers. Furthermore, there are several cancers with mutated P53 where WIP1 acts as a tumor suppressor. Therefore, activating WIP1 phosphatase might also be a therapeutic strategy, depending on the P53 status. To date, no specific, potent WIP1 inhibitors with appropriate pharmacokinetic properties have been reported, nor have WIP1-specific activators. Here, we report the discovery of new WIP1 modulators from a high-throughput screen (HTS) using previously described orthogonal biochemical assays suitable for identifying both inhibitors and activators. The primary HTS was performed against a library of 102â¯277 compounds at a single concentration using a RapidFire mass spectrometry assay. Hits were further evaluated over a range of 11 concentrations with both the RapidFire MS assay and an orthogonal fluorescence-based assay. Further biophysical, biochemical, and cell-based studies of confirmed hits revealed a WIP1 activator and two inhibitors, one competitive and one uncompetitive. These new scaffolds are prime candidates for optimization which might enable inhibitors with improved pharmacokinetics and a first-in-class WIP1 activator.
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BACKGROUND: The activities of MYC, the androgen receptor, and its associated pioneer factors demonstrate substantial reprogramming between early and advanced prostate cancer. Although previous studies have shown a shift in cellular metabolic requirements associated with prostate cancer progression, the epigenetic regulation of these processes is incompletely described. Here, we have integrated chromatin immunoprecipitation sequencing (ChIP-seq) and whole-transcriptome sequencing to identify novel regulators of metabolism in advanced prostate tumors characterized by elevated MYC activity. RESULTS: Using ChIP-seq against MYC, HOXB13, and AR in LNCaP cells, we observed redistribution of co-bound sites suggestive of differential KMT2A activity as a function of MYC expression. In a cohort of 177 laser-capture microdissected foci of prostate tumors, KMT2A expression was positively correlated with MYC activity, AR activity, and HOXB13 expression, but decreased with tumor grade severity. However, KMT2A expression was negatively correlated with these factors in 25 LuCaP patient-derived xenograft models of advanced prostate cancer and 99 laser-capture microdissected foci of metastatic castration-resistant prostate cancer. Stratified by KMT2A expression, ChIP-seq against AR and HOXB13 in 15 LuCaP patient-derived xenografts showed an inverse association with sites involving genes implicated in lipid metabolism, including the arachidonic acid metabolic enzyme PLA2G4F. LuCaP patient-derived xenograft models grown as organoids recapitulated the inverse association between KMT2A expression and fluorine-18 labeled arachidonic acid uptake in vitro. CONCLUSIONS: Our study demonstrates that the epigenetic activity of transcription factor oncogenes exhibits a shift during prostate cancer progression with distinctive phenotypic effects on metabolism. These epigenetically driven changes in lipid metabolism may serve as novel targets for the development of novel imaging agents and therapeutics.
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Cyanine dyes are exceptionally useful probes for a range of fluorescence-based applications, but their photon output can be limited by trans-to-cis photoisomerization. We recently demonstrated that appending a ring system to the pentamethine cyanine ring system improves the quantum yield and extends the fluorescence lifetime. Here, we report an optimized synthesis of persulfonated variants that enable efficient labeling of nucleic acids and proteins. We demonstrate that a bifunctional sulfonated tertiary amide significantly improves the optical properties of the resulting bioconjugates. These new conformationally restricted cyanines are compared to the parent cyanine derivatives in a range of contexts. These include their use in the plasmonic hotspot of a DNA-nanoantenna, in single-molecule Förster-resonance energy transfer (FRET) applications, far-red fluorescence-lifetime imaging microscopy (FLIM), and single-molecule localization microscopy (SMLM). These efforts define contexts in which eliminating cyanine isomerization provides meaningful benefits to imaging performance.
