RESUMEN
S-Adenosyl-l-homocysteine hydrolase (SAHH) is a crucial enzyme that governs S-adenosyl methionine (SAM)-dependent methylation reactions within cells and regulates the intracellular concentration of SAH. Legionella pneumophila, the causative pathogen of Legionnaires' disease, encodes Lpg2021, which is the first identified dimeric SAHH in bacteria and is a promising target for drug development. Here, we report the structure of Lpg2021 in its ligand-free state and in complexes with adenine (ADE), adenosine (ADO), and 3-Deazaneplanocin A (DZNep). X-ray crystallography, isothermal titration calorimetry (ITC), and molecular docking were used to elucidate the binding mechanisms of Lpg2021 to its substrates and inhibitors. Virtual screening was performed to identify potential Lpg2021 inhibitors. This study contributes a novel perspective to the understanding of SAHH evolution and establishes a structural framework for designing specific inhibitors targeting pathogenic Legionella pneumophila SAHH.
Asunto(s)
Adenosilhomocisteinasa , Legionella pneumophila , Simulación del Acoplamiento Molecular , Legionella pneumophila/enzimología , Especificidad por Sustrato , Adenosilhomocisteinasa/metabolismo , Adenosilhomocisteinasa/antagonistas & inhibidores , Adenosilhomocisteinasa/química , Cristalografía por Rayos X , Adenosina/análogos & derivados , Adenosina/metabolismo , Adenosina/química , Adenina/química , Adenina/metabolismo , Adenina/análogos & derivados , Unión Proteica , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , N-Glicosil HidrolasasRESUMEN
S-adenosyl-l-homocysteine (SAH), an amino acid derivative, is a key intermediate metabolite in methionine metabolism, which is normally considered as a harmful by-product and hydrolyzed quickly once formed. AHCY (adenosylhomocysteinase) converts SAH into homocysteine and adenosine. There are two other members in the AHCY family, AHCYL1 (adenosylhomocysteinase like 1) and AHCYL2 (adenosylhomocysteinase like 2). Here we define AHCYL1 function as a SAH sensor to inhibit macroautophagy/autophagy through PIK3C3. The C terminus of AHCYL1 interacts with SAH specifically and the interaction with SAH promotes the binding of the N terminus to the catalytic domain of PIK3C3, resulting in inhibition of PIK3C3. More importantly, this observation was further validated in vivo, indicating that SAH functions as a signaling molecule. Our study uncovers a new axis of SAH-AHCYL1-PIK3C3, which senses the intracellular level of SAH to inhibit autophagy in an MTORC1-independent manner.Abbreviations: ADOX: adenosine dialdehyde; AHCY: adenosylhomocysteinase; AHCYL1: adenosylhomocysteinase like 1; cLEU: cycloleucine; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PtdIns3P: phosphatidylinositol-3-phosphate; SAH: S-adenosyl-l-homocysteine; SAM: S-adenosyl-l-methionine.
Asunto(s)
Autofagia , S-Adenosilhomocisteína , Adenosilhomocisteinasa/química , Adenosilhomocisteinasa/metabolismo , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina , S-Adenosilhomocisteína/química , S-Adenosilhomocisteína/metabolismoRESUMEN
Ketone bodies are bioactive metabolites that function as energy substrates, signaling molecules, and regulators of histone modifications. ß-hydroxybutyrate (ß-OHB) is utilized in lysine ß-hydroxybutyrylation (Kbhb) of histones, and associates with starvation-responsive genes, effectively coupling ketogenic metabolism with gene expression. The emerging diversity of the lysine acylation landscape prompted us to investigate the full proteomic impact of Kbhb. Global protein Kbhb is induced in a tissue-specific manner by a variety of interventions that evoke ß-OHB. Mass spectrometry analysis of the ß-hydroxybutyrylome in mouse liver revealed 891 sites of Kbhb within 267 proteins enriched for fatty acid, amino acid, detoxification, and one-carbon metabolic pathways. Kbhb inhibits S-adenosyl-L-homocysteine hydrolase (AHCY), a rate-limiting enzyme of the methionine cycle, in parallel with altered metabolite levels. Our results illuminate the role of Kbhb in hepatic metabolism under ketogenic conditions and demonstrate a functional consequence of this modification on a central metabolic enzyme.
