RESUMO
Carboxypeptidases (CPs) are a family of hydrolases that cleave one or more amino acids from the C-terminal of peptides or proteins and play indispensable roles in various physiological and pathological processes. However, only a few highly activatable fluorescence probes for CPs have been reported, and there is a need for a flexibly tunable molecular design platform to afford a range of fluorescence probes for CPs for biological and medical research. Here, we focused on the unique activation mechanism of ProTide-based prodrugs and established a modular design platform for CP-targeting florescence probes based on ProTide chemistry. In this design, probe properties such as fluorescence emission wavelength, reactivity/stability, and target CP can be readily tuned and optimized by changing the four probe modules: the fluorophore, the substituent on the phosphorus atom, the linker amino acid at the P1 position, and the substrate amino acid at the P1' position. In particular, switching the linker amino acid at position P1 enabled us to precisely optimize the reactivity for target CPs. As a proof-of-concept, we constructed probes for carboxypeptidase M (CPM) and prostate-specific membrane antigen (also known as glutamate carboxypeptidase II). The developed probes were applicable for the imaging of CP activities in live cells and in clinical specimens from patients. This design strategy should be useful in studying CP-related biological and pathological phenomena.
Assuntos
Carboxipeptidases , Ariloxifosforamidatos , Masculino , Humanos , Fluorescência , Carboxipeptidases/metabolismo , Hidrolases , Aminoácidos , Corantes Fluorescentes/químicaRESUMO
PURPOSE: Anesthesia has been shown to disrupt the circadian rhythm. Recovery of the circadian rhythm after general anesthesia might help alleviate symptoms of insomnia and postoperative delirium. We hypothesized that recovery of the circadian rhythm is faster after total knee arthroplasty (TKA) with desflurane than with sevoflurane. This study compared the influence of sevoflurane versus desflurane anesthesia on the postoperative circadian rhythm of melatonin in adults undergoing TKA. DESIGN: Single-center, prospective, randomized, controlled, open-label study. METHODS: This study involved adult patients undergoing TKA at a university hospital in Japan from May 1, 2018 to December 31, 2019. The primary outcome of the study was the comparison of the effect of sevoflurane and desflurane on the circadian rhythm of salivary melatonin for 3 days postoperatively. The secondary outcomes were postoperative fatigue and sleep quality for 3 days postoperatively. FINDINGS: Twenty-eight patients (American Society of Anesthesiologists physical status of I or II) were scheduled for TKA and randomized to receive sevoflurane (n = 14) or desflurane (n = 14) anesthesia. There was no significant difference in the melatonin concentration between the sevoflurane and desflurane groups. The salivary melatonin concentration after sevoflurane or desflurane anesthesia was significantly higher at 9:00 p.m. on a postoperative day (POD)0 and POD1 than on POD3 (P < .05). Patients in the desflurane group had significantly greater fatigue than those in the sevoflurane group at 7:00 a.m. and 12:00 p.m. on POD3 (P < .05). Patients in the sevoflurane group had a deeper sleep than those in the desflurane group on POD0 (P < .05). In the sevoflurane group, the sleep time during the night of POD2 was longer than that on POD0 (6.1 vs 4.2 hours, P < .05). CONCLUSIONS: Under the current study conditions, desflurane was equivalent to sevoflurane in terms of the postoperative salivary melatonin concentration and sleep disturbance after TKA but not in terms of recovering the postoperative circadian rhythm.
