RESUMO
Sensitive detection of two biological events in vivo has long been a goal in bioluminescence imaging. Antares, a fusion of the luciferase NanoLuc to the orange fluorescent protein CyOFP, has emerged as a bright bioluminescent reporter with orthogonal substrate specificity to firefly luciferase (FLuc) and its derivatives such as AkaLuc. However, the brightness of Antares in mice is limited by the poor solubility and bioavailability of the NanoLuc substrate furimazine. Here, we report a new substrate, hydrofurimazine, whose enhanced aqueous solubility allows delivery of higher doses to mice. In the liver, Antares with hydrofurimazine exhibited similar brightness to AkaLuc with its substrate AkaLumine. Further chemical exploration generated a second substrate, fluorofurimazine, with even higher brightness in vivo. We used Antares with fluorofurimazine to track tumor size and AkaLuc with AkaLumine to visualize CAR-T cells within the same mice, demonstrating the ability to perform two-population imaging with these two luciferase systems.
Assuntos
Furanos/química , Luciferases/química , Medições Luminescentes/métodos , Proteínas Luminescentes/química , Animais , Ensaios Enzimáticos/métodos , Especificidade por SubstratoRESUMO
Nowadays, cancers and dementia, such as Alzheimer's disease, are the most fatal causes of death. Many studies tried to understand the pathogenesis of those diseases clearly and develop a promising way to treat the diseases. Matrix metalloproteinases (MMPs) have been reported to be involved in the pathology of cancers and AD through tumor cell movement and amyloid degradation. Therefore, control of the levels and actions of MMPs, especially MMP-2 and MMP-9, is necessary to care for and/or cure cancer and AD. Various molecules have been examined for their potential application as regulators of MMPs expression and activity. Among the molecules, multiple metal complexes have shown advantages, including simple synthesis, less toxicity and specificity toward MMPs in cancer cells or in the brain. In this review, we summarize the recent studies and knowledge of metal complexes (e.g., Pt-, Ru-, Au-, Fe-, Cu-, Ni-, Zn-, and Sn-complexes) targeting MMPs and their potentials for treating and/or caring the most fatal human diseases, cancers and AD.
Assuntos
Doença de Alzheimer , Complexos de Coordenação , Humanos , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/patologia , Metaloproteinases da Matriz/metabolismo , Encéfalo/metabolismoRESUMO
Fat-soluble vitamins (vitamin A, D, E, and K) assume a pivotal role in maintaining human homeostasis by virtue of their enzymatic functions. The daily inclusion of these vitamins is imperative to the upkeep of various physiological processes including vision, bone health, immunity, and protection against oxidative stress. Current research highlights fat-soluble vitamins as potential therapeutics for human diseases, especially cancer. Fat-soluble vitamins exert their therapeutic effects through multiple pathways, including regulation of matrix metalloproteinases' (MMPs) expression and enzymatic activity. As MMPs have been reported to be involved in the pathology of various diseases, such as cancers, cardiovascular diseases, and neurological disorders, regulating the expression and/or activity of MMPs could be considered as a potent therapeutic strategy. Here, we summarize the properties of fat-soluble vitamins and their potential as promising candidates capable of effectively modulating MMPs through multiple pathways to treat human diseases.
Assuntos
Doenças Cardiovasculares , Vitamina A , Humanos , Vitamina A/farmacologia , Metaloproteinase 2 da Matriz , Vitaminas/uso terapêutico , Vitamina K , Doenças Cardiovasculares/tratamento farmacológico , Vitamina D/uso terapêutico , Vitamina ERESUMO
Fluorescent indicators are used widely to visualize calcium dynamics downstream of membrane depolarization or G-protein-coupled receptor activation, but are poorly suited for non-invasive imaging in mammals. Here, we report a bright calcium-modulated bioluminescent indicator named Orange CaMBI (Orange Calcium-modulated Bioluminescent Indicator). Orange CaMBI reports calcium dynamics in single cells and, in the context of a transgenic mouse, reveals calcium oscillations in whole organs in an entirely non-invasive manner.