Asunto(s)
Ácido 3-Hidroxibutírico/metabolismo , Cuerpos Cetónicos/metabolismo , Hígado/metabolismo , Lisina/metabolismo , Proteómica , Adenosilhomocisteinasa/química , Adenosilhomocisteinasa/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Humanos , Masculino , Ratones Endogámicos C57BL , Modelos Moleculares , NAD/metabolismoRESUMEN
Naegleria fowleri is a pathogenic, thermophilic, free-living amoeba which causes primary amebic meningoencephalitis (PAM). Penetrating the olfactory mucosa, the brain-eating amoeba travels along the olfactory nerves, burrowing through the cribriform plate to its destination: the brain's frontal lobes. The amoeba thrives in warm, freshwater environments, with peak infection rates in the summer months and has a mortality rate of approximately 97%. A major contributor to the pathogen's high mortality is the lack of sensitivity of N. fowleri to current drug therapies, even in the face of combination-drug therapy. To enable rational drug discovery and design efforts we have pursued protein production and crystallography-based structure determination efforts for likely drug targets from N. fowleri. The genes were selected if they had homology to drug targets listed in Drug Bank or were nominated by primary investigators engaged in N. fowleri research. In 2017, 178 N. fowleri protein targets were queued to the Seattle Structural Genomics Center of Infectious Disease (SSGCID) pipeline, and to date 89 soluble recombinant proteins and 19 unique target structures have been produced. Many of the new protein structures are potential drug targets and contain structural differences compared to their human homologs, which could allow for the development of pathogen-specific inhibitors. Five of the structures were analyzed in more detail, and four of five show promise that selective inhibitors of the active site could be found. The 19 solved crystal structures build a foundation for future work in combating this devastating disease by encouraging further investigation to stimulate drug discovery for this neglected pathogen.
Asunto(s)
Descubrimiento de Drogas , Naegleria fowleri/metabolismo , Proteínas Protozoarias/antagonistas & inhibidores , Adenosilhomocisteinasa/antagonistas & inhibidores , Adenosilhomocisteinasa/química , Adenosilhomocisteinasa/metabolismo , Sitios de Unión , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Simulación de Dinámica Molecular , Naegleria fowleri/genética , Fosfoglicerato Mutasa/antagonistas & inhibidores , Fosfoglicerato Mutasa/química , Fosfoglicerato Mutasa/metabolismo , Estructura Cuaternaria de Proteína , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Proteína-Arginina N-Metiltransferasas/química , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteoma , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismoRESUMEN
An antioxidant molecule namely, adenosyl homocysteinase (AHc) was identified from the earlier constructed transcriptome database of Spirulina, where it was cultured in a sulphur deprived condition. From the AHc protein, a small peptide NL13 was identified using bioinformatics tools and was predicted to have antioxidant property. Further, the peptide was synthesised and its antioxidant mechanism was addressed at molecular level. NL13 was subjected to various antioxidant assays including DPPH assay, HARS assay, SARS Assay, NO assay and ABTS assay, where NL13 exhibited significant (P < 0.05) potential antioxidant activity compared to its antioxidant control, Trolox. Cytotoxicity was performed on Human whole blood and the cell viability was performed on VERO fibroblast cells. In both assays, it was found that NL13 did not exhibit any cytotoxic effect towards the cells. Further, the intracellular ROS was performed on Multimode reader followed by imaging on fluorescence microscope which showed scavenging activity even at lower concentration of NL13 (31.2 µM). An effective wound healing property of NL13 on VERO cells was confirmed by analysing the cell migration rate at two different time intervals (24 and 48 h). Overall, the study shows that NL13 peptide scavenges the intracellular oxidative stress.
