RESUMEN
Sec14-like phosphatidylinositol transfer proteins (PITPs) are involved in lipid metabolism and phosphatidylinositol 4-phosphate signaling by transporting phosphatidylinositol (PI) and a secondary ligand between the organellar membranes in eukaryotes. Yeast Sfh2 is a PITP that transfers PI and squalene without phosphatidylcholine transfer activity. To investigate the structural determinants for ligand specificity and transport in Sfh2, crystal structures of Sfh2 in complex with PI and squalene were determined at 1.5 and 2.4â Å resolution, respectively. The inositol head group of PI is recognized by highly conserved residues around the pocket entrance. The acyl chains of PI bind into a large hydrophobic cavity. Squalene is accommodated in the bottom of the cavity entirely by hydrophobic interactions. The binding of PI and squalene are mutually exclusive due to their overlapping binding sites, correlating with the role in lipid exchange. The binding mode of PI is well conserved in Sfh family proteins. However, squalene binding is unique to the Sfh2 homolog due to the specific hydrophobic residues forming a shape-complementary binding pocket. Recombinant apo Sfh2 forms a homodimer in vitro by the hydrophobic interaction of the gating α10-α11 helices in an open conformation. Ligand binding closes the lid and dissociates the dimer into monomers. This study reveals the structural determinants for the recognition of the conserved PI and a secondary ligand, squalene, and provides implications for the lipid-transfer function of Sfh2.
Asunto(s)
Fosfatidilinositoles , Proteínas de Transferencia de Fosfolípidos , Ligandos , Fosfatidilinositoles/química , Fosfatidilinositoles/metabolismo , Proteínas de Transferencia de Fosfolípidos/química , Proteínas de Transferencia de Fosfolípidos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Escualeno/metabolismoRESUMEN
In this work we introduce a novel filtering and molecular modeling pipeline based on a fingerprint and descriptor similarity procedure, coupled with molecular docking and molecular dynamics (MD), to select potential novel quoinone outside inhibitors (QoI) of cytochrome bc1 with the aim of determining the same or different chromophores to usual. The study was carried out using the yeast cytochrome bc1 complex with its docked ligand (stigmatellin), using all the fungicides from FRAC code C3 mode of action, 8617 Drugbank compounds and 401,624 COCONUT compounds. The introduced drug repurposing pipeline consists of compound similarity with C3 fungicides and molecular docking (MD) simulations with final QM/MM binding energy determination, while aiming for potential novel chromophores and perserving at least an amide (R1HN(C=O)R2) or ester functional group of almost all up to date C3 fungicides. 3D descriptors used for a similarity test were based on the 280 most stable Padel descriptors. Hit compounds that passed fingerprint and 3D descriptor similarity condition and had either an amide or an ester group were submitted to docking where they further had to satisfy both Chemscore fitness and specific conformation constraints. This rigorous selection resulted in a very limited number of candidates that were forwarded to MD simulations and QM/MM binding affinity estimations by the ORCA DFT program. In this final step, stringent criteria based on (a) sufficiently high frequency of H-bonds; (b) high interaction energy between protein and ligand through the whole MD trajectory; and (c) high enough QM/MM binding energy scores were applied to further filter candidate inhibitors. This elaborate search pipeline led finaly to four Drugbank synthetic lead compounds (DrugBank) and seven natural (COCONUT database) lead compounds-tentative new inhibitors of cytochrome bc1. These eleven lead compounds were additionally validated through a comparison of MM/PBSA free binding energy for new leads against those obtatined for 19 QoIs.