Assuntos
Cálcio/química , Proteínas Luminescentes/química , Imagem Óptica , Compostos Organometálicos/química , Animais , Medições Luminescentes , Camundongos , Camundongos TransgênicosRESUMO
We found that electron attachment to the van der Waals complex (O2···CO2) turns the weak intermolecular bond into a pseudochemical bond of significant strength. The resulting monomeric molecular anion (O2-CO2)- may be a form of CO4-, the gaseous anionic species suspected to be present in Earth's ionosphere whose chemical characteristics have not been comprehensively identified since its existence was first predicted by Conway in 1962. The measured vertical detachment energy of CO4- is very large (4.56 ± 0.05 eV), while the known electron affinity of its component species is much smaller (0.448 eV, O2) or even negative (-0.6 eV, CO2). These characteristics are correctly borne out by theoretical calculations that show that electron attachment transforms the van der Waals complex to a single contiguous molecular anion, with the formation of a pseudochemical bond between O2 and CO2 through an extended π-orbital system.
RESUMO
Redox-active metal ions, Cu(I/II) and Fe(II/III), are essential biological molecules for the normal functioning of the brain, including oxidative metabolism, synaptic plasticity, myelination, and generation of neurotransmitters. Dyshomeostasis of these redox-active metal ions in the brain could cause Alzheimer's disease (AD). Thus, regulating the levels of Cu(I/II) and Fe(II/III) is necessary for normal brain function. To control the amounts of metal ions in the brain and understand the involvement of Cu(I/II) and Fe(II/III) in the pathogenesis of AD, many chemical agents have been developed. In addition, since toxic aggregates of amyloid-ß (Aß) have been proposed as one of the major causes of the disease, the mechanism of clearing Aß is also required to be investigated to reveal the etiology of AD clearly. Multiple metalloenzymes (e.g., neprilysin, insulin-degrading enzyme, and ADAM10) have been reported to have an important role in the degradation of Aß in the brain. These amyloid degrading enzymes (ADE) could interact with redox-active metal ions and affect the pathogenesis of AD. In this review, we introduce and summarize the roles, distributions, and transportations of Cu(I/II) and Fe(II/III), along with previously invented chelators, and the structures and functions of ADE in the brain, as well as their interrelationships.
Assuntos
Doença de Alzheimer/metabolismo , Amiloide/metabolismo , Encéfalo/metabolismo , Proteína ADAM10/metabolismo , Doença de Alzheimer/etiologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Barreira Hematoencefálica/metabolismo , Quelantes/metabolismo , Cobre/metabolismo , Humanos , Insulisina/metabolismo , Ferro/metabolismo , Proteínas de Membrana/metabolismo , Metais/metabolismo , Neprilisina/metabolismo , Oxirredução , ProteóliseRESUMO
BRAF mutants are categorized into three classes according to dependency on RAS signaling and RAF dimerization-dependency. Class I BRAF V600 mutants (RAS-independent monomer) are sensitive to vemurafenib. In contrast, both class II mutants (RAS-independent dimer) and class III mutants (RAS-dependent heterodimer) are insensitive to vemurafenib. It is not likely that BRAF inhibitors capable of inhibiting all classes of BRAF mutants are currently available. Herein, we report GNF-7 and its novel derivative, SIJ1227 as the first BRAF inhibitors capable of inhibiting all classes of BRAF mutants. Compared with vemurafenib and PLX8394, both GNF-7 and SIJ1227 possess much more strong anti-proliferative activities on melanoma (A375 and C8161) and lung cancer cells (H1755 and H1666) harboring BRAF V600E (class I mutant), BRAF G464E/G469A (class II mutant) and BRAF G466V (class III mutant), respectively. Also, both GNF-7 and SIJ1227 are capable of inhibiting more strongly colony formation than vemurafenib and PLX8394 in 3D soft agar assay using C8161 melanoma cells. In addition, GNF-7 and SIJ1227 suppress more strongly migration/invasion of these cancer cells than vemurafenib and PLX8394. Taken together, both GNF-7 and SIJ1227 are much superior to vemurafenib and PLX8394 in terms of capability to inhibit all classes of BRAF mutants.