Asunto(s)
Adenosilhomocisteinasa/química , Antioxidantes/farmacología , Fibroblastos/citología , Péptidos/farmacología , Spirulina/enzimología , Cicatrización de Heridas/efectos de los fármacos , Animales , Antioxidantes/síntesis química , Antioxidantes/química , Proteínas Bacterianas/química , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular , Chlorocebus aethiops , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Péptidos/síntesis química , Péptidos/química , Especies Reactivas de Oxígeno/metabolismo , Células VeroRESUMEN
S-adenosyl-L-homocysteine hydrolase from Pseudomonas aeruginosa (PaSAHase) coordinates one K+ ion and one Zn2+ ion in the substrate binding area. The cations affect the enzymatic activity and substrate binding but the molecular mechanisms of their action are unknown. Enzymatic and isothermal titration calorimetry studies demonstrated that the K+ ions stimulate the highest activity and strongest ligand binding in comparison to other alkali cations, while the Zn2+ ions inhibit the enzyme activity. PaSAHase was crystallized in the presence of adenine nucleosides and K+ or Rb+ ions. The crystal structures show that the alkali ion is coordinated in close proximity of the purine ring and a 23Na NMR study showed that the monovalent cation coordination site is formed upon ligand binding. The cation, bound in the area of a molecular hinge, orders and accurately positions the amide group of Q65 residue to allow its interaction with the ligand. Moreover, binding of potassium is required to enable unique dynamic properties of the enzyme that ensure its maximum catalytic activity. The Zn2+ ion is bound in the area of a molecular gate that regulates access to the active site. Zn2+ coordination switches the gate to a shut state and arrests the enzyme in its closed, inactive conformation.
Asunto(s)
Adenosilhomocisteinasa/metabolismo , Metales/farmacología , Pseudomonas aeruginosa/enzimología , Adenosilhomocisteinasa/química , Secuencia de Aminoácidos , Sitios de Unión , Cationes , Secuencia Conservada , Inhibidores Enzimáticos/farmacología , Glutamina/metabolismo , Cinética , Ligandos , Potasio/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Especificidad por Sustrato/efectos de los fármacos , Termodinámica , Factores de Tiempo , Zinc/farmacologíaRESUMEN
Deamidation of asparagine (Asn) residues is one of the most common chemical degradation pathways observed in proteins. This reaction must be understood and controlled in therapeutic drug candidates, as chemical changes can affect their efficacy and safety. The analytical tools available for detection of deamidation reaction products, such as isoaspartic acid residues, are either chromatographic or electrophoretic, and require MS detection for absolute identification of peaks. High-throughput measurement of protein degradation has typically been limited to probing the target's physical state using spectroscopic techniques. Here, we describe a high throughput assay for isoaspartate residues using fluorescent detection in a microtiter plate format. The method allows for fast detection of protein deamidation in a cost-efficient manner. The method can be employed even if the target peptide or protein contains free Cys residues. The technique appears to be selective, linear, and accurate.