Asunto(s)
Complejo III de Transporte de Electrones/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Saccharomyces cerevisiae/enzimología , Evaluación Preclínica de Medicamentos , Complejo III de Transporte de Electrones/química , Proteínas de Saccharomyces cerevisiae/químicaRESUMEN
Radiotherapy, the most common therapy for the treatment of solid tumors, exerts its effects by inducing DNA damage. To fully understand the extent and nature of this damage, DNA models that mimic the in vivo situation should be utilized. In a cellular context, genomic DNA constantly interacts with proteins and these interactions could influence both the primary radical processes (triggered by ionizing radiation) and secondary reactions, ultimately leading to DNA damage. However, this is seldom addressed in the literature. In this work, we propose a general approach to tackle these shortcomings. We synthesized a protein-DNA complex that more closely represents DNA in the physiological environment than oligonucleotides solution itself, while being sufficiently simple to permit further chemical analyses. Using click chemistry, we obtained an oligonucleotide-peptide conjugate, which, if annealed with the complementary oligonucleotide strand, forms a complex that mimics the specific interactions between the GCN4 protein and DNA. The covalent bond connecting the oligonucleotide and peptide constitutes a part of substituted triazole, which forms due to the click reaction between the short peptide corresponding to the specific amino acid sequence of GCN4 protein (yeast transcription factor) and a DNA fragment that is recognized by the protein. DNAse footprinting demonstrated that the part of the DNA fragment that specifically interacts with the peptide in the complex is protected from DNAse activity. Moreover, the thermodynamic characteristics obtained using differential scanning calorimetry (DSC) are consistent with the interaction energies calculated at the level of metadynamics. Thus, we present an efficient approach to generate a well-defined DNA-peptide conjugate that mimics a real DNA-peptide complex. These complexes can be used to investigate DNA damage under conditions very similar to those present in the cell.
Asunto(s)
Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/química , ADN de Cadena Simple/química , ADN/química , Péptidos/química , Proteínas de Saccharomyces cerevisiae/química , Secuencia de Aminoácidos , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Sitios de Unión , Rastreo Diferencial de Calorimetría , Catálisis , Cromatografía Líquida de Alta Presión , Química Clic , Cobre/química , ADN/metabolismo , Daño del ADN , ADN de Cadena Simple/metabolismo , Simulación de Dinámica Molecular , Conformación de Ácido Nucleico , Péptidos/metabolismo , Dominios Proteicos , Proteínas de Saccharomyces cerevisiae/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Temperatura de TransiciónRESUMEN
Proteins and RNAs assemble in membrane-less organelles that organize intracellular spaces and regulate biochemical reactions. The ability of proteins and RNAs to form condensates is encoded in their sequences, yet it is unknown which domains drive the phase separation (PS) process and what are their specific roles. Here, we systematically investigated the human and yeast proteomes to find regions promoting condensation. Using advanced computational methods to predict the PS propensity of proteins, we designed a set of experiments to investigate the contributions of Prion-Like Domains (PrLDs) and RNA-binding domains (RBDs). We found that one PrLD is sufficient to drive PS, whereas multiple RBDs are needed to modulate the dynamics of the assemblies. In the case of stress granule protein Pub1 we show that the PrLD promotes sequestration of protein partners and the RBD confers liquid-like behaviour to the condensate. Our work sheds light on the fine interplay between RBDs and PrLD to regulate formation of membrane-less organelles, opening up the avenue for their manipulation.
Asunto(s)
Transición de Fase , Priones/metabolismo , Proteínas/metabolismo , ARN/metabolismo , Sitios de Unión , Recuperación de Fluorescencia tras Fotoblanqueo , Humanos , Proteínas de Unión a Poli(A)/química , Proteínas de Unión a Poli(A)/genética , Proteínas de Unión a Poli(A)/metabolismo , Priones/química , Dominios Proteicos , Proteínas/química , Proteoma , ARN/química , Proteínas con Motivos de Reconocimiento de ARN/química , Proteínas con Motivos de Reconocimiento de ARN/metabolismo , Motivos de Unión al ARN , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
XPA (Xeroderma pigmentosum complementation group A) is a core scaffold protein that plays significant roles in DNA damage verification and recruiting downstream endonucleases in the nucleotide excision repair (NER) pathway. Here, we present the 2.81 Å resolution crystal structure of the DNA-binding domain (DBD) of human XPA in complex with an undamaged splayed-arm DNA substrate with a single pair of non-complementary nucleotides. The structure reveals that two XPA molecules bind to one splayed-arm DNA with a 10-bp duplex recognition motif in a non-sequence-specific manner. XPA molecules bind to both ends of the DNA duplex region with a characteristic ß-hairpin. A conserved tryptophan residue Trp175 packs against the last base pair of DNA duplex and stabilizes the conformation of the characteristic ß-hairpin. Upon DNA binding, the C-terminal last helix of XPA would shift towards the minor groove of the DNA substrate for better interaction. Notably, human XPA is able to bind to the undamaged DNA duplex without any kinks, and XPA-DNA binding does not bend the DNA substrate obviously. This study provides structural basis for the binding mechanism of XPA to the undamaged splayed-arm DNA with a single pair of non-complementary nucleotides.