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Carbocianinas/química , Fotones , Transferencia Resonante de Energía de Fluorescencia , Microscopía Fluorescente , Conformación MolecularRESUMEN
WT P53-Induced Phosphatase 1 (WIP1) is a member of the magnesium-dependent serine/threonine protein phosphatase (PPM) family and is induced by P53 in response to DNA damage. In several human cancers, the WIP1 protein is overexpressed, which is generally associated with a worse prognosis. Although WIP1 is an attractive therapeutic target, no potent, selective, and bioactive small-molecule modulator with favorable pharmacokinetics has been reported. Phosphatase enzymes are among the most challenging targets for small molecules because of the difficulty of achieving both modulator selectivity and bioavailability. Another major obstacle has been the availability of robust and physiologically relevant phosphatase assays that are suitable for high-throughput screening. Here, we describe orthogonal biochemical WIP1 activity assays that utilize phosphopeptides from native WIP1 substrates. We optimized an MS assay to quantify the enzymatically dephosphorylated peptide reaction product in a 384-well format. Additionally, a red-shifted fluorescence assay was optimized in a 1,536-well format to enable real-time WIP1 activity measurements through the detection of the orthogonal reaction product, Pi We validated these two optimized assays by quantitative high-throughput screening against the National Center for Advancing Translational Sciences (NCATS) Pharmaceutical Collection and used secondary assays to confirm and evaluate inhibitors identified in the primary screen. Five inhibitors were further tested with an orthogonal WIP1 activity assay and surface plasmon resonance binding studies. Our results validate the application of miniaturized physiologically relevant and orthogonal WIP1 activity assays to discover small-molecule modulators from high-throughput screens.
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Activadores de Enzimas/química , Fosfopéptidos/química , Proteína Fosfatasa 2C/química , Bibliotecas de Moléculas Pequeñas/química , Activadores de Enzimas/aislamiento & purificación , Activadores de Enzimas/farmacología , Ensayos Analíticos de Alto Rendimiento , Humanos , Proteína Fosfatasa 2C/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/aislamiento & purificación , Bibliotecas de Moléculas Pequeñas/farmacología , Especificidad por Sustrato , Proteína p53 Supresora de Tumor/químicaRESUMEN
Genetic code expansion (GCE) technology allows the specific incorporation of functionalized noncanonical amino acids (ncAAs) into proteins. Here, we investigated the Diels-Alder reaction between trans-cyclooct-2-ene (TCO)-modified ncAAs, and 22 known and novel 1,2,4,5-tetrazine-dye conjugates spanning the entire visible wavelength range. A hallmark of this reaction is its fluorogenicity - the tetrazine moiety can elicit substantial quenching of the dye. We discovered that photoinduced electron transfer (PET) from the excited dye to tetrazine is the main quenching mechanism in red-absorbing oxazine and rhodamine derivatives. Upon reaction with dienophiles quenching interactions are reduced resulting in a considerable increase in fluorescence intensity. Efficient and specific labeling of all tetrazine-dyes investigated permits super-resolution microscopy with high signal-to-noise ratio even at the single-molecule level. The different cell permeability of tetrazine-dyes can be used advantageously for specific intra- and extracellular labeling of proteins and highly sensitive fluorescence imaging experiments in fixed and living cells.