Asunto(s)
Adenosilhomocisteinasa/química , Colorantes Fluorescentes/química , Ensayos Analíticos de Alto Rendimiento/métodos , Amidas/metabolismo , Secuencia de Aminoácidos , Asparagina/química , Asparagina/metabolismo , Pruebas de Enzimas/economía , Pruebas de Enzimas/instrumentación , Pruebas de Enzimas/métodos , Glucagón/química , Ensayos Analíticos de Alto Rendimiento/economía , Ensayos Analíticos de Alto Rendimiento/instrumentación , Concentración de Iones de Hidrógeno , Proteolisis , Sensibilidad y Especificidad , Cloruro de Sodio/químicaRESUMEN
Thermotoga maritima is a hyperthermophilic bacterium but its genome encodes a number of archaeal proteins including S-adenosyl-L-homocysteine hydrolase (SAHase), which regulates cellular methylation reactions. The question of proper folding and activity of proteins of extremophilic origin is an intriguing problem. When expressed in E.coli and purified (as a homotetramer) at room temperature, the hyperthermophilic SAHase from T.maritima was inactive. ITC study indicated that the protein undergoes heat-induced conformational changes, and enzymatic activity assays demonstrated that these changes are required to attain enzymatic activity. To explain the mechanism of thermal activation, two crystal structures of the inactive form of T. maritima SAHase (iTmSAHase) were determined for an incomplete binary complex with the reduced cofactor (NADH), and in a mixture of binary complexes with NADH and with adenosine. In contrast to active SAHases, in iTmSAHase only two of the four subunits contain a bound cofactor, predominantly in its non-reactive, reduced state. Moreover, the closed-like conformation of the cofactor-containing subunits precludes substrate delivery to the active site. The two other subunits cannot be involved in the enzymatic reaction either; although they have an open-like conformation, they do not contain the cofactor, whose binding site may be occupied by an adenosine molecule. The results suggest that this enzyme, when expressed in mesophilic cells, is arrested in the activity-incompatible conformation revealed by its crystal structures.
Asunto(s)
Adenosilhomocisteinasa/genética , Adenosilhomocisteinasa/metabolismo , Escherichia coli/genética , Thermotoga maritima/enzimología , Adenosilhomocisteinasa/química , Adenosilhomocisteinasa/aislamiento & purificación , Sitios de Unión , Coenzimas/metabolismo , Cristalografía por Rayos X , Activación Enzimática , Expresión Génica , Modelos Moleculares , NAD/metabolismo , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Temperatura , Thermotoga maritima/genéticaRESUMEN
We herein describe a novel fluorescent method for the rapid and selective detection of adenosine by utilizing DNA-templated Cu/Ag nanoclusters (NCs) and employing s-adenosylhomocysteine hydrolase (SAHH). SAHH is allowed to promote hydrolysis reaction of s-adenosylhomocysteine (SAH) and consequently produces homocysteine, which would quench the fluorescence signal from DNA-templated Cu/Ag nanoclusters employed as a signaling probe in this study. On the other hand, adenosine significantly inhibits the hydrolysis reaction and prevent the formation of homocysteine. Consequently, highly enhanced fluorescence signal from DNA-Cu/Ag NCs is retained, which could be used to identify the presence of adenosine. By employing this design principle, adenosine was sensitively detected down to 19nM with high specificity over other adenosine analogs such as AMP, ADP, ATP, cAMP, guanosine, cytidine, and urine. Finally, the diagnostic capability of this method was successfully verified by reliably detecting adenosine present in a real human serum sample.
Asunto(s)
Adenosina/aislamiento & purificación , Adenosilhomocisteinasa/química , Técnicas Biosensibles , Adenosina/química , Cobre/química , ADN/química , Fluorescencia , Humanos , Nanopartículas del Metal/química , S-Adenosilhomocisteína , Plata/químicaRESUMEN
S-Adenosyl-L-homocysteine hydrolase (SAHase) is involved in the enzymatic regulation of S-adenosyl-L-methionine (SAM)-dependent methylation reactions. After methyl-group transfer from SAM, S-adenosyl-L-homocysteine (SAH) is formed as a byproduct, which in turn is hydrolyzed to adenosine (Ado) and homocysteine (Hcy) by SAHase. The crystal structure of BeSAHase, an SAHase from Bradyrhizobium elkanii, which is a nitrogen-fixing bacterial symbiont of legume plants, was determined at 1.7â Å resolution, showing the domain organization (substrate-binding domain, NAD(+) cofactor-binding domain and dimerization domain) of the subunits. The protein crystallized in its biologically relevant tetrameric form, with three subunits in a closed conformation enforced by complex formation with the Ado product of the enzymatic reaction. The fourth subunit is ligand-free and has an open conformation. The BeSAHase structure therefore provides a unique snapshot of the domain movement of the enzyme induced by the binding of its natural ligands.