Asunto(s)
Daño del ADN , ADN/química , Modelos Moleculares , Proteína de la Xerodermia Pigmentosa del Grupo A/química , Aminoácidos , Sitios de Unión , Enzimas Reparadoras del ADN/química , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Humanos , Sustancias Macromoleculares/química , Sustancias Macromoleculares/metabolismo , Modelos Biológicos , Conformación Molecular , Unión Proteica , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Relación Estructura-Actividad , Factor de Transcripción TFIIH/química , Factor de Transcripción TFIIH/metabolismo , Proteína de la Xerodermia Pigmentosa del Grupo A/metabolismoRESUMEN
It is a time-consuming and challenging task for affinity measurement of drug lead compounds from a plant extract because of its chemical complexity. In this research, a strategy of ultrafiltration-high-performance liquid chromatography (HPLC) was developed to directly measure dissociation constant (Kd) of compounds from natural product extract to target protein, and the Kd measurement of α-glucosidase ligands from the ethyl acetate fraction of Perilla frutescens (L.) Britt. (PFEA) was performed. The recovery value, binding degree, and signal-to-noise ratio of α-glucosidase ligands from PFEA were first determined according to the ultrafiltration-HPLC results; the Kd values were then calculated using proposed equilibrium. Finally, oleanolic acid (4) and apigenin (8) from PFEA were determined as the high affinity ligands for α-glucosidase, and their Kds were calculated as 44.9⯵M and 88.5⯵M, respectively, which agreed with the isothermal titration calorimetry analysis, kinetic analysis, and computer simulation of molecular docking. These results suggested that the proposed strategy is a simple and convenient method for the direct Kd determination of compounds from natural product extract without using any internal calibrants or internal standards.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Inhibidores de Glicósido Hidrolasas/química , Perilla frutescens/química , Extractos Vegetales/química , Ultrafiltración/métodos , Inhibidores de Glicósido Hidrolasas/aislamiento & purificación , Cinética , Ligandos , Simulación del Acoplamiento Molecular , Extractos Vegetales/aislamiento & purificación , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/química , alfa-Glucosidasas/químicaRESUMEN
Stable recognition of the intron branchpoint (BP) by the U2 snRNP to form the pre-spliceosome is the first ATP-dependent step of splicing. Genetic and biochemical data from yeast indicate that Cus2 aids U2 snRNA folding into the stem IIa conformation prior to pre-spliceosome formation. Cus2 must then be removed by an ATP-dependent function of Prp5 before assembly can progress. However, the location from which Cus2 is displaced and the nature of its binding to the U2 snRNP are unknown. Here, we show that Cus2 contains a conserved UHM (U2AF homology motif) that binds Hsh155, the yeast homolog of human SF3b1, through a conserved ULM (U2AF ligand motif). Mutations in either motif block binding and allow pre-spliceosome formation without ATP. A 2.0 Å resolution structure of the Hsh155 ULM in complex with the UHM of Tat-SF1, the human homolog of Cus2, and complementary binding assays show that the interaction is highly similar between yeast and humans. Furthermore, we show that Tat-SF1 can replace Cus2 function by enforcing ATP dependence of pre-spliceosome formation in yeast extracts. Cus2 is removed before pre-spliceosome formation, and both Cus2 and its Hsh155 ULM binding site are absent from available cryo-EM structure models. However, our data are consistent with the apparent location of the disordered Hsh155 ULM between the U2 stem-loop IIa and the HEAT repeats of Hsh155 that interact with Prp5. We propose a model in which Prp5 uses ATP to remove Cus2 from Hsh155 such that extended base-pairing between U2 snRNA and the intron BP can occur.
Asunto(s)
Adenosina Trifosfato/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteína Nuclear Pequeña U2/química , Ribonucleoproteína Nuclear Pequeña U2/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Secuencias de Aminoácidos , Sitios de Unión , Secuencia Conservada , Cristalografía por Rayos X , ARN Helicasas DEAD-box/metabolismo , Humanos , Modelos Moleculares , Mutación , Unión Proteica , Empalme del ARN , Proteínas de Unión al ARN/genética , Ribonucleoproteína Nuclear Pequeña U2/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Pre-mRNA splicing must occur with extremely high fidelity. Spliceosomes assemble onto pre-mRNA guided by specific sequences (5' splice site, 3' splice site, and branchpoint). When splice sites are mutated, as in many hereditary diseases, the spliceosome can aberrantly select nearby pseudo- or "cryptic" splice sites, often resulting in nonfunctional protein. How the spliceosome distinguishes authentic splice sites from cryptic splice sites is poorly understood. We performed a Caenorhabditis elegans genetic screen to find cellular factors that affect the frequency with which the spliceosome uses cryptic splice sites and identified two alleles in core spliceosome component Prp8 that alter cryptic splicing frequency. Subsequent complementary genetic and structural analyses in yeast implicate these alleles in the stability of the spliceosome's catalytic core. However, despite a clear effect on cryptic splicing, high-throughput mRNA sequencing of these prp-8 mutant C. elegans reveals that overall alternative splicing patterns are relatively unchanged. Our data suggest the spliceosome evolved intrinsic mechanisms to reduce the occurrence of cryptic splicing and that these mechanisms are distinct from those that impact alternative splicing.
