RESUMEN
Dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is one of the drug target kinases involved in neurological disorders. DYRK1A phosphorylates substrate proteins related to disease progression in an intermolecular manner. Meanwhile, DYRK1A intramolecularly phosphorylates its own residues on key segments during folding process, which is required for its activation and stabilization. To reproduce the autophosphorylation in vitro, DYRK1A was expressed in Escherichia coli-based cell-free protein synthesis system. Although this system was useful for investigating autophosphorylation of serine residue at position 97 (Ser97) in DYRK1A, only a small fraction of the synthesized protein was successfully autophosphorylated. In this study, we found that the addition of DnaK, a bacterial HSP70 chaperone, to cell-free expression of DYRK1A promoted its Ser97 autophosphorylation. Structure prediction with AlphaFold2 indicates that Ser97 forms a hydrogen bond within an α-helix structure, indicating a possibility that DnaK unfolds the α-helix and maintains the structure around Ser97 in a conformation susceptible to phosphorylation. In addition, DnaK promoted phosphorylation of DYRK1B and HIPK2, but not DYRK2 and DYRK4, suggesting a sequence selectivity in the action of DnaK. This study provides a facile method for promoting autophosphorylation of DYRK family kinases in cell-free protein expression.
Asunto(s)
Escherichia coli , Procesamiento Proteico-Postraduccional , Fosforilación , Escherichia coli/genética , Biosíntesis de ProteínasRESUMEN
A concise route for dibenzoazacyclooctynes (DIBACs) synthesis was developed based on Pictet-Spengler reaction and a novel cobalt decomplexation method established for dibenzo-fused cyclooctyne-cobalt complexes. The method allowed for the facile preparation of functionalized DIBACs, including bisDIBAC, which served as an efficient bisreactive linker for protein modification via the double-click reaction.
RESUMEN
The kinase DYRK1A phosphorylates substrate proteins that are involved in the progression of many diseases. DYRK1A also phosphorylates its own residues on key elements intramolecularly to activate and stabilize itself during the folding process. Once the folding process of DYRK1A has completed, it can no longer catalyzes the intramolecular reaction, suggesting that a transitional intermediate state that catalyzes the autophosphorylation exists. In the previous study, we identified a small molecule, designated as FINDY, that selectively inhibits the folding intermediate of DYRK1A. Although evidence has suggested that FINDY targets the ATP-binding pocket of DYRK1A, it remains elusive as to whether the DYRK1A kinase domain could be purified as a complex with FINDY. In this study, we successfully expressed and purified the kinase domain of DYRK1A in complex with FINDY. The DYRK1A kinase domain was expressed as a fusion protein with a hexahistidine tag and ZZ-domain (His-ZZ-DYRK1A) at 6 °C by using a cold shock induction system in Escherichia coli cells. The cells were incubated with FINDY. The cell pellets were gently extracted on ice and subjected to immobilized-metal affinity chromatography. The amount of FINDY in the elution fraction was measured by UV absorbance specific for FINDY. The eluate contained FINDY with the ratio of FINDY to DYRK1A protein being 0.15 in quadruplicate experiments. Thus, this study demonstrates the direct interaction between the DYRK1A kinase domain and FINDY, paving the way for structural determination of the complex.
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Proteínas Serina-Treonina Quinasas , Proteínas Tirosina Quinasas , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/genéticaRESUMEN
DYRK1A phosphorylates proteins involved in neurological disorders in an intermolecular manner. Meanwhile, during the protein folding process of DYRK1A, a transitional folding intermediate catalyzes the intramolecular autophosphorylation required for the "one-off" inceptive activation and stabilization. In our previous study, a small molecule termed FINDY (1) was identified, which inhibits the folding intermediate-catalyzed intramolecular autophosphorylation of DYRK1A but not the folded state-catalyzed intermolecular phosphorylation. However, the structural features of FINDY (1) responsible for this intermediate-selective inhibition remain elusive. In this study, structural derivatives of FINDY (1) were designed and synthesized according to its predicted binding mode in the ATP pocket of DYRK1A. Quantitative structure-activity relationship (QSAR) of the derivatives revealed that the selectivity against the folding intermediate is determined by steric hindrance between the bulky hydrophobic moiety of the derivatives and the entrance to the pocket. In addition, a potent derivative 3 was identified, which inhibited the folding intermediate more strongly than FINDY (1); it was designated as dp-FINDY. Although dp-FINDY (3) did not inhibit the folded state, as well as FINDY (1), it inhibited the intramolecular autophosphorylation of DYRK1A in an in vitro cell-free protein synthesis assay. Furthermore, dp-FINDY (3) destabilized endogenous DYRK1A in HEK293 cells. This study provides structural insights into the folding intermediate-selective inhibition of DYRK1A and expands the chemical options for the design of a kinase inhibitor.