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Colorantes Fluorescentes/química , Código Genético , Microscopía Confocal/instrumentación , Imagen Óptica/instrumentación , Animales , Células COS , Chlorocebus aethiops , Colorantes/química , Reacción de Cicloadición , Ciclooctanos/química , Células HEK293 , Compuestos Heterocíclicos/química , Compuestos Heterocíclicos con 1 Anillo/química , Humanos , Microscopía Confocal/métodos , Imagen Óptica/métodos , Rodaminas , Coloración y EtiquetadoRESUMEN
Hepatocellular carcinoma (HCC) is a common cause of cancer-related death worldwide. As obesity and diabetes become more prevalent, the contribution of non-alcoholic fatty liver disease (NAFLD) to HCC is rising. Recently, we reported intrahepatic CD4+ T cells are critical for anti-tumor surveillance in NAFLD. Lipid accumulation in the liver is the hallmark of NAFLD, which may perturb T cell function. We sought to investigate how the lipid-rich liver environment influences CD4+ T cells by focusing on carnitine palmitoyltransferase (CPT) family members, which control the mitochondrial ß-oxidation of fatty acids and act as key molecules in lipid catabolism. Linoleic acid (C18:2) co-localized within the mitochondria along with a corresponding increase in CPT gene upregulation. This CPT upregulation can be recapitulated by feeding mice with a high-C18:2 diet or the NAFLD promoting methionine-choline-deficient (MCD) diet. Using an agonist and antagonist, the induction of CPT genes was found to be mediated by peroxisome proliferator-activated receptor alpha (PPAR-α). CPT gene upregulation increased mitochondrial reactive oxygen species (ROS) and led to cell apoptosis. In vivo, using liver-specific inducible MYC transgenic mice fed MCD diet, blocking CPT with the pharmacological inhibitor perhexiline decreased apoptosis of intrahepatic CD4+ T cells and inhibited HCC tumor formation. These results provide useful information for potentially targeting the CPT family to rescue intrahepatic CD4+ T cells and to aid immunotherapy for NAFLD-promoted HCC.
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Apoptosis/genética , Linfocitos T CD4-Positivos/patología , Carcinogénesis/patología , Carcinoma Hepatocelular/genética , Carnitina O-Palmitoiltransferasa/genética , Ácido Linoleico/farmacología , Neoplasias Hepáticas/genética , Regulación hacia Arriba/genética , Células 3T3 , Animales , Apoptosis/efectos de los fármacos , Linfocitos T CD4-Positivos/efectos de los fármacos , Carcinogénesis/genética , Carcinoma Hepatocelular/patología , Carnitina O-Palmitoiltransferasa/antagonistas & inhibidores , Carnitina O-Palmitoiltransferasa/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Ácido Linoleico/química , Neoplasias Hepáticas/patología , Ratones , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Modelos Biológicos , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/patología , PPAR alfa/metabolismo , Perhexilina/farmacología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Both agonists and antagonists of the UDP-activated P2Y6 receptor (P2Y6R) have been proposed for therapeutic use, in conditions such as cancer, inflammation, neurodegeneration and diabetes. Uracil nucleotides containing a South-bicyclo[3.1.0]hexane ((S)-methanocarba) ring system in place of the ribose ring were synthesized and shown to be potent P2Y6R agonists in a calcium mobilization assay. The (S)-methanocarba modification was compatible with either a 5-iodo or 4-methoxyimino group on the pyrimidine, but not with a α,ß-methylene 5´-diphosphate. (S)-Methanocarba dinucleotide potency was compatible with a N4-methoxy modification on the proximal nucleoside that is assumed to bind at the P2Y6R similarly to UDP; (N)-methanocarba was preferred on the distal nucleoside moiety. This suggests that the distal dinucleotide P2Y6R binding site prefers a ribose-like group that can attain a (N) conformation, rather than (S). Dinucleotide binding was modeled by homology modeling, docking and molecular dynamics simulations, which suggested the same ribose conformational preferences found empirically.
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Glutamine (Gln) and its analogues may serve as imaging agents for tumor diagnosis using positron emission tomography (PET), especially for tumors with negative [(18)F]FDG scan. We report the first automated synthesis of [(18)F](2S,4R)-4-fluoroglutamine ([(18)F]FGln) on a GE TRACERlab™ FX-N Pro module. [(18)F]FGln was obtained in 80±3min with a radiochemical yield of 21±3% (n=5, uncorrected). The radiochemical purity was >98%, and optical purity 90±5%. The synthesis is highly reproducible with good chemical purity, radiochemical yield, and is suitable for translation to cGMP production.
