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1.
Am J Physiol Renal Physiol ; 327(2): F208-F223, 2024 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-38870264

RESUMEN

Increased dietary phosphate consumption intensifies renal phosphate burden. Several mechanisms for phosphate-induced renal tubulointerstitial fibrosis have been reported. Considering the dual nature of phosphate as both a potential renal toxin and an essential nutrient for the body, kidneys may possess inherent protective mechanisms against phosphate overload, rather than succumbing solely to injury. However, there is limited understanding of such mechanisms. To identify these mechanisms, we conducted single-cell RNA sequencing (scRNA-seq) analysis of the kidneys of control and dietary phosphate-loaded (Phos) mice at a time point when the Phos group had not yet developed tubulointerstitial fibrosis. scRNA-seq analysis identified the highest number of differentially expressed genes in the clusters belonging to proximal tubular epithelial cells (PTECs). Based on these differentially expressed genes, in silico analyses suggested that the Phos group activated peroxisome proliferator-activated receptor-α (PPAR-α) and fatty acid ß-oxidation (FAO) in the PTECs. This activation was further substantiated through various experiments, including the use of an FAO activity visualization probe. Compared with wild-type mice, Ppara knockout mice exhibited exacerbated tubulointerstitial fibrosis in response to phosphate overload. Experiments conducted with cultured PTECs demonstrated that activation of the PPAR-α/FAO pathway leads to improved cellular viability under high-phosphate conditions. The Phos group mice showed a decreased serum concentration of free fatty acids, which are endogenous PPAR-α agonists. Instead, experiments using cultured PTECs revealed that phosphate directly activates the PPAR-α/FAO pathway. These findings indicate that noncanonical metabolic reprogramming via endogenous activation of the PPAR-α/FAO pathway in PTECs is essential to counteract phosphate toxicity.NEW & NOTEWORTHY This study revealed the activation of peroxisome proliferator-activated receptor-α and fatty acid ß-oxidation in proximal tubular epithelial cells as an endogenous mechanism to protect the kidney from phosphate toxicity. These findings highlight noncanonical metabolic reprogramming as a potential target for suppressing phosphate toxicity in the kidneys.


Asunto(s)
Túbulos Renales Proximales , PPAR alfa , Fosfatos , Animales , Túbulos Renales Proximales/metabolismo , Túbulos Renales Proximales/patología , Túbulos Renales Proximales/efectos de los fármacos , PPAR alfa/metabolismo , PPAR alfa/genética , Fosfatos/metabolismo , Fosfatos/toxicidad , Fibrosis , Ratones Endogámicos C57BL , Masculino , Ratones , Células Epiteliales/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Ácidos Grasos/metabolismo , Ratones Noqueados , Oxidación-Reducción
2.
Biochem Biophys Res Commun ; 720: 150077, 2024 Aug 06.
Artículo en Inglés | MEDLINE | ID: mdl-38759303

RESUMEN

Hericenone C is one of the most abundant secondary metabolites derived from Hericium erinaceus, under investigation for medicinal properties. Here, we report that Hericenone C inhibits the second phase of formalin-induced nociceptive behavior in mice. As the second phase is involved in inflammation, in a mechanistic analysis on cultured cells targeting NF-κB response element (NRE): luciferase (Luc)-expressing cells, lipopolysaccharide (LPS)-induced NRE::Luc luciferase activity was found to be significantly inhibited by Hericenone C. Phosphorylation of p65, which is involved in the inflammatory responses of the NF-κB signaling pathway, was also induced by LPS and significantly reduced by Hericenone C. Additionally, in mice, the number of CD11c-positive cells increased in the paw during the peak of the second phase of the formalin test, which decreased upon Hericenone C intake. Our findings confirm the possibility of Hericenone C as a novel therapeutic target for pain-associated inflammation.