Asunto(s)
Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Tiazoles/farmacología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Estructura Molecular , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Relación Estructura-Actividad , Tiazoles/química , Quinasas DyrKRESUMEN
Metabolic activities are altered in cancer cells compared with those in normal cells, and the cancer-specific pathway becomes a potential therapeutic target. Higher cellular glucose consumption, which leads to lower glucose levels, is a hallmark of cancer cells. In an objective screening for chemicals that induce cell death under low-glucose conditions, we discovered a compound, denoted as ALESIA (Anticancer Ligand Enhancing Starvation-induced Apoptosis). By our shedding assay of transforming growth factor α in HEK293A cells, ALESIA was determined to act as a sphingosine-1-phosphate receptor 3-G12-biased agonist that promotes nitric oxide production and oxidative stress. The oxidative stress triggered by ALESIA resulted in the exhaustion of glucose, cellular NADPH deficiency, and then cancer cell death. Intraperitoneal administration of ALESIA improved the survival of mice with peritoneally disseminated rhabdomyosarcoma, indicating its potential as a new type of anticancer drug for glucose starvation therapy.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Glucosa/metabolismo , Neoplasias/tratamiento farmacológico , Receptores de Esfingosina-1-Fosfato/agonistas , Animales , Antineoplásicos/química , Línea Celular , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Desnudos , Neoplasias/metabolismo , Neoplasias/patología , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Óxido Nítrico/antagonistas & inhibidores , Óxido Nítrico/biosíntesis , Estrés Oxidativo/efectos de los fármacos , Receptores de Esfingosina-1-Fosfato/metabolismoRESUMEN
Drug discovery using small molecule inhibitors is reaching a stalemate due to low selectivity, adverse off-target effects and inevitable failures in clinical trials. Conventional chemical screening methods may miss potent small molecules because of their use of simple but outdated kits composed of recombinant enzyme proteins. Non-canonical inhibitors targeting a hidden pocket in a protein have received considerable research attention. Kii and colleagues identified an inhibitor targeting a transient pocket in the kinase DYRK1A during its folding process and termed it FINDY. FINDY exhibits a unique inhibitory profile; that is, FINDY does not inhibit the fully folded form of DYRK1A, indicating that the FINDY-binding pocket is hidden in the folded form. This intriguing pocket opens during the folding process and then closes upon completion of folding. In this review, we discuss previously established kinase inhibitors and their inhibitory mechanisms in comparison with FINDY. We also compare the inhibitory mechanisms with the growing concept of "cryptic inhibitor-binding sites." These sites are buried on the inhibitor-unbound surface but become apparent when the inhibitor is bound. In addition, an alternative method based on cell-free protein synthesis of protein kinases may allow the discovery of small molecules that occupy these mysterious binding sites. Transitional folding intermediates would become alternative targets in drug discovery, enabling the efficient development of potent kinase inhibitors.