Assuntos
Artroplastia do Joelho , Desflurano , Sevoflurano , Adulto , Humanos , Anestésicos Inalatórios/farmacologia , Artroplastia do Joelho/efeitos adversos , Desflurano/farmacologia , Melatonina/metabolismo , Estudos Prospectivos , Sevoflurano/farmacologia , Transtornos do Sono-Vigília , Saliva/químicaRESUMO
Synthetic chemical fluorescent dyes promise to be useful for many applications in biology. Covalent, targeted labeling, such as with a SNAP-tag, uses synthetic dyes to label specific proteins in vivo for studying processes such as endocytosis or for imaging via super-resolution microscopy. Despite its potential, such chemical tagging has not been used effectively in plants. A major drawback has been the limited knowledge regarding cell wall and membrane permeability of the available synthetic dyes. Of 31 synthetic dyes tested here, 23 were taken up into BY-2 cells, while eight were not. This creates sets of dyes that can serve to measure endocytosis. Three of the dyes that were able to enter the cells, SNAP-tag ligands of diethylaminocoumarin, tetramethylrhodamine, and silicon-rhodamine 647, were used to SNAP-tag α-tubulin. Successful tagging was verified by live cell imaging and visualization of microtubule arrays in interphase and during mitosis in Arabidopsis (Arabidopsis thaliana) seedlings. Fluorescence activation-coupled protein labeling with DRBG-488 was used to observe PIN-FORMED2 (PIN2) endocytosis and delivery to the vacuole as well as preferential delivery of newly synthesized PIN2 to the actively forming cell plate during mitosis. Together, the data demonstrate that specific self-labeling of proteins can be used effectively in plants to study a wide variety of cellular and biological processes.
Assuntos
Proteínas de Arabidopsis/metabolismo , Corantes Fluorescentes/farmacocinética , Células Vegetais/química , Arabidopsis/citologia , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Endocitose , Corantes Fluorescentes/química , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , O(6)-Metilguanina-DNA Metiltransferase/química , Células Vegetais/efeitos dos fármacos , Células Vegetais/metabolismo , Plantas Geneticamente Modificadas , Rodaminas/química , Rodaminas/farmacocinética , Plântula , Imagem com Lapso de Tempo , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismoRESUMO
Parkin promotes cell survival by removing damaged mitochondria via mitophagy. However, although some studies have suggested that Parkin induces cell death, the regulatory mechanism underlying the dual role of Parkin remains unknown. Herein, we report that mitochondrial ubiquitin ligase (MITOL/MARCH5) regulates Parkin-mediated cell death through the FKBP38-dependent dynamic translocation from the mitochondria to the ER during mitophagy. Mechanistically, MITOL mediates ubiquitination of Parkin at lysine 220 residue, which promotes its proteasomal degradation, and thereby fine-tunes mitophagy by controlling the quantity of Parkin. Deletion of MITOL leads to accumulation of the phosphorylated active form of Parkin in the ER, resulting in FKBP38 degradation and enhanced cell death. Thus, we have shown that MITOL blocks Parkin-induced cell death, at least partially, by protecting FKBP38 from Parkin. Our findings unveil the regulation of the dual function of Parkin and provide a novel perspective on the pathogenesis of PD.
Assuntos
Mitofagia , Ubiquitina-Proteína Ligases , Sobrevivência Celular , Células HeLa , Humanos , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Fluorogenic probes for bioimaging have become essential tools for life science and medicine, and the key to their development is a precise understanding of the mechanisms available for fluorescence off/on control, such as photoinduced electron transfer (PeT) and Förster resonance energy transfer (FRET). Here we establish a new molecular design strategy to rationally develop activatable fluorescent probes, which exhibit a fluorescence off/on change in response to target biomolecules, by controlling the twisted intramolecular charge transfer (TICT) process. This approach was developed on the basis of a thorough investigation of the fluorescence quenching mechanism of N-phenyl rhodamine dyes (commercially available as the QSY series) by means of time-dependent density functional theory (TD-DFT) calculations and photophysical evaluation of their derivatives. To illustrate and validate this TICT-based design strategy, we employed it to develop practical fluorogenic probes for HaloTag and SNAP-tag. We further show that the TICT-controlled fluorescence off/on mechanism is generalizable by synthesizing a Si-rhodamine-based fluorogenic probe for HaloTag, thus providing a palette of chemical dyes that spans the visible and near-infrared range.
Assuntos
Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes , Corantes Fluorescentes/química , Rodaminas , IonóforosRESUMO
We established a methodology for initiating cross-linking of antibodies selectively on the cell surface through intermolecular copper-free click reactions facilitated by increased effective concentrations of antibodies binding to target antigens. Upon cross-linking of tetrazine- and bicyclononyne-modified trastuzumab on the surface of HER2-overexpressing cells, increased antibody uptake and activation of intracellular signaling were observed. Our findings demonstrate that the cross-linking reaction can significantly alter the biophysical properties of proteins, activating their unique functionalities on targeted cells to realize an increased cargo delivery and synthetic manipulation of cellular signaling.