Asunto(s)
Epidermis , Formaldehído , Animales , Fosforilación/efectos de los fármacos , Ratones , Masculino , Epidermis/metabolismo , Epidermis/efectos de los fármacos , Factor de Transcripción ReIA/metabolismo , Antígenos CD11/metabolismo , Nocicepción/efectos de los fármacos , Humanos
3.
Biol Pharm Bull ; 47(3): 641-651, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38508744

RESUMEN

Recently, mitochondrial dysfunction has gained attention as a causative factor in the pathogenesis and progression of age-related macular degeneration (AMD). Mitochondrial damage plays a key role in metabolism and disrupts the balance of intracellular metabolic pathways, such as oxidative phosphorylation (OXPHOS) and glycolysis. In this study, we focused on oxidized low-density lipoprotein (ox-LDL), a major constituent of drusen that accumulates in the retina of patients with AMD, and investigated whether it could be a causative factor for metabolic alterations in retinal pigment epithelial (RPE) cells. We found that prolonged exposure to ox-LDL induced changes in fatty acid ß-oxidation (FAO), OXPHOS, and glycolytic activity and increased the mitochondrial reactive oxygen species production in RPE cells. Notably, the effects on metabolic alterations varied with the concentration and duration of ox-LDL treatment. In addition, we addressed the limitations of using ARPE-19 cells for retinal disease research by highlighting their lower barrier function and FAO activity compared to those of induced pluripotent stem cell-derived RPE cells. Our findings can aid in the elucidation of mechanisms underlying the metabolic alterations in AMD.


Asunto(s)
Degeneración Macular , Epitelio Pigmentado de la Retina , Humanos , Epitelio Pigmentado de la Retina/metabolismo , Lipoproteínas LDL/metabolismo , Estrés Oxidativo , Células Epiteliales , Pigmentos Retinianos/metabolismo , Pigmentos Retinianos/farmacología
4.
Am J Physiol Endocrinol Metab ; 325(5): E552-E561, 2023 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-37729022

RESUMEN

Mitochondrial fatty acid ß-oxidation (FAO) plays a key role in energy homeostasis. Several FAO evaluation methods are currently available, but they are not necessarily suitable for capturing the dynamics of FAO in vivo at a cellular-level spatial resolution and seconds-level time resolution. FAOBlue is a coumarin-based probe that undergoes ß-oxidation to produce a fluorescent substrate, 7-hydroxycoumarin-3-(N-(2-hydroxyethyl))-carboxamide (7-HC). After confirming that 7-HC could be specifically detected using multiphoton microscopy at excitation/emission wavelength = 820/415-485 nm, wild-type C57BL/6 mice were randomly divided into control, pemafibrate, fasting (24 or 72 h), and etomoxir groups. These mice received a single intravenous injection of FAOBlue. FAO activities in the liver of these mice were visualized using multiphoton microscopy at 4.2 s/frame. These approaches could visualize the difference in FAO activities between periportal and pericentral hepatocytes in the control, pemafibrate, and fasting groups. FAO velocity, which was expressed by the maximum slope of the fluorescence intensity curve, was accelerated in the pemafibrate and 72-h fasting groups both in the periportal and the pericentral hepatocytes in comparison with the control group. Our approach revealed differences in the FAO activation mode by the two stimuli, i.e., pemafibrate and fasting, with pemafibrate accelerating the time of first detection of FAO-derived fluorescence. No increase in the fluorescence was observed in etomoxir-pretreated mice, confirming that FAOBlue specifically detected FAO in vivo. Thus, FAOBlue is useful for visualizing in vivo liver FAO dynamics at the single-cell-level spatial resolution and seconds-level time resolution.NEW & NOTEWORTHY Fatty acid ß-oxidation (FAO) plays a key role in energy homeostasis. Here, the authors established a strategy for visualizing FAO activity in vivo at the cellular-level spatial resolution and seconds-level time resolution in mice. Quantitative analysis revealed spatiotemporal heterogeneity in hepatic FAO dynamics. Our method is widely applicable because it is simple and uses a multiphoton microscope to observe the FAOBlue-injected mice.