Asunto(s)
Empalme Alternativo , Sitios de Empalme de ARN , Ribonucleoproteína Nuclear Pequeña U4-U6/genética , Ribonucleoproteína Nuclear Pequeña U5/genética , Proteínas de Saccharomyces cerevisiae/genética , Alelos , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Aminoácidos , Animales , Caenorhabditis elegans , Secuencia Conservada , Frecuencia de los Genes , Sitios Genéticos , Modelos Moleculares , Conformación Proteica , Precursores del ARN , Ribonucleoproteína Nuclear Pequeña U4-U6/química , Ribonucleoproteína Nuclear Pequeña U5/química , Proteínas de Saccharomyces cerevisiae/química , EmpalmosomasRESUMEN
Alcohol dehydrogenase from Saccharomyces cerevisiae was immobilized on different inorganic support materials, i.e. powders of Al2O3, SiC, TiO2 and YSZ-8, by covalent bonding and physical adsorption. The raw powders were characterized by scanning electron microscopy, BET surface area, particle size distribution and ζ-potential measurements. Enzyme activity retention, storage stability and recyclability were evaluated on the basis of the measured support material properties. Preliminary experiments showed that the buffer selection was a critical factor. The properties of both the enzyme and the powders varied considerably between the buffers used; namely Tris-HCl (100 mM, pH 7) and MES (40 mM, pH 6.5) buffers. The enzyme activity was higher and more stable in the MES buffer, whereas the commonly used Tris buffer was problematic due to apparent incompatibility with formaldehyde. In MES, the order of decreasing activity of covalently bonded enzyme was on SiC > YSZ-8 > Al2O3 > TiO2. The lower performance of TiO2 was ascribed to the negative ζ-potential of the material, which impeded an efficient immobilization. Particle agglomeration, caused by low colloidal stability of the particles in MES buffer, hampered the storage stability of the immobilized systems. The results from this study show the advantages and limitations of using nanoparticles as immobilization supports, and highlight which properties of nanoparticles must be considered to ensure an efficient immobilization.
Asunto(s)
Alcohol Deshidrogenasa/química , Enzimas Inmovilizadas/química , Compuestos Inorgánicos/química , Proteínas de Saccharomyces cerevisiae/química , Adsorción , Alcohol Deshidrogenasa/metabolismo , Alcohol Deshidrogenasa/ultraestructura , Óxido de Aluminio/química , Tampones (Química) , Estabilidad de Enzimas , Enzimas Inmovilizadas/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Microscopía Electrónica de Rastreo , Nanopartículas/química , Polvos , Proteínas de Saccharomyces cerevisiae/metabolismo , Propiedades de Superficie , Titanio/químicaRESUMEN
Pollen viability depends on dynamic vacuolar changes during pollen development involving increases and decreases of vacuolar volume through water and osmolite accumulation and vacuolar fission. Mutations in FAB1A to FAB1D, the genes encoding phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2]-converting kinases, are male gametophyte lethal in Arabidopsis (Arabidopsis thaliana) due to defective vacuolar fission after pollen mitosis I, suggesting a key role of the phospholipid in dynamic vacuolar organization. However, other genetic components that regulate the production of PI(3,5)P2 and its involvement in pollen germination and tube growth are unknown. Here, we identified and characterized Arabidopsis VAC14, a homolog of the yeast and metazoan VAC14s that are crucial for the production of PI(3,5)P2VAC14 is constitutively expressed and highly present in developing pollen. Loss of function of VAC14 was male gametophyte lethal due to defective pollen development. Ultrastructural studies showed that vacuolar fission after pollen mitosis I was compromised in vac14 mutant microspores, which led to pollen abortion. We further showed that inhibiting the production of PI(3,5)P2 or exogenous application of PI(3,5)P2 mimicked or rescued the pollen developmental defect of the vac14 mutant, respectively. Genetic interference and pharmacological approaches suggested a role of PI(3,5)P2 in pollen germination and tube growth. Our results provide insights into the function of VAC14 and, by inference, that of PI(3,5)P2 in plant cells.