Assuntos
Compostos Aza/imunologia , Compostos Bicíclicos com Pontes/imunologia , Reagentes de Ligações Cruzadas/química , Trastuzumab/imunologia , Células 3T3 , Animais , Compostos Aza/química , Compostos Bicíclicos com Pontes/química , Linhagem Celular Tumoral , Humanos , Camundongos , Estrutura Molecular , Receptor ErbB-2/química , Receptor ErbB-2/imunologia , Propriedades de Superfície , Trastuzumab/químicaRESUMO
Methyl transfer reactions play important roles in many biological phenomena, wherein the methylation cofactor S-adenosyl-l-methionine (SAM) serves as the important currency to orchestrate those reactions. We have developed a fluorescent-probe-based high-throughput screening (HTS) system to search for the compounds that control cellular SAM levels. HTS with a drug repositioning library revealed the importance of catechol-O-methyltransferase (COMT) and its substrates in controlling the SAM concentrations and histone methylation levels in colorectal tumor cells.
Assuntos
Catecóis/farmacologia , Epigênese Genética , Redes e Vias Metabólicas , S-Adenosilmetionina/metabolismo , Animais , Catecol O-Metiltransferase/metabolismo , Células HT29 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos NusRESUMO
Ca2+ is one of the most important second messengers in cells. A far-red to near-infrared (NIR) Ca2+ fluorescent probe is useful for multi-color imaging in GFP or YFP-expressing biosamples. Here we developed a cytosolically localized far-red to NIR rhodamine-based fluorescent probe for Ca2+, CaSiR-2 AM, while rhodamine dyes are basically localized to mitochondria or lysosomes in cells.
Assuntos
Cálcio , Corantes Fluorescentes , Íons , Lisossomos , RodaminasRESUMO
Folate receptors (FRs) are membrane proteins involved in folic acid uptake, and the alpha isoform (FR-α) is overexpressed in ovarian and endometrial cancer cells. For fluorescence imaging of FRs inâ vivo, the near-infrared (NIR) region (650-900â nm), in which tissue penetration is high and autofluorescence is low, is optimal, but existing NIR fluorescent probes targeting FR-α show high non-specific tissue adsorption, and require prolonged washout to visualize tumors. We have designed and synthesized a new NIR fluorescent probe, FolateSiR-1, utilizing a Si-rhodamine fluorophore having a carboxy group at the benzene moiety, coupled to a folate ligand moiety through a negatively charged tripeptide linker. This probe exhibits very low background fluorescence and afforded a tumor-to-background ratio (TBR) of up to 83 in FR-expressing tumor-bearing mice within 30â min. Thus, FolateSiR-1 has the potential to contribute to the research in the field of biology and the clinical medicine.
Assuntos
Corantes Fluorescentes/química , Receptores de Folato com Âncoras de GPI/metabolismo , Regulação Neoplásica da Expressão Gênica , Imagem Molecular/métodos , Razão Sinal-Ruído , Animais , Linhagem Celular Tumoral , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , Ácido Fólico/metabolismo , Humanos , Camundongos , Rodaminas/síntese química , Rodaminas/química , Rodaminas/metabolismo , Fatores de TempoRESUMO
We have developed a novel method to globally monitor the enzymatic activities of biological samples based on performing the global activity analysis on a proteome separated by native electrophoresis. The study of the alteration in peptide-metabolizing enzymatic activity in colorectal tumor specimens led us to the discovery of elevated thimet oligopeptidase activity, which contributed to the faster consumption of immune-stimulating peptide neurotensin.