Asunto(s)
Adenosilhomocisteinasa/química , Proteínas Bacterianas/química , Bradyrhizobium/química , NAD/química , Subunidades de Proteína/química , S-Adenosilhomocisteína/química , S-Adenosilmetionina/química , Adenosina/química , Adenosina/metabolismo , Adenosilhomocisteinasa/genética , Adenosilhomocisteinasa/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Biocatálisis , Bradyrhizobium/enzimología , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Homocisteína/química , Homocisteína/metabolismo , Modelos Moleculares , NAD/metabolismo , Unión Proteica , Multimerización de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , S-Adenosilhomocisteína/metabolismo , S-Adenosilmetionina/metabolismoRESUMEN
S-adenosyl-L-homocysteine hydrolase (SAH hydrolase or SAHH) is a highly conserved enzyme that catalyses the reversible hydrolysis of SAH to L-homocysteine (HCY) and adenosine (ADO). High-resolution crystal structures have been reported for bacterial and plant SAHHs, but not mammalian SAHHs. Here, we report the first high-resolution crystal structure of mammalian SAHH (mouse SAHH) in complex with a reaction product (ADO) and with two reaction intermediate analogues-3'-keto-aristeromycin (3KA) and noraristeromycin (NRN)-at resolutions of 1.55, 1.55, and 1.65 Å. Each of the three structures constitutes a structural snapshot of one of the last three steps of the five-step process of SAH hydrolysis by SAHH. In the NRN complex, a water molecule, which is an essential substrate for ADO formation, is structurally identified for the first time as the candidate donor in a Michael addition by SAHH to the 3'-keto-4',5'-didehydroadenosine reaction intermediate. The presence of the water molecule is consistent with the reaction mechanism proposed by Palmer &Abeles in 1979. These results provide insights into the reaction mechanism of the SAHH enzyme.
Asunto(s)
Adenosilhomocisteinasa/química , Adenosilhomocisteinasa/metabolismo , Adenosina/análogos & derivados , Adenosina/química , Adenosina/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Biocatálisis , Cristalografía por Rayos X , Hidrólisis , Ratones , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , S-Adenosilhomocisteína/metabolismo , Alineación de Secuencia , Especificidad por SustratoRESUMEN
S-adenosylhomocysteine (SAH) hydrolase catalyzes the reversible hydrolysis of SAH into adenosine and homocysteine by using NAD(+) as a cofactor. The enzyme from Thermotoga maritima (tmSAHH) has great potentials in industrial applications because of its hyperthermophilic properties. Here, two crystal structures of tmSAHH in complex with NAD(+) show both open and closed conformations despite the absence of bound substrate. Each subunit of the tetrameric enzyme is composed of three domains, namely the catalytic domain, the NAD(+)-binding domain and the C-terminal domain. The NAD(+) binding mode is clearly observed and a substrate analogue can also be modeled into the active site, where two cysteine residues in mesophilic enzymes are replaced by serine and threonine in tmSAHH. Notably, the C-terminal domain of tmSAHH lacks the second loop region of mesophilic SAHH, which is important in NAD(+) binding, and thus exposes the bound cofactor to the solvent. The difference explains the higher NAD(+) requirement of tmSAHH because of the reduced affinity. Furthermore, the feature of missing loop is consistently observed in thermophilic bacterial and archaeal SAHHs, and may be related to their thermostability.