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Polen/crecimiento & desarrollo , Vacuolas/metabolismo , Aminopiridinas/farmacología , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Flores/genética , Regulación de la Expresión Génica de las Plantas , Compuestos Heterocíclicos con 3 Anillos/farmacología , Proteínas de la Membrana/química , Mutación , Fosfatos de Fosfatidilinositol/metabolismo , Plantas Modificadas Genéticamente , Polen/citología , Polen/efectos de los fármacos , Proteínas de Saccharomyces cerevisiae/química , Homología de Secuencia de Aminoácido , Vacuolas/genéticaRESUMEN
In the fission yeast Schizosaccharomyces pombe, acquisition of exogenous heme is largely mediated by the cell membrane-associated Shu1. Here, we report that Str3, a member of the major facilitator superfamily of transporters, promotes cellular heme import. Using a strain that cannot synthesize heme de novo (hem1Δ) and lacks Shu1, we found that the heme-dependent growth deficit of this strain is rescued by hemin supplementation in the presence of Str3. Microscopic analyses of a hem1Δ shu1Δ str3Δ mutant strain in the presence of the heme analog zinc mesoporphyrin IX (ZnMP) revealed that ZnMP fails to accumulate within the mutant cells. In contrast, Str3-expressing hem1Δ shu1Δ cells could take up ZnMP at a 10-µm concentration. The yeast Saccharomyces cerevisiae cannot efficiently transport exogenously supplied hemin. However, heterologous expression of Str3 from S. pombe in S. cerevisiae resulted in ZnMP accumulation within S. cerevisiae cells. Moreover, hemin-agarose pulldown assays revealed that Str3 binds hemin. In contrast, an Str3 mutant in which Tyr and Ser residues of two putative heme-binding motifs (530YX3Y534 and 552SX4Y557) had been replaced with alanines exhibited a loss of affinity for hemin. Furthermore, this Str3 mutant failed to rescue the heme-dependent growth deficit of a hem1Δ shu1Δ str3Δ strain. Further analysis by absorbance spectroscopy disclosed that a predicted extracellular loop region in Str3 containing the two putative heme-binding motifs interacts with hemin, with a KD of 6.6 µm Taken together, these results indicate that Str3 is a second cell-surface membrane protein for acquisition of exogenous heme in S. pombe.
Asunto(s)
Proteínas Portadoras/química , Hemo/química , Proteínas de Schizosaccharomyces pombe/química , Schizosaccharomyces/química , Secuencias de Aminoácidos , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Hemo/genética , Hemo/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Especificidad de la EspecieRESUMEN
The pyruvate dehydrogenase complex (PDC) bridges glycolysis and the citric acid cycle. In human, PDC deficiency leads to severe neurodevelopmental delay and progressive neurodegeneration. The majority of cases are caused by variants in the gene encoding the PDC subunit E1α. The molecular effects of the variants, however, remain poorly understood. Using yeast as a eukaryotic model system, we have studied the substitutions A189V, M230V, and R322C in yeast E1α (corresponding to the pathogenic variants A169V, M210V, and R302C in human E1α) and evaluated how substitutions of single amino acid residues within different functional E1α regions affect PDC structure and activity. The E1α A189V substitution located in the heterodimer interface showed a more compact conformation with significant underrepresentation of E1 in PDC and impaired overall PDC activity. The E1α M230V substitution located in the tetramer and heterodimer interface showed a relatively more open conformation and was particularly affected by low thiamin pyrophosphate concentrations. The E1α R322C substitution located in the phosphorylation loop of E1α resulted in PDC lacking E3 subunits and abolished overall functional activity. Furthermore, we show for the E1α variant A189V that variant E1α accumulates in the Hsp60 chaperonin, but can be released upon ATP supplementation. Our studies suggest that pathogenic E1α variants may be associated with structural changes of PDC and impaired folding of E1α.