Assuntos
Neoplasias Colorretais/enzimologia , Metaloendopeptidases/análise , Proteoma/análise , Proteômica/métodos , Sequência de Aminoácidos , Cromatografia Líquida , Eletroforese , Humanos , Metaloendopeptidases/química , Neurotensina/química , Biblioteca de Peptídeos , Espectrometria de Massas em TandemRESUMO
Photoacoustic (PA) imaging is a novel imaging modality that combines the high contrast of optical imaging and the deep tissue penetration of ultrasound. PA imaging contrast agents targeting various biological phenomena have been reported, but the development of activatable PA probes, which show a PA signal only in the presence of target molecules, remains challenging in spite of their potential usefulness for real-time PA imaging of specific biomolecules in vivo. To establish a simple design strategy for activatable PA probes, we first designed and synthesized a silicon-rhodamine based near-infrared nonfluorescent dye, wsSiNQ660 (water-soluble SiNQ660), as a scaffold and demonstrated that it offers a high conversion efficiency from light to ultrasound compared to typical near-infrared fluorescent dyes. Importantly, absorption off/on strategies previously established for rhodamine-based fluorescent probes are also applicable to this nonfluorescent dye scaffold. We validated this approach by synthesizing an activatable PA probe for hypochlorous acid (HOCl) and confirmed that it enables three-dimensional imaging of HOCl in mouse subcutis.
Assuntos
Ácido Hipocloroso/análise , Compostos de Organossilício/química , Rodaminas/química , Animais , Desenho de Fármacos , Humanos , Ácido Hipocloroso/química , Raios Infravermelhos , Masculino , Camundongos Endogâmicos BALB C , Compostos de Organossilício/síntese química , Compostos de Organossilício/efeitos da radiação , Técnicas Fotoacústicas/métodos , Rodaminas/síntese química , Rodaminas/efeitos da radiação , Tela Subcutânea/químicaRESUMO
We have developed an activatable red fluorescence probe for dipeptidylpeptidase-IV (DPP-IV) by precisely controlling the photoinduced electron transfer (PeT) process of a red fluorescent scaffold, SiR600. The developed probe exhibited an extremely low background signal and showed significant fluorescence activation upon reaction with DPP-IV, enabling sensitive detection of esophageal cancer in clinical specimens from cancer patients.
Assuntos
Dipeptidil Peptidase 4/metabolismo , Neoplasias Esofágicas/diagnóstico , Corantes Fluorescentes/química , Dipeptidil Peptidase 4/química , Neoplasias Esofágicas/enzimologia , Humanos , Sensibilidade e Especificidade , Espectrometria de FluorescênciaRESUMO
We designed a ratiometric carbohydrate sensor consisting of the boron dipyrromethene fluorophore substituted with boronic acid at the 2-position, based upon the strong substituent dependency of the absorbance/fluorescence wavelengths of BODIPY. The substituent is in equilibrium between the boronic acid B(OH)2 and boronate (B(OH)3-) forms, which have different absorbance/fluorescence wavelengths in the visible region. Reaction of the boronic acid moiety with hydroxy groups of carbohydrate affords a cyclic ester and shifts the equilibrium in favor of the boronate (B(OR)3-) form, resulting in a carbohydrate-concentration-dependent change of the fluorescence ratio. Thus, the sensor, BA-BODIPY, can ratiometrically detect carbohydrate at a pH near the pKa of cyclic ester formation.
Assuntos
Compostos de Boro/química , Carboidratos/análise , Desenvolvimento de Medicamentos , Compostos de Boro/síntese química , Concentração de Íons de Hidrogênio , Estrutura Molecular , Espectrometria de FluorescênciaRESUMO
We have developed a platform for activatable fluorescent substrates of glucose transporters (GLUTs). We firstly conjugated fluorescein to glucosamine via an amide or methylene linker at the C-2 position of d-glucosamine, but the resulting compounds, FLG1 and FLG2, showed no uptake into MIN6 cells. So, we changed the fluorophore moiety to a fluorescein analogue, 2-Me TokyoGreen, which is less negatively charged. TokyoGreen-conjugated glucosamines TGG1 and TGG2 were successfully taken up into cells via GLUT. We further derivatized TGG1 and TGG2, and among the synthesized compounds, 2-Me-4-OMe TGG showed weak fluorescence under the acidic conditions of the extracellular environment inside tumors and in gastric cancers, and strong fluorescence at the intracellular physiological pH, under the control of a photoinduced electron transfer (PeT) process. This fluorogenic platform should be useful for developing a range of activatable fluorescent substrates targeting GLUTs, as well as derivatives that would be fluorescently activated by various intracellular enzymes, such as esterases, ß-galactosidase and bioreductases.