Assuntos
Antineoplásicos/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/patologia , Simulação de Acoplamento Molecular , Mutação , Proteínas Proto-Oncogênicas B-raf/química , Pirimidinonas/farmacologia , Vemurafenib/farmacologiaRESUMO
A robust method for simultaneous visualization of all four cell cycle phases in living cells is highly desirable. We developed an intensiometric reporter of the transition from S to G2 phase and engineered a far-red fluorescent protein, mMaroon1, to visualize chromatin condensation in mitosis. We combined these new reporters with the previously described Fucci system to create Fucci4, a set of four orthogonal fluorescent indicators that together resolve all cell cycle phases.
Assuntos
Ciclo Celular/fisiologia , Proteínas Luminescentes/química , Imagem Molecular/métodos , Proteínas Recombinantes de Fusão/química , Imagem com Lapso de Tempo/métodos , Animais , Técnicas de Cultura de Células , Cromatina/metabolismo , Fase G2/fisiologia , Células HEK293 , Células HeLa , Humanos , Proteínas Luminescentes/genética , Camundongos , Mitose , Modelos Moleculares , Células NIH 3T3 , Proteínas Recombinantes de Fusão/genética , Fase S/fisiologia , Proteína Vermelha FluorescenteRESUMO
Anaplastic lymphoma kinase (ALK) has been recognised as a promising molecular target of targeted therapy for NSCLC. We performed SAR study of pyrazolo[3,4-b]pyridines to override crizotinib resistance caused by ALK-L1196M mutation and identified a novel and potent L1196M inhibitor, 10g. 10g displayed exceptional enzymatic activities (<0.5 nM of IC50) against ALK-L1196M as well as against ALK-wt. In addition, 10g is an extremely potent inhibitor of ROS1 (<0.5 nM of IC50) and displays excellent selectivity over c-Met. Moreover, 10g strongly suppresses proliferation of ALK-L1196M-Ba/F3 and H2228 cells harbouring EML4-ALK via apoptosis and the ALK signalling blockade. The results of molecular docking studies reveal that, in contrast to crizotinib, 10g engages in a favourable interaction with M1196 in the kinase domain of ALK-L1196M and hydrogen bonding with K1150 and E1210. This SAR study has provided a useful insight into the design of novel and potent inhibitors against ALK gatekeeper mutant.
Assuntos
Quinase do Linfoma Anaplásico/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Pirazóis/farmacologia , Piridinas/farmacologia , Quinase do Linfoma Anaplásico/metabolismo , Apoptose/efeitos dos fármacos , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Linhagem Celular Transformada , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida , Cristalografia por Raios X , Inibidores Enzimáticos/química , Humanos , Concentração Inibidora 50 , Simulação de Acoplamento Molecular , Espectroscopia de Prótons por Ressonância Magnética , Pirazóis/química , Piridinas/química , Transdução de Sinais/efeitos dos fármacos , Espectrometria de Massas por Ionização por Electrospray , Relação Estrutura-AtividadeRESUMO
The human FGF receptors (FGFRs) play critical roles in various human cancers, and several FGFR inhibitors are currently under clinical investigation. Resistance usually results from selection for mutant kinases that are impervious to the action of the drug or from up-regulation of compensatory signaling pathways. Preclinical studies have demonstrated that resistance to FGFR inhibitors can be acquired through mutations in the FGFR gatekeeper residue, as clinically observed for FGFR4 in embryonal rhabdomyosarcoma and neuroendocrine breast carcinomas. Here we report on the use of a structure-based drug design to develop two selective, next-generation covalent FGFR inhibitors, the FGFR irreversible inhibitors 2 (FIIN-2) and 3 (FIIN-3). To our knowledge, FIIN-2 and FIIN-3 are the first inhibitors that can potently inhibit the proliferation of cells dependent upon the gatekeeper mutants of FGFR1 or FGFR2, which confer resistance to first-generation clinical FGFR inhibitors such as NVP-BGJ398 and AZD4547. Because of the conformational flexibility of the reactive acrylamide substituent, FIIN-3 has the unprecedented ability to inhibit both the EGF receptor (EGFR) and FGFR covalently by targeting two distinct cysteine residues. We report the cocrystal structure of FGFR4 with FIIN-2, which unexpectedly exhibits a "DFG-out" covalent binding mode. The structural basis for dual FGFR and EGFR targeting by FIIN3 also is illustrated by crystal structures of FIIN-3 bound with FGFR4 V550L and EGFR L858R. These results have important implications for the design of covalent FGFR inhibitors that can overcome clinical resistance and provide the first example, to our knowledge, of a kinase inhibitor that covalently targets cysteines located in different positions within the ATP-binding pocket.