Asunto(s)
Sustitución de Aminoácidos , Piruvato Deshidrogenasa (Lipoamida)/genética , Enfermedad por Deficiencia del Complejo Piruvato Deshidrogenasa/genética , Complejo Piruvato Deshidrogenasa/genética , Proteínas de Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Microscopía Confocal , Pliegue de Proteína , Piruvato Deshidrogenasa (Lipoamida)/química , Piruvato Deshidrogenasa (Lipoamida)/metabolismo , Complejo Piruvato Deshidrogenasa/química , Complejo Piruvato Deshidrogenasa/metabolismo , Enfermedad por Deficiencia del Complejo Piruvato Deshidrogenasa/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Dioscorea opposita Thunb, commonly known as "yam" that has a long dietary therapy history for diabetes, is widely consumed as a botanical dietary supplement and widely cultivated in China. In this work, a method for rapid screening of α-glucosidase inhibitors from Dioscorea opposita Thunb peel extract was developed using α-glucosidase functionalized magnetic nanoparticles (αG-MNPs) as a solid phase extraction absorbent in combination with high performance liquid chromatography-mass spectrometry (HPLC-MS). Two α-glucosidase inhibitors were selectively extracted and identified as batatasin I and 2,4-dimethoxy-6,7-dihydroxyphenanthrene. Their α-glucosidase inhibitory activities (IC50 = 2.55 mM and 0.40 mM, respectively) were significantly higher than that of acarbose (as control). Taking advantage of the specificity in enzyme binding and the convenience of magnetic separation, this method has great potential for rapid and fast screening of α-glucosidase inhibitors from complex natural resources.
Asunto(s)
Dioscorea/química , Inhibidores de Glicósido Hidrolasas/química , Extractos Vegetales/química , Cromatografía Líquida de Alta Presión , Inhibidores de Glicósido Hidrolasas/aislamiento & purificación , Nanopartículas de Magnetita/química , Extractos Vegetales/aislamiento & purificación , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Proteínas de Saccharomyces cerevisiae/química , Espectrometría de Masas en Tándem , alfa-Glucosidasas/químicaRESUMEN
CONTEXT: Previous studies have shown that extracts of Zizyphus rugosa Lam. (Rhamnaceae) bark contained phytoconstituents with antidiabetic potential to lower blood glucose levels in diabetic rats. However, there has been no report on the active compounds in this plant as potential antidiabetic inhibitors. OBJECTIVE: We evaluated the α-glucosidase inhibitory and antioxidant activities of Z. rugosa extract. Moreover, the active phytochemical constituents were isolated and characterized. MATERIALS AND METHODS: The α-glucosidase inhibition of crude ethanol extract obtained from the bark of Z. rugosa was assayed as well as the antioxidant activity. Active compounds (1-6) were isolated, the structures were determined, and derivatives (2a-2 l) were prepared. All compounds were tested for their α-glucosidase inhibitory (yeast and rat intestine) and antioxidant (DPPH) activities. RESULTS: The active α-glucosidase inhibitors (1-6) were isolated from Z. rugosa bark and 12 derivatives (2a-2 l) were prepared. Compound 2 showed the most powerful yeast α-glucosidase inhibitory activity (IC50 16.3 µM), while compounds 3 and 4 display only weak inhibition toward rat intestinal α-glucosidase. Moreover, compound 6 showed the most potent antioxidant activity (IC50 42.8 µM). The molecular docking results highlighted the role of the carboxyl moiety of 2 for yeast α-glucosidase inhibition through H-bonding. DISCUSSION AND CONCLUSIONS: These results suggest the potential of Z. rugosa bark for future application in the treatment of diabetes and active compounds 1 and 2 have emerged as promising molecules for therapy.