Asunto(s)
Adenosilhomocisteinasa/química , Modelos Moleculares , Thermotoga maritima/enzimología , Adenosilhomocisteinasa/metabolismo , Cristalización , NAD/química , NAD/metabolismo , Unión Proteica , Conformación Proteica , Difracción de Rayos XRESUMEN
Detection of salt- and analyte-induced aggregation of gold nanoparticles (AuNPs) mostly relies on costly and bulky analytical instruments. To response this drawback, a portable, miniaturized, sensitive, and cost-effective detection technique is urgently required for rapid field detection and monitoring of target analyte via the use of AuNP-based sensor. This study combined a miniaturized spectrometer with a 532-nm laser to develop a laser-induced Rayleigh scattering technique, allowing the sensitive and selective detection of Rayleigh scattering from the aggregated AuNPs. Three AuNP-based sensing systems, including salt-, thiol- and metal ion-induced aggregation of the AuNPs, were performed to examine the sensitivity of laser-induced Rayleigh scattering technique. Salt-, thiol-, and metal ion-promoted NP aggregation were exemplified by the use of aptamer-adsorbed, fluorosurfactant-stabilized, and gallic acid-capped AuNPs for probing K(+), S-adenosylhomocysteine hydrolase-induced hydrolysis of S-adenosylhomocysteine, and Pb(2+), in sequence. Compared to the reported methods for monitoring the aggregated AuNPs, the proposed system provided distinct advantages of sensitivity. Laser-induced Rayleigh scattering technique was improved to be convenient, cheap, and portable by replacing a diode laser and a miniaturized spectrometer with a laser pointer and a smart-phone. Using this smart-phone-based detection platform, we can determine whether or not the Pb(2+) concentration exceed the maximum allowable level of Pb(2+) in drinking water.
Asunto(s)
Agua Potable/química , Oro/química , Plomo/análisis , Nanopartículas del Metal/química , Nefelometría y Turbidimetría/métodos , Contaminantes Químicos del Agua/análisis , Adenosilhomocisteinasa/química , Cationes Bivalentes , Teléfono Celular , Ácido Gálico/química , Humanos , Rayos Láser , Luz , Límite de Detección , Nefelometría y Turbidimetría/instrumentación , S-Adenosilhomocisteína/química , Dispersión de RadiaciónRESUMEN
S-Adenosylhomocysteine hydrolase (SAHH) catalyzes the reversible conversion of S-adenosylhomocysteine into adenosine and homocysteine. The SAHH from Thermotoga maritima (TmSAHH) was expressed in Escherichia coli and the recombinant protein was purified and crystallized. TmSAHH crystals belonging to space group C2, with unit-cell parameters a=106.3, b=112.0, c=164.9â Å, ß=103.5°, were obtained by the sitting-drop vapour-diffusion method and diffracted to 2.85â Å resolution. Initial phase determination by molecular replacement clearly indicated that the crystal contains one homotetramer per asymmetric unit. Further refinement of the crystal structure is in progress.
Asunto(s)
Adenosilhomocisteinasa/química , Proteínas Bacterianas/química , Thermotoga maritima/enzimología , Adenosilhomocisteinasa/aislamiento & purificación , Proteínas Bacterianas/aislamiento & purificación , Cristalización , Difracción de Rayos XRESUMEN
In this work, an ultrasensitive peroxydisulfate electrochemiluminescence (ECL) immunosensor using in situ generation of L-homocysteine (L-Hcys) for signal amplification was successfully constructed for detection of carcinoembryonic antigen (CEA). In the reaction of biological methylation, S-adenosyl-L-homocysteine hydrolase (SAHH) catalyzed the reversible hydrolysis of S-adenosyl-L-homocysteine (SAH) to produce L-Hcys, which was inducted into ECL system to construct the immunosensor for signal amplification in this work. Simultaneously, Gold and palladium nanoparticles functionalized multi-walled carbon nanotubes (Au-PdNPs@MWCNTs) were prepared, which were introduced to immobilize the secondary antibody (Ab2) and SAHH with high loading amount and good biological activity due to their improved surface area and excellent biocompatibility. Then the proposed ECL immunosensor was developed by a sandwich-type format using Au-PdNPs@MWCNTs-SAHH-Ab2 as tracer and graphene together with AuNPs as substrate. Besides the enhancement of Au-PdNPs, the enzymatic catalysis reaction also amplified the ECL signal dramatically, which was achieved by efficient catalysis of the SAHH towards the hydrolysis of SAH to generate improved amount of L-Hcys in situ. Furthermore, due to the special interaction between Au-PdNPs and -SH or -NH2 in L-Hcys, L-Hcys would gradually accumulate on the surface of the immunosensor, which greatly enhanced the concentration of L-Hcys on the immunosensor surface and further improved the ECL intensity. With the amplification factors above, a wide linear ranged from 0.1 pg mL(-1) to 80 ng mL(-1) was acquired with a relatively low detection limit of 33 fg mL(-1) for CEA.