Asunto(s)
Butiratos , Mitocondrias , Ratones , Animales , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Butiratos/metabolismo , Oxidación-Reducción , Ácidos Grasos/metabolismo
5.
J Am Chem Soc ; 145(14): 8248-8260, 2023 04 12.
Artículo en Inglés | MEDLINE | ID: mdl-37011039

RESUMEN

Detection of metabolic activity enables us to reveal the inherent metabolic state of cells and elucidate mechanisms underlying cellular homeostasis and growth. However, a fluorescence approach for the study of metabolic pathways is still largely unexplored. Herein, we have developed a new chemical probe for the fluorescence-based detection of fatty acid ß-oxidation (FAO), a key process in lipid catabolism, in cells and tissues. This probe serves as a substrate of FAO and forms a reactive quinone methide (QM) as a result of metabolic reactions. The liberated QM is covalently captured by intracellular proteins, and subsequent bio-orthogonal ligation with a fluorophore enables fluorescence analysis. This reaction-based sensing allowed us to detect FAO activity in cells at a desired emission wavelength using diverse analytical techniques including fluorescence imaging, in-gel fluorescence activity-based protein profiling (ABPP), and fluorescence-activated cell sorting (FACS). The probe was able to detect changes in FAO activity induced by chemical modulators in cultured cells. The probe was further employed for fluorescence imaging of FAO in mouse liver tissues and revealed the metabolic heterogeneity of FAO activity in hepatocytes by the combination of FACS and gene expression analysis, highlighting the utility of our probe as a chemical tool for fatty acid metabolism research.


Asunto(s)
Ácidos Grasos , Hepatocitos , Ratones , Animales , Oxidación-Reducción , Fluorescencia , Hepatocitos/metabolismo , Ácidos Grasos/metabolismo
6.
Commun Chem ; 4(1): 104, 2021 Jul 06.
Artículo en Inglés | MEDLINE | ID: mdl-36697807

RESUMEN

Despite continuous and active development of fluorescent metal-ion probes, their molecular design for ratiometric detection is restricted by the limited choice of available sensing mechanisms. Here we present a multicolor and ratiometric fluorescent sensing platform for metal ions based on the interaction between the metal ion and the aromatic ring of a fluorophore (arene-metal-ion, AM, coordination). Our molecular design provided the probes possessing a 1,9-bis(2'-pyridyl)-2,5,8-triazanonane as a flexible metal ion binding unit attached to a tricyclic fluorophore. This architecture allows to sense various metal ions, such as Zn(II), Cu(II), Cd(II), Ag(I), and Hg(II) with emission red-shifts. We showed that this probe design is applicable to a series of tricyclic fluorophores, which allow ratiometric detection of the metal ions from the blue to the near-infrared wavelengths. X-ray crystallography and theoretical calculations indicate that the coordinated metal ion has van der Waals contact with the fluorophore, perturbing the dye's electronic structure and ring conformation to induce the emission red-shift. A set of the probes was useful for the differential sensing of eight metal ions in a one-pot single titration via principal component analysis. We also demonstrate that a xanthene fluorophore is applicable to the ratiometric imaging of metal ions under live-cell conditions.

7.
Molecules ; 25(9)2020 May 05.
Artículo en Inglés | MEDLINE | ID: mdl-32380657

RESUMEN

Self-immolative linker is a useful building block of molecular probes, with broad applications in the fields of enzyme activity analysis, stimuli-responsive material science, and drug delivery. This manuscript presents N-methyl dimethyl methyl (i.e., trimethyl) carbamate as a new class of self-immolative linker for the fluorescence detection of enzyme reactions. The trimethyl carbamate was shown to spontaneously undergo intramolecular cyclization upon formation of a carboxylate group, to liberate a fluorophore with the second time rapid reaction kinetics. Interestingly, the auto-cleavage reaction of trimethyl carbamate was also induced by the formation of hydroxyl and amino groups. Fluorescent probes with a trimethyl carbamate could be applicable for fluorescence monitoring of the enzyme reactions catalyzed by esterase, ketoreductase, and aminotransferase, and for fluorescence imaging of intracellular esterase activity in living cells, hence demonstrating the utility of this new class of self-immolative linker.