Asunto(s)
Antioxidantes/farmacología , Inhibidores de Glicósido Hidrolasas/farmacología , Simulación del Acoplamiento Molecular , Corteza de la Planta/química , Extractos Vegetales/farmacología , Proteínas de Saccharomyces cerevisiae/metabolismo , Ziziphus/química , alfa-Glucosidasas/metabolismo , Antioxidantes/química , Antioxidantes/aislamiento & purificación , Compuestos de Bifenilo/química , Etanol/química , Inhibidores de Glicósido Hidrolasas/química , Inhibidores de Glicósido Hidrolasas/aislamiento & purificación , Cinética , Fitoterapia , Picratos/química , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Plantas Medicinales , Unión Proteica , Conformación Proteica , Proteínas de Saccharomyces cerevisiae/química , Solventes/química , Relación Estructura-Actividad , alfa-Glucosidasas/químicaRESUMEN
Prions are a group of proteins that can adopt a spectrum of metastable conformations in vivo. These alternative states change protein function and are self-replicating and transmissible, creating protein-based elements of inheritance and infectivity. Prion conformational flexibility is encoded in the amino acid composition and sequence of the protein, which dictate its ability not only to form an ordered aggregate known as amyloid but also to maintain and transmit this structure in vivo. But, while we can effectively predict amyloid propensity in vitro, the mechanism by which sequence elements promote prion propagation in vivo remains unclear. In yeast, propagation of the [PSI+] prion, the amyloid form of the Sup35 protein, has been linked to an oligopeptide repeat region of the protein. Here, we demonstrate that this region is composed of separable functional elements, the repeats themselves and a repeat proximal region, which are both required for efficient prion propagation. Changes in the numbers of these elements do not alter the physical properties of Sup35 amyloid, but their presence promotes amyloid fragmentation, and therefore maintenance, by molecular chaperones. Rather than acting redundantly, our observations suggest that these sequence elements make complementary contributions to prion propagation, with the repeat proximal region promoting chaperone binding to and the repeats promoting chaperone processing of Sup35 amyloid.
Asunto(s)
Proteínas Amiloidogénicas/metabolismo , Amiloidosis/metabolismo , Factores de Terminación de Péptidos/metabolismo , Priones/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenina/metabolismo , Proteínas Amiloidogénicas/química , Proteínas Amiloidogénicas/genética , Amiloidosis/genética , Amiloidosis/patología , Luciferasas , Chaperonas Moleculares/metabolismo , Factores de Terminación de Péptidos/química , Factores de Terminación de Péptidos/genética , Reacción en Cadena de la Polimerasa , Priones/genética , Unión Proteica , Pliegue de Proteína , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Análisis de Secuencia de ProteínaRESUMEN
Phytochemical investigation of the natural products from Xanthium strumarium led to the isolation of fourteen compounds including seven caffeoylquinic acid (CQA) derivatives. The individual compounds were screened for inhibition of α-glucosidase, protein tyrosine phosphatase 1ß (PTP1ß), advanced glycation end products (AGEs), and ABTS⺠radical scavenging activity using in vitro assays. Among the isolated compounds, methyl-3,5-di-caffeoyquinic acid exhibited significant inhibitory activity against α-glucosidase (18.42 µM), PTP1ß (1.88 µM), AGEs (82.79 µM), and ABTS⺠(6.03 µM). This effect was marked compared to that of the positive controls (acarbose 584.79 µM, sumarin 5.51 µM, aminoguanidine 1410.00 µM, and trolox 29.72 µM respectively). In addition, 3,5-di-O-CQA (88.14 µM) and protocatechuic acid (32.93 µM) had a considerable inhibitory effect against α-glucosidase and ABTSâº. Based on these findings, methyl-3,5-di-caffeoyquinic acid was assumed to be potentially responsible for the anti-diabetic actions of X. strumarium.
Asunto(s)
Benzotiazoles/química , Inhibidores de Glicósido Hidrolasas/química , Proteína Tirosina Fosfatasa no Receptora Tipo 1 , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimología , Ácidos Sulfónicos/química , Xanthium/química , alfa-Glucosidasas/química , Animales , Bovinos , Complicaciones de la Diabetes/tratamiento farmacológico , Complicaciones de la Diabetes/metabolismo , Productos Finales de Glicación Avanzada/antagonistas & inhibidores , Productos Finales de Glicación Avanzada/química , Inhibidores de Glicósido Hidrolasas/uso terapéutico , Humanos , Proteína Tirosina Fosfatasa no Receptora Tipo 1/antagonistas & inhibidores , Proteína Tirosina Fosfatasa no Receptora Tipo 1/química , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Proteínas de Saccharomyces cerevisiae/química , Albúmina Sérica BovinaRESUMEN
It is important to investigate the inhibition of α-glucosidase due to its correlation with type 2 diabetes. Morin was found to exert significant inhibition activity on α-glucosidase in a reversible mixed-type manner with an IC50 value of (4.48 ± 0.04) µM. Analyses of fluorescence and circular dichroism spectra indicated that the formation of the morin-α-glucosidase complex was driven mainly by hydrophobic forces and hydrogen bonding, and caused the conformational changes of α-glucosidase. The phase diagrams of fluorescence showed that the conformational change process was monophasic without intermediates. Molecular docking indicated that morin mainly interacted with amino acid residues located close to the active site of α-glucosidase, which may move to cover the active pocket to reduce the binding of the substrate and then inhibit the catalytic activity. Morin was also found to exhibit inhibition in the generation of advanced glycation end products which was related to the long term complications of diabetes.