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Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Sitios de Unión , Descubrimiento de Drogas/métodos , Humanos , Unión Proteica , Proteínas Recombinantes/farmacologíaRESUMEN
The hepatitis E virus (HEV) is a causative agent of hepatitis E. HEV virions in circulating blood and culture media are quasi-enveloped, while those in feces are nonenveloped. The capsid (ORF2) protein associated with an enveloped HEV virion is reported to comprise the translation product of leucine 14/methionine 16 to 660 (C-terminal end). However, the nature of the ORF2 protein associated with fecal HEV remains unclear. In the present study, we compared the molecular size of the ORF2 protein among fecal HEV, cell-culture-generated HEV (HEVcc), and detergent-treated protease-digested HEVcc. The ORF2 proteins associated with fecal HEV were C-terminally truncated and showed the same size as those of the detergent-treated protease-digested HEVcc virions (60 kDa), in contrast to those of the HEVcc (68 kDa). The structure prediction of the ORF2 protein (in line with previous studies) demonstrated that the C-terminal region (54 amino acids) of an ORF2 protein is in flux, suggesting that proteases target this region. The nonenveloped nondigested HEV structure prediction indicates that the C-terminal region of the ORF2 protein moves to the surface of the virion and is unnecessary for HEV infection. Our findings clarify the maturation of nonenveloped HEV and will be useful for studies on the HEV lifecycle.
RESUMEN
Efficient consecutive 1,2,3-triazole formations using multiazide platforms are disclosed. On the basis of unique clickability of the 1-adamantyl azido group, a four-step synthesis of tetrakis(triazole)s was achieved from a tetraazide platform molecule. This method was applied to a convergent synthesis of tetrafunctionalized probes in a modular synthetic manner.
RESUMEN
Essential components of the human circadian clock, BMAL1 and CLOCK, which are intrinsically disordered transcription factors, were expressed and subjected to a fluorescent in vitro binding assay using an E-box DNA fragment. Screening of a chemical library identified 5,8-quinoxalinedione (1), which was found to inhibit binding of the heterodimer BMAL1/CLOCK to E-box at low micromolar concentrations.
Asunto(s)
Factores de Transcripción ARNTL/antagonistas & inhibidores , Proteínas CLOCK/antagonistas & inhibidores , Relojes Circadianos , ADN/metabolismo , Proteínas Intrínsecamente Desordenadas/antagonistas & inhibidores , Quinoxalinas/farmacología , Factores de Transcripción ARNTL/química , Factores de Transcripción ARNTL/metabolismo , Proteínas CLOCK/química , Proteínas CLOCK/metabolismo , ADN/química , Relación Dosis-Respuesta a Droga , Humanos , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/metabolismo , Estructura Molecular , Unión Proteica/efectos de los fármacosRESUMEN
The irradiation of red light-emitting-diode light (λ = 660 nm) to 3-acyl-2-methoxyindolizines in the presence of a catalytic amount of methylene blue triggered the photooxidation of the indolizine ring, resulting in a nearly quantitative release of alcohols or carboxylic acids within a few minutes. The method was applicable for photouncaging various functional molecules such as a carboxylic anticancer drug and a phenolic fluorescent dye from the corresponding indolizine conjugates, including an insulin-indolizine-dye conjugate.
RESUMEN
Highly sensitive protein quantification enables the detection of a small number of protein molecules that serve as markers/triggers for various biological phenomena, such as cancer. Here, we describe the development of a highly sensitive protein quantification system called HaloTag protein barcoding. The method involves covalent linking of a target protein to a unique molecule counting oligonucleotide at a 1:1 conjugation ratio based on an azido-cycloalkyne click reaction. The sensitivity of the HaloTag-based barcoding was remarkably higher than that of a conventional luciferase assay. The HaloTag system was successfully validated by analyzing a set of protein-protein interactions, with the identification rate of 44% protein interactions between positive reference pairs reported in the literature. Desmoglein 3, the target antigen of pemphigus vulgaris, an IgG-mediated autoimmune blistering disease, was used in a HaloTag protein barcode assay to detect the anti-DSG3 antibody. The dynamic range of the assay was over 104-times wider than that of a conventional enzyme-linked immunosorbent assay (ELISA). The technology was used to detect anti-DSG3 antibody in patient samples with much higher sensitivity compared to conventional ELISA. Our detection system, with its superior sensitivity, enables earlier detection of diseases possibly allowing the initiation of care/treatment at an early disease stage.