Asunto(s)
Carbamatos/síntesis química , Cumarinas/química , Colorantes Fluorescentes/síntesis química , Células A549 , Carbamatos/química , Ciclización , Pruebas de Enzimas , Colorantes Fluorescentes/química , Humanos , Estructura Molecular
8.
Chem Commun (Camb) ; 56(20): 3023-3026, 2020 Mar 10.
Artículo en Inglés | MEDLINE | ID: mdl-32048639

RESUMEN

Detection of metabolic activity in living cells facilitates the understanding of the cell mechanism. Here, we report a fluorescent probe that can detect fatty acid beta oxidation (FAO) in living cells. This probe is metabolically degraded by the sequential enzyme reactions of FAO and can visualize the FAO activity with turn-on fluorescence.


Asunto(s)
Ácidos Grasos/análisis , Colorantes Fluorescentes/química , Imagen Óptica , Ácidos Grasos/metabolismo , Células Hep G2 , Humanos , Estructura Molecular , Oxidación-Reducción
9.
iScience ; 22: 256-268, 2019 Dec 20.
Artículo en Inglés | MEDLINE | ID: mdl-31786521

RESUMEN

Electron microscopy (EM) is a technology that enables visualization of single proteins at a nanometer resolution. However, current protein analysis by EM mainly relies on immunolabeling with gold-particle-conjugated antibodies, which is compromised by large size of antibody, precluding precise detection of protein location in biological samples. Here, we develop a specific chemical labeling method for EM detection of proteins at single-molecular level. Rational design of α-helical peptide tag and probe structure provided a complementary reaction pair that enabled specific cysteine conjugation of the tag. The developed chemical labeling with gold-nanoparticle-conjugated probe showed significantly higher labeling efficiency and detectability of high-density clusters of tag-fused G protein-coupled receptors in freeze-fracture replicas compared with immunogold labeling. Furthermore, in ultrathin sections, the spatial resolution of the chemical labeling was significantly higher than that of antibody-mediated labeling. These results demonstrate substantial advantages of the chemical labeling approach for single protein visualization by EM.

10.
Bioorg Med Chem Lett ; 27(15): 3486-3489, 2017 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-28576616

RESUMEN

We report the discovery of a highly reactive peptide tag for the specific cysteine conjugation of proteins. Screening of cysteine-containing peptides using ELISA-type screening yielded a 19-amino acid tag (DCPPPDDAADDAADDAADD), named DCP3 tag, which enabled the rapid and selective labeling of the tag-fused protein with a synthetic zinc complex on the surface of living cells.


Asunto(s)
Cisteína/química , Imagen Óptica , Péptidos/química , Proteínas/análisis , Secuencia de Aminoácidos , Complejos de Coordinación/química , Ensayo de Inmunoadsorción Enzimática/métodos , Células HEK293 , Humanos , Proteínas de Unión a Maltosa/análisis , Imagen Óptica/métodos , Receptores Acoplados a Proteínas G/análisis , Zinc/química
11.
Chem Sci ; 8(2): 1134-1140, 2017 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-28451253

RESUMEN

Hydropersulfide (R-SSH) is an important class of reactive sulfur species (RSS) involved in a variety of physiological processes in mammals. A fluorescent probe capable of real-time detection of hydropersulfide levels in living cells would be a versatile tool to elucidate its roles in cell signalling and redox homeostasis. In this paper, we report a ratiometric fluorescent probe for hydropersulfide sensing, based on a fluorescence resonance energy transfer (FRET) mechanism. This sensing mechanism involves a nucleophilic reaction of a hydropersulfide with the pyronine-unit of the probe, which modulates the intramolecular FRET efficiency to induce a dual-emission change. The reversible nature of this reaction allows us to detect increases and decreases of hydropersulfide levels in a real-time manner. The probe fluorometrically sensed highly reactive hydropersulfides, such as H2S2 and Cys-SSH, while the fluorescence response to biologically abundant cysteine and glutathione was negligible. Taking advantage of the reversible and selective sensing properties, this probe was successfully applied to the ratiometric imaging of concentration dynamics of endogenously produced hydropersulfides in living cells.