Assuntos
Corantes Fluorescentes/química , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Animais , Linhagem Celular , Fluoresceína/química , Glucosamina/análogos & derivados , Glucosamina/metabolismo , Glucose/análogos & derivados , Glucose/metabolismo , Camundongos , Microscopia de FluorescênciaRESUMO
Elevated intraocular pressure (IOP) is the major cause of glaucoma, which is the second leading cause of blindness. However, current glaucoma treatments cannot completely regulate IOP and progression of glaucoma. Our group recently found that autotaxin (ATX) activity in human aqueous humor (AH) was positively correlated with increased IOP in various subtypes of glaucoma. To develop new IOP-lowering treatments, we generated a novel ATX inhibitor as an ophthalmic drug by high-throughput screening, followed by inhibitor optimization. Administration of the optimized ATX inhibitor (Aiprenon) reduced IOP in laser-treated mice exhibiting elevated IOP and higher level of ATX activity in AH and normal mice in vivo. The stimulation of ATX induced outflow resistance in the trabecular pathway; however, administration of Aiprenon recovered the outflow resistance in vitro. The in vitro experiments implied that the IOP-lowering effect of Aiprenon could be correlated with the altered cellular behavior of trabecular meshwork (TM) and Schlemm's canal endothelial (SC) cells. Overall, our findings showed that ATX had major impact in regulating IOP as a target molecule, and potent ATX inhibitors such as Aiprenon could be a promising therapeutic approach for lowering IOP.
Assuntos
Pressão Intraocular/efeitos dos fármacos , Hipertensão Ocular/tratamento farmacológico , Inibidores de Fosfodiesterase/uso terapêutico , Diester Fosfórico Hidrolases/efeitos dos fármacos , Animais , Humor Aquoso , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos , Células Endoteliais/efeitos dos fármacos , Glaucoma/metabolismo , Glaucoma/fisiopatologia , Humanos , Macaca fascicularis , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Estrutura Molecular , Hipertensão Ocular/induzido quimicamente , Inibidores de Fosfodiesterase/química , Malha Trabecular/efeitos dos fármacosRESUMO
In biological systems, the pH in intracellular organelles or tissues is strictly regulated, and differences of pH are deeply related to key biological events such as protein degradation, intracellular trafficking, renal failure, and cancer. Ratiometric fluorescence imaging is useful for determination of precise pH values, but existing fluorescence probes have substantial limitations, such as inappropriate p Ka for imaging in the physiological pH range, inadequate photobleaching resistance, and insufficiently long excitation and emission wavelengths. Here we report a versatile scaffold for ratiometric fluorescence pH probes, based on asymmetric rhodamine. To demonstrate its usefulness for biological applications, we employed it to develop two probes. (1) SiRpH5 has suitable p Ka and water solubility for imaging in acidic intracellular compartments; by using transferrin tagged with SiRpH5, we achieved time-lapse imaging of pH in endocytic compartments during protein trafficking for the first time. (2) Me-pEPPR is a near-infrared (NIR) probe; by using dextrin tagged with Me-pEPPR, we were able to image extracellular pH of renal tubules and tumors in situ. These chemical tools should be useful for studying the influence of intra- and extracellular pH on biological processes, as well as for in vivo imaging.