Asunto(s)
Biomarcadores de Tumor/análisis , Técnicas Electroquímicas , Homocisteína/análisis , Mediciones Luminiscentes , Adenosilhomocisteinasa/química , Adenosilhomocisteinasa/metabolismo , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , Oro/química , Grafito/química , Humanos , Nanopartículas del Metal/química , Nanotubos de Carbono/química , Nanotubos de Carbono/ultraestructura , Neoplasias/diagnóstico , Paladio/químicaRESUMEN
ß-Sultams are potent electrophiles that modify nucleophilic residues in selected enzyme active sites. We here identify and characterize some of the specific bacterial targets and show a unique inhibition of the azoreductase family.
Asunto(s)
Bacterias/enzimología , Proteínas Bacterianas/metabolismo , Sulfonamidas/metabolismo , Adenosilhomocisteinasa/química , Adenosilhomocisteinasa/metabolismo , Proteínas Bacterianas/química , Dominio Catalítico , Cromatografía Líquida de Alta Presión , Cristalografía por Rayos X , Unión Proteica , Proteína Disulfuro Isomerasas/química , Proteína Disulfuro Isomerasas/metabolismo , Sulfonamidas/química , Espectrometría de Masas en TándemRESUMEN
Novel biomarker verification assays are urgently required to improve the efficiency of biomarker development. Benefitting from lower development costs, multiple reaction monitoring (MRM) has been used for biomarker verification as an alternative to immunoassay. However, in general MRM analysis, only one sample can be quantified in a single experiment, which restricts its application. Here, a Hyperplex-MRM quantification approach, which combined mTRAQ for absolute quantification and iTRAQ for relative quantification, was developed to increase the throughput of biomarker verification. In this strategy, equal amounts of internal standard peptides were labeled with mTRAQ reagents Δ0 and Δ8, respectively, as double references, while 4-plex iTRAQ reagents were used to label four different samples as an alternative to mTRAQ Δ4. From the MRM trace and MS/MS spectrum, total amounts and relative ratios of target proteins/peptides of four samples could be acquired simultaneously. Accordingly, absolute amounts of target proteins/peptides in four different samples could be achieved in a single run. In addition, double references were used to increase the reliability of the quantification results. Using this approach, three biomarker candidates, ademosylhomocysteinase (AHCY), cathepsin D (CTSD), and lysozyme C (LYZ), were successfully quantified in colorectal cancer (CRC) tissue specimens of different stages with high accuracy, sensitivity, and reproducibility. To summarize, we demonstrated a promising quantification method for high-throughput verification of biomarker candidates.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Neoplasias Colorrectales/metabolismo , Espectrometría de Masas en Tándem/normas , Adenosilhomocisteinasa/química , Adenosilhomocisteinasa/metabolismo , Adulto , Anciano , Secuencia de Aminoácidos , Calibración , Catepsina D/química , Catepsina D/metabolismo , Neoplasias Colorrectales/diagnóstico , Femenino , Humanos , Masculino , Persona de Mediana Edad , Muramidasa/química , Muramidasa/metabolismo , Fragmentos de Péptidos/química , Estándares de Referencia , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Coloración y EtiquetadoRESUMEN
Mycobacterium tuberculosis modulates expression of various metabolism-related genes to adapt in the adverse host environment. The gene coding for M. tuberculosis S-adenosylhomocysteine hydrolase (Mtb-SahH) is essential for optimal growth and the protein product is involved in intermediary metabolism. However, the relevance of SahH in mycobacterial physiology is unknown. In this study, we analyze the role of Mtb-SahH in regulating homocysteine concentration in surrogate host Mycobacterium smegmatis. Mtb-SahH catalyzes reversible hydrolysis of S-adenosylhomocysteine to homocysteine and adenosine and we demonstrate that the conserved His363 residue is critical for bi-directional catalysis. Mtb-SahH is regulated by serine/threonine phosphorylation of multiple residues by M. tuberculosis PknB. Major phosphorylation events occur at contiguous residues Thr219, Thr220 and Thr221, which make pivotal contacts with cofactor NADâº. Consequently, phosphorylation negatively modulates affinity of enzyme towards NAD⺠as well as SAH-synthesis. Thr219, Thr220 and Thr221 are essential for enzyme activity, and therefore, responsible for SahH-mediated regulation of homocysteine.