12.
Chem Commun (Camb) ; 52(49): 7715-8, 2016 Jun 08.
Artículo en Inglés | MEDLINE | ID: mdl-27229298

RESUMEN

A new glutathione (GSH)-responsive traceless tag that facilitates intracellular delivery of small molecule chemical probes has been developed.

13.
Comb Chem High Throughput Screen ; 19(5): 378-83, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27055752

RESUMEN

Control of fluorescent dye localization in live cells is crucial for fluorescence imaging. Here, we describe quantitative structure activity relation (QSAR) models for predicting intracellular localization of fluorescent dyes. For generating the QSAR models, electric charge (Z) calculated by pKa, conjugated bond number (CBN), the largest conjugated fragment (LCF), molecular weight (MW) and log P were used as parameters. We identified the intracellular localization of 119 BODIPY dyes in live NIH3T3 cells, and assessed the accuracy of our models by comparing their predictions with the observed dye localizations. As predicted by the models, no BODIPY dyes localized in nuclei or plasma membranes. The accuracy of the model for localization in fat droplets was 92%, with the models for cytosol and lysosomes showing poorer agreement with observed dye localization, albeit well above chance levels. Overall therefore the utility of QSAR models for predicting dye localization in live cells was clearly demonstrated.


Asunto(s)
Colorantes Fluorescentes/farmacocinética , Relación Estructura-Actividad Cuantitativa , Animales , Compuestos de Boro , Interacciones Hidrofóbicas e Hidrofílicas , Espacio Intracelular/metabolismo , Gotas Lipídicas/metabolismo , Ratones , Células 3T3 NIH
14.
Chempluschem ; 81(11): 1209-1215, 2016 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-31964103

RESUMEN

Reported is a new fluorescent probe based on boron-dipyrromethene dye (BODIPY) for late endosome staining in live cells. Among the 14 pH-responsive BODIPY-based far-red/near-infrared dyes used, it was found that a (4-methyl-1-piperazinyl)phenyl-substituted BODIPY could stain late endosomes in three different cell lines. This BODIPY dye was applicable for measurement of the fusion time of late endosomes and lysosomes, thereby demonstrating the utility of this dye for functional analyses of late endosomes in live cells.

15.
Bioorg Med Chem Lett ; 24(13): 2855-8, 2014 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-24835629

RESUMEN

Selective protein labeling with a small molecular probe is a versatile method for elucidating protein functions under live-cell conditions. In this Letter, we report the design of the binuclear Ni(II)-iminodiacetic acid (IDA) complex for selective recognition and covalent labeling of His-tag-fused proteins. We found that the Ni(II)-IDA complex 1-2Ni(II) binds to the His6-tag (HHHHHH) with a strong binding affinity (Kd=24 nM), the value of which is 16-fold higher than the conventional Ni(II)-NTA complex (Kd=390 nM). The strong binding affinity of the Ni(II)-IDA complex was successfully used in the covalent labeling and fluorescence bioimaging of a His-tag fused GPCR (G-protein coupled receptor) located on the surface of living cells.