Assuntos
Fluorescência , Corantes Fluorescentes/química , Neoplasias/diagnóstico por imagem , Imagem Óptica , Animais , Células COS , Linhagem Celular Tumoral , Chlorocebus aethiops , Corantes Fluorescentes/farmacocinética , Humanos , Concentração de Íons de Hidrogênio , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos ICR , Camundongos Nus , Estrutura Molecular , Neoplasias/patologia , Neoplasias Experimentais/diagnóstico por imagem , Neoplasias Experimentais/patologia , Solubilidade , Água/químicaRESUMO
Carboxypeptidases (CPs) are a family of hydrolases that cleave one or more amino acids from the C-terminal of peptides or proteins. However, methodology to monitor the activities of CPs is poorly developed. Here, we present the first versatile design strategy to obtain activatable fluorescent probes for CPs by utilizing intramolecular spirocyclization of rhodamine to translate the "aliphatic carboxamide to aliphatic carboxylate" structural conversion catalyzed by CPs into dynamic fluorescence activation. Based on this novel strategy, we developed probes for carboxypeptidases A and B. One of these probes was able to detect pancreatic juice leakage in mice ex vivo, suggesting that its suitability for intraoperative diagnosis of pancreatic fistula. This design strategy should be broadly applicable to CPs, as well as other previously untargetable enzymes, enabling development of fluorescent probes to study various pathological and biological processes.
RESUMO
The Escherichia coli lacZ gene encoding ß-galactosidase is a widely used reporter, but few synthetic substrates are available for detecting its activity with single-cell resolution in living samples. Our recently reported fluorogenic substrate SPiDER-ßGal is suitable for this purpose, but its hydrolysis product shows green fluorescence emission, and a red-shifted analogue is therefore required for use in combination with green fluorescent protein (GFP) markers. Herein, we describe the development of a red-shifted fluorogenic substrate for ß-galactosidase, SPiDER-Red-ßGal, based on a silicon rhodol scaffold and a carboxylic group as the intramolecular nucleophile. LacZ-positive cells were successfully labeled with SPiDER-Red-ßGal at single-cell resolution in living samples, which enabled us to visualize different cell types in combination with GFP markers.
Assuntos
Escherichia coli/citologia , Corantes Fluorescentes/química , Óperon Lac/genética , Análise de Célula Única , beta-Galactosidase/química , Escherichia coli/genética , Escherichia coli/metabolismo , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/metabolismo , beta-Galactosidase/metabolismoRESUMO
Photodynamic therapy (PDT) utilizes photoirradiation in the presence of photosensitizers to ablate cancer cells via generation of singlet oxygen (1O2), but it is important to minimize concomitant injury to normal tissues. One approach for achieving this is to use activatable photosensitizers that can generate 1O2 only under specific conditions. Here, we report a novel photosensitizer that is selectively activated under hypoxia, a common condition in solid tumors. We found that introducing an azo moiety into the conjugated system of a seleno-rosamine dye effectively hinders the intersystem crossing process that leads to 1O2 generation. We show that the azo group is reductively cleaved in cells under hypoxia, enabling production of 1O2 to occur. In PDT in vitro, cells under mild hypoxia, within the range typically found in solid tumors (up to about 5% O2), were selectively ablated, leaving adjacent normoxic cells intact. This simple and practical azo-based strategy should be widely applicable to design a range of activatable photosensitizers.
Assuntos
Compostos Azo/farmacologia , Hipóxia Celular/efeitos dos fármacos , Fotoquimioterapia , Fármacos Fotossensibilizantes/farmacologia , Compostos Azo/síntese química , Compostos Azo/química , Linhagem Celular Tumoral , Humanos , Fármacos Fotossensibilizantes/síntese química , Fármacos Fotossensibilizantes/químicaRESUMO
Cellular homeostasis is maintained by a complex network of reactions catalyzed by enormous numbers of enzymatic activities (the enzymome), which serve to determine the phenotypes of cells. Here, we focused on the enzymomics of proteases and peptidases because these enzymes are an important class of disease-related proteins. We describe a system that (A) simultaneously evaluates metabolic activities of peptides using a series of exogenous peptide substrates and (B) identifies the enzymes that metabolize the specified peptide substrate with high throughput. We confirmed that the developed system was able to discover cell-type-specific and disease-related exo- and endopeptidase activities and identify the responsible enzymes. For example, we found that the activity of the endopeptidase neurolysin is highly elevated in human colorectal tumor tissue samples. This simple but powerful enzymomics platform should be widely applicable to uncover cell-type-specific reactions and altered enzymatic functions with potential value as biomarkers or drug targets in various disease states and to investigate the mechanisms of the underlying pathologies.