Assuntos
Antineoplásicos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos , Substituição de Aminoácidos , Antineoplásicos/química , Antineoplásicos/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Cristalografia por Raios X , Resistencia a Medicamentos Antineoplásicos/genética , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/química , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Mutação de Sentido Incorreto , Neoplasias/enzimologia , Neoplasias/genética , Neoplasias/patologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/química , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/química , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/química , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/metabolismo , Relação Estrutura-AtividadeRESUMO
A combination of DNA stretching method and super-resolution nanoscopy allows an accurate and precise measurement of the length of DNA fragments ranging widely in size from 117 to 23,130 bp. BstEII- and HindIII-treated λDNA fragments were stained with an intercalating dye and then linearly stretched on a coverslip by dynamic molecular combing. The image of individual DNA fragments was obtained by stimulated emission depletion nanoscopy. For DNA fragments longer than â¼1000 bp, the measured lengths of DNA fragments were consistently within â¼0.5 to 1.0 % of the reference values, raising the possibility of this method in a wide range of applications including facile detection for copy number variations and trinucleotide repeat disorder.
Assuntos
DNA/análise , Microscopia Confocal/instrumentação , Microscopia de Fluorescência/instrumentação , Nanotecnologia/instrumentação , Desenho de Equipamento , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Simulação de Dinâmica Molecular , Nanotecnologia/métodosRESUMO
BACKGROUND: The zoonotic transmission of highly pathogenic avian influenza viruses and the global pandemic of H1N1 influenza in 2009 signified the need for a wider coverage of therapeutic options for the control of influenza. METHODS: An in-house compound library was screened using a cytopathic effect inhibition assay. Selected hits were then tested in vivo and used as a core skeleton for derivative synthesis. RESULTS: The hit compound (BMD-2601505) was effective [50% effective concentration (EC50) of 60-70 µM] in reducing the death rate of cells infected with human influenza A and B viruses as well as avian influenza A virus. Furthermore, BMD-2601505 reduced the weight loss and increased the survival after lethal infection. The compound was further modified to enhance its antiviral potency. Results show that one derivative with bromobenzene moiety was most effective (EC50 of 22-37 µM) against the influenza viruses tested. CONCLUSION: We identified a small benzamide compound exhibiting antiviral activity against influenza viruses. The results warrant further evaluation of antiviral activities against drug-resistant influenza isolates.