Asunto(s)
Diseño de Fármacos , Histidina/química , Iminoácidos/química , Níquel/química , Compuestos Organometálicos/química , Proteínas Recombinantes de Fusión/química , Células HEK293 , Humanos , Estructura Molecular , Compuestos Organometálicos/síntesis química , Compuestos Organometálicos/farmacología , Coloración y Etiquetado , Relación Estructura-Actividad
16.
Inorg Chem ; 53(4): 1816-23, 2014 Feb 17.
Artículo en Inglés | MEDLINE | ID: mdl-24131471

RESUMEN

Protein-labeling methods serve as essential tools for analyzing functions of proteins of interest under complicated biological conditions such as in live cells. These labeling methods are useful not only to fluorescently visualize proteins of interest in biological systems but also to conduct protein and cell analyses by harnessing the unique functions of molecular probes. Among the various labeling methods available, an appropriate binding pair consisting of a short peptide and a de novo designed small molecular probe has attracted attention because of its wide utility and versatility. Interestingly, most peptide tag/probe pairs exploit metal-ligand coordination interactions as the main binding force responsible for their association. Herein, we provide an overview of the recent progress of these coordination-chemistry-based protein-labeling methods and their applications for fluorescence imaging and functional analysis of cellular proteins, while highlighting our originally developed labeling methods. These successful examples clearly exemplify the utility and versatility of metal coordination chemistry in protein functional analysis.


Asunto(s)
Complejos de Coordinación/química , Colorantes Fluorescentes/química , Sondas Moleculares/química , Péptidos/química , Proteínas/química , Coloración y Etiquetado
17.
Chem Commun (Camb) ; 49(44): 5022-4, 2013 Jun 04.
Artículo en Inglés | MEDLINE | ID: mdl-23612601

RESUMEN

A new method for in-cell protein labeling was developed. This method employed a binding-induced nucleophilic reaction between the Cys-appended His-tag and the Ni(II)-NTA containing an α-chloroacetamide. Using this method, not only labeling of His-tag fused proteins but also the detection of a protein-protein interaction was achieved inside living cells.


Asunto(s)
Proteínas Fluorescentes Verdes/metabolismo , Histidina/metabolismo , Oligopéptidos/metabolismo , Acetamidas/química , Línea Celular Tumoral , Cisteína/química , Cisteína/metabolismo , Escherichia coli/metabolismo , Colorantes Fluorescentes/química , Proteínas Fluorescentes Verdes/genética , Células HeLa , Histidina/genética , Humanos , Cinética , Níquel/química , Oligopéptidos/genética , Mapas de Interacción de Proteínas , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteína 1A de Unión a Tacrolimus/genética , Proteína 1A de Unión a Tacrolimus/metabolismo
18.
Chem Commun (Camb) ; 48(4): 594-6, 2012 Jan 14.
Artículo en Inglés | MEDLINE | ID: mdl-22113378

RESUMEN

A Zn(II) complex (Zn(II)-Ida) was designed as the new fluorescent probe for His-tag fused proteins. Thanks to the tight binding ability to histidine-rich sequences and bright fluorescence property of the Cy5-appended Zn(II)-Ida probes, selective and clear fluorescent imaging of the His-tag fused G-protein coupled receptors on live cell surfaces was carried out.


Asunto(s)
Carbocianinas/química , Complejos de Coordinación/química , Colorantes Fluorescentes/química , Histidina/análisis , Oligopéptidos/análisis , Receptores Acoplados a Proteínas G/análisis , Zinc/química , Células HEK293 , Humanos , Microscopía Fluorescente
19.
J Am Chem Soc ; 132(27): 9301-9, 2010 Jul 14.
Artículo en Inglés | MEDLINE | ID: mdl-20568758

RESUMEN

Selective protein labeling with a small molecular probe is a versatile method for elucidating protein functions in living cells. In this paper, we report a covalent labeling method of tag-fused G-protein coupled receptor (GPCR) proteins expressing on cell surfaces utilizing small functional molecules. This method employs the selective and rapid reaction of a peptide tag and a molecular probe, which comprises the cysteine-containing short CA6D4x2 tag (CAAAAAADDDDGDDDD) and a tetranuclear Zn(II)-DpaTyr probe containing a reactive alpha-chloroacetyl moiety. The covalent labeling of tag-fused GPCRs such as bradykinin receptor (B2R) and acetylcholine receptor (m1AchR) selectively proceeded under physiological conditions during short incubation (10-30 min) with Zn(II)-DpaTyr probes bearing various functional groups. Labeling with fluorophore-appended Zn(II)-DpaTyr probes enabled visualization of the GPCRs on the surface of HEK293 cells by fluorescence. Labeling with the biotin-appended probe allowed introduction of a biotin unit into the GPCRs. This biotin label was utilized for fluorescence bioimaging studies and postlabeling blotting analysis of the labeled GPCRs by use of the specific biotin-streptavidin interaction. The utility of this labeling method was demonstrated in several function analyses of GPCRs, such as fluorescence visualization of the stimuli-responsive internalization of GPCRs and pH change in endosomes containing the internalized GPCRs.