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Radioisótopos de Flúor , Glutamina/análogos & derivados , Radiofármacos/síntesis química , Automatización/instrumentación , Automatización/métodos , Técnicas de Química Sintética/instrumentación , Técnicas de Química Sintética/métodos , Estabilidad de Medicamentos , Glutamina/síntesis química , Humanos , Neoplasias/diagnóstico por imagen , Tomografía de Emisión de Positrones/métodos , Tecnología Radiológica/instrumentación , Tecnología Radiológica/métodosRESUMEN
Nanoscopy has now become a real procedure in fluorescence microscopy of living cells. The STED/RESOLFT family of nanoscopy approaches has the best prospects for delivering high speed imaging, but the history of STED includes a continuing struggle to reduce the deactivation power applied, along with difficulties in achieving simultaneous multicolor images. In this manuscript, we present a concept for a similar real-time nanoscopy, using a new class of bipartite probes that separate the luminescent and quenching functions into two coupled molecules. In particular, the STAQ (Superresolution via Transiently Activated Quencher) example we show herein employs the excited state absorbance (not ground state) of the partner to accept energy from and quench the luminescent dye. The result is that much less deactivation power is needed for superresolved (â¼50 nm) imaging. Moreover, the TAQ partner excited by the "donut" beam is shown to quench several different visible dyes via the same mechanism, opening the door to easier multicolor imaging. We demonstrate three dyes sharing the same deactivation and show examples of superresolved multicolor images. We suggest STAQ will facilitate the growth of real-time nanoscopy by reducing confounding photodamage within living cells while expanding the nanoscopist's palette.
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Colorantes Fluorescentes/análisis , Procesamiento de Imagen Asistido por Computador/métodos , Microscopía Fluorescente/métodos , Coloración y Etiquetado/métodos , Color , LuminiscenciaRESUMEN
INTRODUCTION: The small molecule 2-(5-(dimethylamino)naphthalene-1-sulfonamido)-2-(fluoromethyl)butanoic acid (NST732) is a member of the ApoSense family of compounds, capable of selective targeting, binding and accumulation within cells undergoing apoptotic cell death. It has application in molecular imaging and blood clotting particularly for monitoring antiapoptotic drug treatments. We are investigating a fluorine-18-radiolabeled analog of this compound for positron emission tomography studies. METHODS: We prepared the tosylate precursor methyl 2-(5-(dimethylamino)naphthalene-1-sulfonamido)-2-(tosyloxymethyl)butanoate (4) to synthesize fluorine-18-labeled NST732. Fluorination reaction of the tosylate precursor in 1:1 acetonitrile:dimethylsulfoxide with tetrabutyl ammonium fluoride proceeds through an aziridine intermediate (4A) to afford two regioisomers: 2-(5-(dimethylamino)naphthalene-1-sulfonamido)-2-fluorobutanoate (5) and methyl 2-(5-(dimethylamino)naphthalene-1-sulfonamido)-2-(fluoromethyl)butanoate (6). Acid hydrolysis of the fluoromethylbutanoate (6) isomer produced NST732. As the fluorination reaction of the tosylate precursor proceeds through an aziridine intermediate (4A) and the fluorination conceivably could be done directly on the aziridine, we have separately prepared an aziridine precursor (4A). Fluorine-18 labeling of the aziridine precursor (4A) was performed with [(18)F]tetrabutyl ammonium fluoride to afford the same two regioisomers (5 and 6). The [18F]2-((5-dimethylamino)naphthalene-1-sulfonamido)methyl)-2-fluorobutanoic acid (NST732) was then obtained by the hydrolysis of corresponding [18F]-labeled ester (6) with 6 N hydrochloric acid. RESULTS: Two regioisomers obtained from the fluorination reaction of aziridine were easily separated by high-performance liquid chromatography. The total radiochemical yield was 15%±3% (uncorrected, n=18) from the aziridine precursor in a 70-min synthesis time with a radiochemical purity>99%. CONCLUSION: Fluorine-18-labeled ApoSense compound [18F]NST732 is prepared in moderate yield by direct fluorination of an aziridine precursor.