Assuntos
Antivirais/farmacologia , Benzamidas/farmacologia , Vírus da Influenza A/efeitos dos fármacos , Vírus da Influenza B/efeitos dos fármacos , Animais , Antivirais/administração & dosagem , Antivirais/química , Benzamidas/administração & dosagem , Benzamidas/química , Bromobenzenos/administração & dosagem , Bromobenzenos/química , Bromobenzenos/farmacologia , Cães , Feminino , Humanos , Influenza Humana/tratamento farmacológico , Influenza Humana/virologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/tratamento farmacológico , Infecções por Orthomyxoviridae/virologia , Compostos de Fenilureia/administração & dosagem , Compostos de Fenilureia/química , Compostos de Fenilureia/farmacologia , Bibliotecas de Moléculas Pequenas , Zoonoses/tratamento farmacológico , Zoonoses/virologiaRESUMO
We investigated electron attachment to three dihalobenzene molecules, bromochlorobenzene (BCB), bromoiodobenzene (BIB) and chloroiodobenzene (CIB), by molecular beam photoelectron spectroscopy. The most prominent product of electron attachment in the anion mass spectra was the atomic fragment of the less electronegative halogen of the two, i.e., Br(-) for BCB and I(-) for BIB and CIB. Photoelectron spectroscopy and ab initio calculations suggested that the approaching electron prefers to attack the less electronegative atom, a seemingly counterintuitive finding but consistent with the mass spectrometric result. For the iodine-containing species BIB and CIB, the photoelectron spectrum consists of bands from both the molecular anion and atomic I(-), the latter of which is produced by photodissociation of the former. Molecular orbital analysis revealed that a large degree of orbital energy reordering takes place upon electron attachment. These phenomena were shown to be readily explained by simple molecular orbital theory and the electronegativity of the halogen atoms.
Assuntos
Elétrons , Hidrocarbonetos Halogenados/química , Hidrocarbonetos Halogenados/classificação , Teoria Quântica , Espectroscopia FotoeletrônicaRESUMO
The Castanopsis cuspidata var. sieboldii (CCS) plant grows predominantly in temperate regions of Asian countries, such as South Korea. Research on CCS has so far concentrated on the nutritional analysis, antioxidant activity, and anti-inflammation properties of its branches. However, the isolation of compounds and structural elucidation of effective single molecules remain unexplored, necessitating further exploration of CCS branches. Therefore, this study demonstrates the antioxidant and antimelanogenic activity of a single substance of ethyl gallate (EG) isolated from CCS branch extracts. Notably, the antimelanogenic (whitening) activity of EG extracted from CCS branches remains unexplored. Tyrosinase inhibition, kinetic enzyme assays, and molecular docking studies were conducted using mushroom tyrosinase in order to examine the antioxidant mechanism and antimelanin activity of EG in B16F10 melanoma cells. Nontoxic EG concentrations were found to be below 5 µg/mL. While EG significantly reduced the levels of whitening-associated proteins, p-CREB, and p-PKA, it dose-dependently inhibited the expression of TYR, TRP-1, TRP-2, and transcription factor (MITF). In addition, EG downregulated melanogenetic gene expression and activated autophagy signals. Therefore, EG extracted from CCS branches could serve as a novel functional cosmetic material with antimelanogenic and autophagy-enhancing activity.
RESUMO
Hepatocellular carcinoma (HCC) is disease with a high mortality rate and limited treatment options. Alterations of fibroblast growth factor receptor 4 (FGFR4) has been regarded as an oncogenic driver for HCC and a promising target for HCC therapeutics. Herein, we report that GNF-7, a multi-targeted kinase inhibitor, and its derivatives including SIJ1263 (IC50 < 1 nM against FGFR4) are highly potent FGFR4 inhibitors and are capable of strongly suppressing proliferation of HCC cells and Ba/F3 cells transformed with wtFGFR4 or mtFGFR4. Compared with known FGFR4 inhibitors, both GNF-7 and SIJ1263 possess much higher (up to 100-fold) anti-proliferative activities via FGFR signaling blockade and apoptosis on HCC cells. Especially, SIJ1263 is 80-fold more potent (GI50 = 24 nM) on TEL-FGFR4 V550E Ba/F3 cells than BLU9931, which suggests that SIJ1263 would be effective for overriding drug resistance. In addition, both substances strongly suppress migration/invasion and colony formation of HCC cells. It is worth noting that SIJ1263 is superior to GNF-7 with regards to the fact that activities of SIJ1263 are higher than those of GNF-7 in all assays performed in this study. Collectively, this study provides insight into designing highly potent FGFR4 inhibitors capable of potentially overcoming drug-resistance for the treatment of HCC patients.