Asunto(s)
Anticuerpos Antiidiotipos/aislamiento & purificación , Desmogleína 3/aislamiento & purificación , Dominios y Motivos de Interacción de Proteínas/genética , Proteínas/aislamiento & purificación , Anticuerpos Antiidiotipos/genética , Anticuerpos Antiidiotipos/inmunología , Enfermedades Autoinmunes/diagnóstico , Enfermedades Autoinmunes/inmunología , Química Clic , Cicloparafinas/química , Desmogleína 3/genética , Desmogleína 3/inmunología , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Luciferasas/química , Oligonucleótidos , Proteínas/genética , Proteínas/inmunologíaRESUMEN
Periostin is a secretory matricellular protein with a multi-domain structure that is composed of an amino-terminal EMI domain, a tandem repeat of four FAS 1 domains, and a carboxyl-terminal domain (CTD). Periostin has been suggested to function as a scaffold for assembly of several extracellular matrix proteins as well as its accessory proteins (Fig. 3.1, Table 3.1), which underlies highly sophisticated extracellular architectures. This scaffold function is likely due to periostin's multi-domain structure, in which the adjacent domains in periostin interact with different kinds of proteins, put these interacting proteins in close proximity, and promote intermolecular interactions between these proteins, leading to their assembly into a large complex. In this chapter, I introduce the proteins that interact with each of the adjacent domains in periostin, and discuss how the multi-domain structure of periostin functions as a scaffold for the assembly of the interacting proteins, and how it underlies construction of highly sophisticated extracellular architectures.
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Moléculas de Adhesión Celular/química , Proteínas de la Matriz Extracelular/química , Humanos , Dominios ProteicosRESUMEN
In physiological condition, periostin is expressed in limited tissues such as periodontal ligament, periosteum, and heart valves. Periostin protein is mainly localized on extracellular collagen bundles and in matricellular space. On the other hand, in pathological condition, expression of periostin is induced in disordered tissues of human patients. In tumor development and progression, periostin is elevated mainly in its microenvironment and stromal tissue rich in extracellular matrix. Tumor stromal fibroblasts highly express periostin and organize the tumor-surrounding extracellular matrix architecture. In fibrosis in lung, liver, and kidney, proliferating activated fibroblasts express periostin and replace normal functional tissues with dense connective tissues. In inflammation and allergy, inflammatory cytokines such as IL-4 and IL-13 induce expression of periostin that plays important roles in pathogenesis of these diseases. The elevated levels of periostin in human patients could be detected not only in tissue biopsy samples but also in peripheral bloods using specific antibodies against periostin, because periostin secreted from the disordered tissues is transported into blood vessels and circulates in the cardiovascular system. In this chapter, I introduce the elevated expression of periostin in pathological conditions, and discuss how periostin could be utilized as a biomarker in disease diagnosis.
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Biomarcadores/análisis , Moléculas de Adhesión Celular/análisis , Enfermedad , Matriz Extracelular , Fibroblastos , Fibrosis , HumanosRESUMEN
Hepatitis E, which is caused by hepatitis E virus (HEV), is generally a self-limiting, acute, and rarely fatal disease. It is sometimes fulminant and lethal, especially during pregnancy. Indeed, it occasionally takes a chronic course in immunocompromised individuals. To cure hepatitis E patients, the broad-spectrum antivirals (ribavirin and pegylated interferon α) are used. However, this treatment is insufficient and unsafe in some patients due to embryoteratogenic effects, leukopenia, and thrombocytopenia. In this study, we constructed an HEV replication reporter system with Gaussia luciferase for comprehensively screening anti-HEV drug candidates, and developed a cell-culture system using cells robustly producing HEV to validate the efficacy of anti-HEV drug candidates. We screened anti-HEV drug candidates from United States Food and Drug Administration-approved drugs using the established HEV replication reporter system, and investigated the selected candidates and type III interferons (interferon λ1-3) using the cell-culture system. In conclusion, we constructed an HEV replicon system for anti-HEV drug screening and a novel cell-culture system to strictly evaluate the replication-inhibitory activities of the obtained anti-HEV candidates. Our findings suggested that interferon λ1-3 might be effective for treating hepatitis E.
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Antivirales/farmacología , Evaluación Preclínica de Medicamentos/métodos , Virus de la Hepatitis E/efectos de los fármacos , Interferones/farmacología , Técnicas de Cultivo de Célula , Línea Celular , Genes Reporteros , Virus de la Hepatitis E/fisiología , Humanos , Replicón/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Interferón lambdaRESUMEN
A facile method for preparing various functional cycloalkynes, including proteins incorporated with a cycloalkyne moiety, from the corresponding azides is developed. Treatment of diynes bearing strained and terminal alkyne moieties with a copper salt enabled terminal alkyne-selective click conjugation with azides, whereas a more azidophilic strained alkyne moiety was transiently protected from the click reaction via complexation with copper.