Asunto(s)
Técnicas de Sonda Molecular , Sondas Moleculares/química , Receptores Acoplados a Proteínas G/química , Biotina , Línea Celular , Cisteína , Endocitosis , Endosomas/metabolismo , Fluoresceínas , Humanos , Oligopéptidos , Compuestos Organometálicos , Receptores Acoplados a Proteínas G/metabolismo , Coloración y Etiquetado
20.
Chem Asian J ; 5(4): 877-86, 2010 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-20143369

RESUMEN

A complementary recognition pair of a short-peptide tag and a small molecular probe is a versatile molecular tool for protein detection, handling, and purification, and so forth. In this manuscript, we report that the binuclear Ni(II)-DpaTyr (DpaTyr=bis((dipicolylamino)methyl)tyrosine) complex serves as a strong binding probe for an oligo-aspartate tag tethered to a protein. Among various binuclear metal complexes of M-DpaTyr (M=Zn(II), Ni(II), Mn(II), Cu(II), Cd(II), Co(III), and Fe(III)), we have found that Ni(II)-DpaTyr (1-2Ni(II)) displays a strong-binding affinity (apparent binding constant: K(app) approximately 10(5) M(-1)) for an oligo-aspartate peptide under neutral aqueous conditions (50 mM HEPES, 100 mM NaCl, pH 7.2). Detailed isothermal-titration calorimetry (ITC) studies reveal that the tri-aspartate D3-tag (DDD) is an optimal sequence recognized by 1-2Ni(II) in a 1:1 binding stoichiometry. On the other hand, other metal complexes of DpaTyr, except for Ni(II)- and Zn(II)-DpaTyr, show a negligible binding affinity for the oligo-aspartate peptide. The binding affinity was greatly enhanced in the pair between the dimer of Ni(II)-DpaTyr and the repeated D3 tag peptide (D3x2), such as DDDXXDDD, on the basis of the multivalent coordination interaction between them. Most notably, a remarkably high-binding affinity (K(app)=2x10(9) M(-1)) was achieved between the Ni(II)-DpaTyr dimer 4-4Ni(II) and the D3x2 tag peptide (DDDNGDDD). This affinity is approximately 100-fold stronger than that observed in the binding pair of the Zn(II)-DpaTyr (4-4Zn(II)) and the D4x2 tag (DDDDGDDDD), a useful tag-probe pair previously reported by us. The recognition pair of the Ni(II)-DpaTyr probe and the D3x2 tag can also work effectively on a protein surface, that is, 4-4Ni(II) is strongly bound to the FKBP12 protein tethered with the D3x2 tag (DDDNGDDD) with a large K(app) value of 5x10(8) M(-1). Taking advantage of the strong-binding affinity, this pair was successfully applied to the selective inactivation of the tag-fused beta-galactosidase by using the chromophore-assisted light inactivation (CALI) technique under crude conditions, such as cell lysate.


Asunto(s)
Ácido Aspártico/química , Complejos de Coordinación/química , Níquel/química , Oligopéptidos/química , Proteínas/química , Secuencia de Aminoácidos , Calorimetría , Complejos de Coordinación/síntesis química , Dimerización , Colorantes Fluorescentes/química , Unión Proteica , Proteína 1A de Unión a Tacrolimus/química , beta-Galactosidasa/química
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