Assuntos
Antineoplásicos/farmacologia , Pirimidinonas/farmacologia , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Pirimidinonas/química , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/genética , Relação Estrutura-AtividadeRESUMO
Cystic fibrosis transmembrane conductance regulator (CFTR) is a cell-surface anion channel that permeates chloride and bicarbonate ions. The most frequent mutation of CFTR that causes cystic fibrosis is the deletion of phenylalanine at position 508 (ΔF508), which leads to defects in protein folding and cellular trafficking to the plasma membrane. The lack of the cell-surface CFTR results in a reduction in the lifespan due to chronic lung infection with progressive deterioration of lung function. Hsc70 plays a crucial role in degradation of mutant CFTR by the ubiquitin-proteasome system. To date, various Hsc70 inhibitors and transcription regulators have been tested to determine whether they correct the defective activity of mutant CFTR. However, they exhibited limited or questionable effects on restoring the chloride channel activity in cystic fibrosis cells. Herein, we show that a small molecule apoptozole (Az) has high cellular potency to promote membrane trafficking of mutant CFTR and its chloride channel activity in cystic fibrosis cells. Results from affinity chromatography and ATPase activity assay indicate that Az inhibits the ATPase activity of Hsc70 by binding to its ATPase domain. In addition, a ligand-directed protein labeling and molecular modeling studies also suggest the binding of Az to an ATPase domain, in particular, an ATP-binding pocket. It is proposed that Az suppresses ubiquitination of ΔF508-CFTR maybe by blocking interaction of the mutant with Hsc70 and CHIP, and, as a consequence, it enhances membrane trafficking of the mutant.
Assuntos
Adenosina Trifosfatases/metabolismo , Benzamidas/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Proteínas de Choque Térmico HSC70/metabolismo , Imidazóis/metabolismo , Mutação , Adenosina Trifosfatases/química , Benzamidas/química , Sítios de Ligação , Linhagem Celular , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Humanos , Imidazóis/química , Espectroscopia de Ressonância Magnética , Transporte Proteico , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , UbiquitinaçãoRESUMO
BACKGROUND: Sialic-acid-binding immunoglobulin-like lectins (Siglecs) are the best-characterized immunoglobulin-type lectins. There is a growing amount of data linking Siglec and autoimmune diseases. The recently identified Siglec-9 inhibits T cell receptor (TCR)-mediated signaling which has been demonstrated by site-directed mutagenesis. In human Siglec-9, at least 8 nonsynonymous SNPs have been detected without functional studies. This study examined the SNP(s) related to TCR-mediated signaling. METHODS: Since the functions of Siglecs are modulated by their interaction with sialic-acid-containing carbohydrate groups, a molecular modeling analysis of carbohydrate binding interactions and an RBC binding analysis were performed using the 8 SNPs. The TCR-mediated signaling was analyzed with the downstream signaling molecules ZAP-70 and IL-2. RESULTS: This study revealed that an A391C polymorphism is the only mutant related to the binding. Jurkat T cells transfected with the A391C mutant reduced the inhibition of ZAP-70 phosphorylation and IL-2 production compared to cells transfected with the wild type. CONCLUSIONS: Siglec-9 A391C was the only polymorphism related to TCR-mediated signaling in human Siglec-9, resulting in less inhibition compared to the wild type.