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Alquinos/química , Azidas/química , Química Clic/métodos , Colorantes Fluorescentes/química , Proteínas/química , Alquinos/síntesis química , Azidas/síntesis química , Catálisis , Cobre/química , Ciclización , Reacción de Cicloadición/métodos , Colorantes Fluorescentes/síntesis química , Células HEK293 , Humanos , Imagen Óptica , Proteínas/análisis , Proteínas/síntesis químicaRESUMEN
We identified â¼30-mer amyloid-ß protein (Aß) assemblies, termed amylospheroids, from brains of patients with Alzheimer disease (AD) as toxic entities responsible for neurodegeneration and showed that Na+,K+-ATPase α3 (NAKα3) is the sole target of amylospheroid-mediated neurodegeneration. However, it remains unclear where in neurons amylospheroids form and how they reach their targets to induce neurodegeneration. Here, we present an in vitro culture system designed to chronologically follow amylospheroid formation in mature neurons expressing amyloid precursor protein bearing early-onset AD mutations. Amylospheroids were found to accumulate mainly in the trans-Golgi network of excitatory neurons and were initially transported in axons. Proteasome inhibition dramatically increased amylospheroid amounts in trans-Golgi by increasing Aß levels and induced dendritic transport. Amylospheroids were secreted and caused the degeneration of adjacent NAKα3-expressing neurons. Interestingly, the ASPD-producing neurons later died non-apoptotically. Our findings demonstrate a link between ASPD levels and proteasome function, which may have important implications for AD pathophysiology.
RESUMEN
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
RESUMEN
Efficient formation of water- and air-stable aza-ylides has been achieved using the Staudinger reaction between electron-deficient aromatic azides such as 2,6-dichlorophenyl azide and triarylphosphines. The reaction proceeds rapidly and has been successfully applied to chemical modification of proteins in living cells.
Asunto(s)
Compuestos Aza/química , Azidas/química , Química Clic , Glutatión Transferasa/química , Células HEK293 , Humanos , Microscopía Fluorescente , Fosfinas/químicaRESUMEN
Oxytocin (OXT) and arginine vasopressin (AVP) are structurally similar neuropeptide hormones that function as neurotransmitters in the brain, and have opposite key roles in social behaviors. These peptides bind to their G protein-coupled receptors (OXTR and AVPRs), inducing calcium ion-dependent signaling pathways and endocytosis of these receptors. Because selective agonists and antagonists for these receptors have been developed as therapeutic and diagnostic agents for diseases such as psychiatric disorders, facile methods are in demand for the evaluation of selectivity between these receptors. In this study, we developed a quantitative assay for OXT- and AVP-induced endocytosis of their receptors. The mutated Oplophorus luciferase, nanoKAZ, was fused to OXTR and AVPRs to enable rapid quantification of agonist-induced endocytosis by bioluminescence reduction. Agonist stimulation significantly decreases bioluminescence of nanoKAZ-fused receptors in living cells. Using this system, we evaluated clinically used OXTR antagonist atosiban and a reported pyrazinyltriazole derivative, hereby designated as PF13. Atosiban acted as an antagonist of AVPR1a, as well as an agonist for AVPR1b, whereas PF13 antagonized OXTR more selectively than atosiban, as reported previously. This paper shows a strategy for quantification of agonist-induced endocytosis of OXTR and AVPRs, and confirms its potent utility in the evaluation of agonists and antagonists.
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Endocitosis/efectos de los fármacos , Luciferasas/metabolismo , Mediciones Luminiscentes/métodos , Receptores de Oxitocina/metabolismo , Receptores de Vasopresinas/metabolismo , Vasotocina/análogos & derivados , Animales , Células CHO , Cricetulus , Células HEK293 , Humanos , Oxidación-Reducción/efectos de los fármacos , Vasotocina/farmacologíaRESUMEN
A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.