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Aminobutiratos/química , Aminobutiratos/síntesis química , Aziridinas/química , Ácido Butírico/química , Ácido Butírico/síntesis química , Técnicas de Química Sintética/métodos , Sulfonamidas/química , Sulfonamidas/síntesis química , Técnicas de Química Sintética/normas , Radioquímica , Estándares de ReferenciaRESUMEN
Alpha-galactosidase A hydrolyzes the terminal alpha-galactosyl moieties from glycolipids and glycoproteins in lysosomes. Mutations in alpha-galactosidase cause lysosomal accumulation of the glycosphingolipid, globotriaosylceramide, which leads to Fabry disease. Small-molecule chaperones that bind to mutant enzyme proteins and correct their misfolding and mistrafficking have emerged as a potential therapy for Fabry disease. We have synthesized a red fluorogenic substrate, resorufinyl alpha-D-galactopyranoside, for a new alpha-galactosidase enzyme assay. This assay can be measured continuously at lower pH values, without the addition of a stop solution, due to the relatively low pK(a) of resorufin (approximately 6). In addition, the assay emits red fluorescence, which can significantly reduce interferences due to compound fluorescence and dust/lint as compared to blue fluorescence. Therefore, this new red fluorogenic substrate and the resulting enzyme assay can be used in high-throughput screening to identify small-molecule chaperones for Fabry disease.
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Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/metabolismo , Galactósidos/química , Galactósidos/síntesis química , alfa-Galactosidasa/metabolismo , 1-Desoxinojirimicina/análogos & derivados , 1-Desoxinojirimicina/farmacología , Cromatografía en Capa Delgada , Fluorescencia , Colorantes Fluorescentes/química , Concentración de Iones de Hidrógeno , Cinética , Oxazinas/química , Sensibilidad y Especificidad , alfa-Galactosidasa/antagonistas & inhibidores , alfa-Galactosidasa/químicaRESUMEN
Mutations in alpha-glucosidase cause accumulation of glycogen in lysosomes, resulting in Pompe disease, a lysosomal storage disorder. Small molecule chaperones that bind to enzyme proteins and correct the misfolding and mistrafficking of mutant proteins have emerged as a new therapeutic approach for the lysosomal storage disorders. In addition, alpha-glucosidase is a therapeutic target for type II diabetes, and alpha-glucosidase inhibitors have been used in the clinic as alternative treatments for this disease. We have developed a new fluorogenic substrate for the alpha-glucosidase enzyme assay, resorufin alpha-d-glucopyranoside. The enzyme reaction product of this new substrate emits at a peak of 590 nm, reducing the interference from fluorescent compounds seen with the existing fluorogenic substrate, 4-methylumbelliferyl-alpha-D-glucopyranoside. Also, the enzyme kinetic assay can be carried out continuously without the addition of stop solution due to the lower pK(a) of the product of this substrate. Therefore, this new fluorogenic substrate is a useful tool for the alpha-glucosidase enzyme assay and will facilitate compound screening for the development of new therapies for Pompe disease.
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Benzoxazinas/análisis , Colorantes Fluorescentes/análisis , Glucósidos/análisis , Oxazinas/análisis , alfa-Glucosidasas/metabolismo , Benzoxazinas/síntesis química , Benzoxazinas/química , Colorantes Fluorescentes/química , Glucósidos/síntesis química , Glucósidos/química , Enfermedad del Almacenamiento de Glucógeno Tipo II/diagnóstico , Humanos , Cinética , Mutación , Oxazinas/síntesis química , Oxazinas/química , Espectrometría de FluorescenciaRESUMEN
Fluorescent tagging is a powerful tool for imaging proteins in living cells. However, the steric effects imposed by fluorescent tags impair the behavior of many proteins. Here, we report a novel technique, Instant with DTT, EDT, And Low temperature (IDEAL)-labeling, for rapid and specific FlAsH-labeling of tetracysteine-tagged cell surface proteins by using prion protein (PrP) and amyloid precursor protein (APP) as models. In prion-infected cells, FlAsH-labeled tetracysteine-tagged PrP converted from the normal isoform (PrPsen) to the disease-associated isoform (PrPres), suggesting minimal steric effects of the tag. Pulse-chase analysis of PrP and APP by fluorescent gel imaging demonstrated the utility of IDEAL labeling in investigating protein metabolism by identifying an as-yet-unrecognized C-terminal fragment (C3) of PrPsen and by characterizing the kinetics of PrPres and APP metabolism. C3 generation and N-terminal truncation of PrPres were inhibited by the anti-prion compound E64, a cysteine protease inhibitor. Surprisingly, E64 did not inhibit the synthesis of new PrPres, providing insight into the mechanism by which E64 reduces steady-state PrPres levels in prion-infected cells. To expand the versatility of tetracysteine tagging, we created new Alexa Fluor- and biotin-conjugated tetracysteine-binding molecules that were applied to imaging PrP endocytosis and ultrastructural localization. IDEAL-labeling extends the use of biarsenical derivatives to extracellular proteins and beyond microscopic imaging.