Asunto(s)
Antioxidantes/metabolismo , Suplementos Dietéticos , Flavonoides/metabolismo , Productos Finales de Glicación Avanzada/antagonistas & inhibidores , Inhibidores de Glicósido Hidrolasas/metabolismo , Hipoglucemiantes/metabolismo , Acarbosa/química , Acarbosa/metabolismo , Acarbosa/farmacología , Antioxidantes/química , Antioxidantes/farmacología , Sitios de Unión , Dominio Catalítico , Dicroismo Circular , Análisis por Conglomerados , Suplementos Dietéticos/análisis , Flavonoides/química , Glucósidos/química , Glucósidos/metabolismo , Productos Finales de Glicación Avanzada/metabolismo , Inhibidores de Glicósido Hidrolasas/química , Inhibidores de Glicósido Hidrolasas/farmacología , Glicosilación/efectos de los fármacos , Humanos , Hipoglucemiantes/química , Hipoglucemiantes/farmacología , Cinética , Conformación Molecular , Simulación del Acoplamiento Molecular , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Espectrometría de Fluorescencia , Espectrofotometría , Termodinámica , alfa-Glucosidasas/química , alfa-Glucosidasas/metabolismoRESUMEN
The bioactive flavonoid fisetin (FS) is a diet-derived antioxidant that is being increasingly investigated for its health-promoting effects. Unfortunately, the poor physicochemical and pharmacokinetic properties affect and limit the clinical application. In this study, novel polymeric nanoparticles (NPs), based on Poly-(ε-caprolactone) (PCL) and PLGA-PEG-COOH, encapsulating FS were formulated as suitable oral controlled release systems. Results showed NPs having a mean diameter of 140-200nm, and a percent loading of FS ranging from 70 to 82%. In vitro release studies revealed that NPs are able to protect and preserve the release of FS in gastric simulated conditions, also controlling the release in the intestinal medium. Moreover, the DPPH and ABTS scavenging capacity of FS, as well as α-glucosidase inhibition activity, that resulted about 20-fold higher than commercial Acarbose, were retained during nanoencapsulation process. In summary, our developed NPs can be proposed as an attractive delivery system to control the release of antioxidant and anti-hyperglycemic FS for nutraceutical and/or therapeutic application.
Asunto(s)
Antioxidantes , Suplementos Dietéticos , Flavonoides , Inhibidores de Glicósido Hidrolasas , Nanocápsulas/química , Antioxidantes/química , Antioxidantes/farmacocinética , Flavonoides/química , Flavonoides/farmacocinética , Flavonoles , Inhibidores de Glicósido Hidrolasas/química , Inhibidores de Glicósido Hidrolasas/farmacocinética , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , alfa-Glucosidasas/química , alfa-Glucosidasas/metabolismoRESUMEN
Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein's function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento/métodos , Mapeo de Interacción de Proteínas/métodos , Tetrahidrofolato Deshidrogenasa/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Tetrahidrofolato Deshidrogenasa/químicaRESUMEN
Polyphenols and caffeoylquinic acid (CQA) derivatives (3-CQA, 4-CQA, 5-CQA, 3,4-diCQA, 3,5-diCQA, and 4,5-diCQA) were prepared from Ilex kudingcha C.J. Tseng, and their effects and mechanisms on the activities of α-glucosidase from Saccharomyces cerevisiae were investigated in the present study. As results, the IC50 values for CQA derivatives were 0.16-0.39 mg/mL, and the inhibition mode of CQA derivatives was noncompetitive. On the basis of fluorescence spectroscopy and circular dichroism spectroscopy data, the binding constants and number of binding sites were calculated to be 10(6)-10(8) M(-1) and 1.42-1.87, respectively. CQA derivatives could bind to the enzyme mainly through hydrophobic interaction, altering the microenvironment and molecular conformation of the enzyme, thus decreasing the catalytic activity. To the authors' knowledge, this is the first report on α-glucosidase inhibitory mechanism by CQA derivatives from I. kudingcha, and the findings suggest a potential use of kudingcha as functional foods for the prevention and treatment of diabetes and related symptoms.