Asunto(s)
Adenosilhomocisteinasa/metabolismo , Homocisteína/metabolismo , Mycobacterium tuberculosis/enzimología , Adenosilhomocisteinasa/química , Activación Enzimática , Histidina/química , Hidrólisis , Cinética , Redes y Vías Metabólicas , Modelos Moleculares , Mycobacterium/enzimología , Mycobacterium/metabolismo , Fosforilación , Conformación Proteica , Reproducibilidad de los ResultadosRESUMEN
S-Adenosylhomocysteine hydrolase (SahH) is known as an ubiquitous player in methylation-based process that maintains the intracellular S-adenosylhomocysteine (SAH) and S-adenosylmethionine (SAM) equilibrium. Given its crucial role in central metabolism in both eukaryotes and prokaryotes, it is assumed that SahH must be regulated, albeit little is known regarding molecular mechanisms governing its activity. We report here that SahH from Mycobacterium tuberculosis can be phosphorylated by mycobacterial Ser/Thr protein kinases and that phosphorylation negatively affects its enzymatic activity. Mass spectrometric analyses and site-directed mutagenesis identified Thr2 and Thr221 as the two phosphoacceptors. SahH_T2D, SahH_T221D and SahH_T2D/T221D, designed to mimic constitutive phosphorylation, exhibited markedly decreased activity compared to the wild-type enzyme. Both residues are fully conserved in other mycobacterial SahH orthologues, suggesting that SahH phosphorylation on Thr2 and Thr221 may represent a novel and presumably more general mechanism of regulation of the SAH/SAM balance in mycobacteria.
Asunto(s)
Adenosilhomocisteinasa/metabolismo , Proteínas Bacterianas/metabolismo , Mycobacterium tuberculosis/enzimología , Procesamiento Proteico-Postraduccional , Serina/metabolismo , Treonina/metabolismo , Adenosilhomocisteinasa/química , Adenosilhomocisteinasa/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Secundaria de Proteína , Serina/química , Serina/genética , Treonina/química , Treonina/genéticaRESUMEN
The methylation of DNA, RNA, and proteins plays crucial roles in numerous biological processes, including epigenetic control, virus replication, and cell differentiation. In mammals, the rate-limiting step of the S-adenosylmethionine-dependent methylation process is exclusively controlled by S-adenosylhomocysteine (S-AdoHcy) hydrolase (SAHH). SAHH hydrolyzes S-AdoHcy to adenosine and homocysteine (Hcy) and is therefore a potential therapeutic target for various diseases, including cancer, malaria, and viral diseases. However, a simple and highly sensitive assay for the evaluation of SAHH activity, particularly for drug discovery, had not yet been developed. Here we present the development of a fluorescence-based assay for the measurement of SAHH activity in biological samples. We combined the advantages of the detection of fluorescent thiol groups in Hcy by ThioGlo1 with the S-AdoHcy-driven enzyme-coupled reaction. Our results confirmed the reliability of the proposed assay for the measurement of the SAHH activity of purified SAHH and showed the potential of this assay for the measurement of the SAHH activity of biological samples. Therefore, the proposed SAHH activity assay may be utilized in clinical laboratories and in high-throughput screenings for the identification of new SAHH inhibitors with potentially beneficial effects on numerous pathologies.