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Precursor de Proteína beta-Amiloide , Arsenicales/metabolismo , Colorantes Fluorescentes/metabolismo , Proteínas de la Membrana , Priones , Coloración y Etiquetado/métodos , Secuencia de Aminoácidos , Precursor de Proteína beta-Amiloide/química , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Arsenicales/química , Biotina/química , Biotina/metabolismo , Línea Celular , Colorantes Fluorescentes/química , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Ratones , Datos de Secuencia Molecular , Estructura Molecular , Priones/química , Priones/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismoRESUMEN
We have shown previously that a potent synthetic antagonist of growth factor receptor-bound protein 2 (Grb2) Src homology 2 (SH2) domain binding (1) blocks growth factor stimulated motility, invasion, and angiogenesis in cultured cell models, as well as tumor metastasis in animals. To characterize the selectivity of 1 for the SH2 domain of Grb2 over other proteins containing similar structural binding motifs, we synthesized a biotinylated derivative (3) that retained high affinity Grb2 SH2 domain binding and potent biological activity. To investigate the selectivity of 1 and 3 for Grb2, the biotinylated antagonist 3 was used to immobilize target proteins from cell extracts for subsequent identification by mass spectrometry. Non-specific binding was identified in parallel using a biotinylated analogue that lacked a single critical binding determinant. The mechanism of action of the antagonist was further characterized by immunoprecipitation, immunoblotting, and light microscopy. This approach to defining protein binding antagonist selectivity and molecular basis of action should be widely applicable in drug development.
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Biotina/farmacología , Proteína Adaptadora GRB2/antagonistas & inhibidores , Dominios Homologos src/efectos de los fármacos , Sitios de Unión , Biotina/análogos & derivados , Biotina/química , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Estructura Molecular , Relación Estructura-ActividadRESUMEN
Mitochondrial NADH fluorescence has been a useful tool in evaluating mitochondrial energetics both in vitro and in vivo. Mitochondrial NADH fluorescence is enhanced several-fold in the matrix through extended fluorescence lifetimes (EFL). However, the actual binding sites responsible for NADH EFL are unknown. We tested the hypothesis that NADH binding to Complex I is a significant source of mitochondrial NADH fluorescence enhancement. To test this hypothesis, the effect of Complex I binding on NADH fluorescence efficiency was evaluated in purified protein, and in native gels of the entire porcine heart mitochondria proteome. To avoid the oxidation of NADH in these preparations, we conducted the binding experiments under anoxic conditions in a specially designed apparatus. Purified intact Complex I enhanced NADH fluorescence in native gels approximately 10-fold. However, no enhancement was detected in denatured individual Complex I subunit proteins. In the Clear and Ghost native gels of the entire mitochondrial proteome, NADH fluorescence enhancement was localized to regions where NADH oxidation occurred in the presence of oxygen. Inhibitor and mass spectroscopy studies revealed that the fluorescence enhancement was specific to Complex I proteins. No fluorescence enhancement was detected for MDH or other dehydrogenases in this assay system, at physiological mole fractions of the matrix proteins. These data suggest that NADH associated with Complex I significantly contributes to the overall mitochondrial NADH fluorescence signal and provides an explanation for the well established close correlation of mitochondrial NADH fluorescence and the metabolic state.