Assuntos
Antígenos CD/genética , Lectinas/genética , Receptores de Antígenos de Linfócitos T/imunologia , Antígenos CD/imunologia , Western Blotting , Eritrócitos/imunologia , Citometria de Fluxo , Humanos , Interleucina-2/imunologia , Células Jurkat , Lectinas/imunologia , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ácido N-Acetilneuramínico/imunologia , Fosforilação/imunologia , Polimorfismo de Nucleotídeo Único , RNA Mensageiro/química , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Lectinas Semelhantes a Imunoglobulina de Ligação ao Ácido Siálico , Transdução de Sinais , Transfecção , Proteína-Tirosina Quinase ZAP-70/imunologiaRESUMO
Cyclic adenosine monophosphate (cAMP) plays a key role in signal transduction pathways as a second messenger. Studies on the cAMP dynamics provided useful scientific insights for drug development and treatment of cAMP-related diseases such as some cancers and prefrontal cortex disorders. For example, modulation of cAMP-mediated intracellular signaling pathways by anti-tumor drugs could reduce tumor growth. However, most early stage tools used for measuring the cAMP level in living organisms require cell disruption, which is not appropriate for live cell imaging or animal imaging. Thus, in the last decades, tools were developed for real-time monitoring of cAMP distribution or signaling dynamics in a non-invasive manner. Genetically-encoded sensors based on fluorescent proteins and luciferases could be powerful tools to overcome these drawbacks. In this review, we discuss the recent genetically-encoded cAMP sensors advances, based on single fluorescent protein (FP), Föster resonance energy transfer (FRET), single luciferase, and bioluminescence resonance energy transfer (BRET) for real-time non-invasive imaging.
Assuntos
Técnicas Biossensoriais , AMP Cíclico/análise , Proteínas Luminescentes , Animais , Transdução de SinaisRESUMO
Autophagy plays an important role in maintaining tumor cell progression and survival in response to metabolic stress. Thus, the regulation of autophagy can be used as a strategy for anticancer therapy. Here, we report dutomycin (DTM) as a novel autophagy enhancer that eventually induces apoptosis due to excessive autophagy. Also, human serine protease inhibitor B6 (SERPINB6) was identified as a target protein of DTM, and its novel function which is involved in autophagy was studied for the first time. We show that DTM directly binds SERPINB6 and then activates intracellular serine proteases, resulting in autophagy induction. Inhibitory effects of DTM on the function of SERPINB6 were confirmed through enzyme- and cell-based approaches, and SERPINB6 was validated as a target protein using siRNA-mediated knockdown and an overexpression test. In a zebrafish xenograft model, DTM showed a significant decrease in tumor area. Furthermore, the present findings will be expected to contribute to the expansion of novel basic knowledge about the correlation of cancer and autophagy by promoting active further research on SERPINB6, which was not previously considered the subject of cancer biology.
Assuntos
Antraciclinas/uso terapêutico , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Serpinas/metabolismo , Animais , Antraciclinas/metabolismo , Antraciclinas/farmacologia , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Células HeLa , Humanos , Serina Proteases/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Peixe-ZebraRESUMO
Various amyloidogenic proteins have been suggested to be involved in the onset and progression of neurodegenerative diseases (ND) such as Alzheimer's disease (AD) and Parkinson's disease (PD). Particularly, the aggregation of misfolded amyloid-ß and hyperphosphorylated tau and α-synuclein are linked to the pathogenesis of AD and PD, respectively. In order to care the diseases, multiple small molecules have been developed to regulate the aggregation pathways of these amyloid proteins. In addition to controlling the aggregation of amyloidogenic proteins, maintaining the levels of the proteins in the brain by amyloid degrading enzymes (ADE; neprilysin (NEP), insulin-degrading enzyme (IDE), asparagine endopeptidase (AEP), and ADAM10) is also essential to cure AD and PD. Therefore, numerous biological molecules and chemical agents have been investigated as either inducer or inhibitor against the levels and activities of ADE. Although the side effect of enhancing the activity of ADE could occur, the removal of amyloidogenic proteins could result in a relatively good strategy to treat AD and PD. Furthermore, since the causes of ND are diverse, various multifunctional (multitarget) chemical agents have been designed to control the actions of multiple risk factors of ND, including amyloidogenic proteins, metal ions, and reactive oxygen species. Many of them, however, were invented without considerations of regulating ADE levels and actions. Incorporation of previously created molecules with the chemical agents handling ADE could be a promising way to treat AD and PD. This review introduces the ADE and molecules capable of modulating the activity and expression of ADE.