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1.
PLoS Comput Biol ; 20(3): e1011881, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38442111

RESUMEN

Antibody-based therapeutics must not undergo chemical modifications that would impair their efficacy or hinder their developability. A commonly used technique to de-risk lead biotherapeutic candidates annotates chemical liability motifs on their sequence. By analyzing sequences from all major sources of data (therapeutics, patents, GenBank, literature, and next-generation sequencing outputs), we find that almost all antibodies contain an average of 3-4 such liability motifs in their paratopes, irrespective of the source dataset. This is in line with the common wisdom that liability motif annotation is over-predictive. Therefore, we have compiled three computational flags to prioritize liability motifs for removal from lead drug candidates: 1. germline, to reflect naturally occurring motifs, 2. therapeutic, reflecting chemical liability motifs found in therapeutic antibodies, and 3. surface, indicative of structural accessibility for chemical modification. We show that these flags annotate approximately 60% of liability motifs as benign, that is, the flagged liabilities have a smaller probability of undergoing degradation as benchmarked on two experimental datasets covering deamidation, isomerization, and oxidation. We combined the liability detection and flags into a tool called Liability Antibody Profiler (LAP), publicly available at lap.naturalantibody.com. We anticipate that LAP will save time and effort in de-risking therapeutic molecules.


Asunto(s)
Anticuerpos , Secuenciación de Nucleótidos de Alto Rendimiento , Anticuerpos/uso terapéutico , Probabilidad
2.
Inorg Chem ; 58(17): 11782-11792, 2019 Sep 03.
Artículo en Inglés | MEDLINE | ID: mdl-31433630

RESUMEN

Reproduction of the dominant vector of Zika and dengue diseases, Aedes aegypti mosquito, is controlled by an active heterodimer complex composed of the 20-hydroxyecdysone receptor (EcR) and ultraspiracle protein. Although A. aegypti EcR shares the structural and functional organization with other nuclear receptors, its C-terminus has an additional long F domain (AaFEcR). Recently, we showed that the full length AaFEcR is intrinsically disordered with the ability to specifically bind divalent metal ions. Here, we describe the details of the exhaustive structural and thermodynamic properties of Zn2+- and Cu2+-complexes with the AaFEcR domain, based on peptide models of its two putative metal binding sites (Ac-HGPHPHPHG-NH2 and Ac-QQLTPNQQQHQQQHSQLQQVHANGS-NH2). Unexpectedly, only in the presence of increasing concentrations of Cu2+ ions, the Ac-HGPHPHPHG-NH2 peptide gained a metal ion-induced poly-l-proline type II helical structure, which is unique for members of the family of nuclear receptors.


Asunto(s)
Aedes/efectos de los fármacos , Antivirales/farmacología , Cobre/farmacología , Compuestos Organometálicos/farmacología , Péptidos/farmacología , Receptores de Esteroides/antagonistas & inhibidores , Animales , Antivirales/química , Sitios de Unión/efectos de los fármacos , Cobre/química , Dengue/tratamiento farmacológico , Dengue/metabolismo , Estructura Molecular , Compuestos Organometálicos/química , Péptidos/química , Receptores de Esteroides/metabolismo , Termodinámica , Infección por el Virus Zika/tratamiento farmacológico , Infección por el Virus Zika/metabolismo
3.
Biol Chem ; 399(5): 467-484, 2018 04 25.
Artículo en Inglés | MEDLINE | ID: mdl-29337690

RESUMEN

FK506-binding proteins (FKBPs) belong to a distinct class of immunophilins that interact with immunosuppressants. They use their peptidyl-prolyl isomerase (PPIase) activity to catalyze the cis-trans conversion of prolyl bonds in proteins during protein-folding events. FKBPs also act as a unique group of chaperones. The Drosophila melanogaster peptidyl-prolyl cis-trans isomerase FK506-binding protein of 39 kDa (FKBP39) is thought to act as a transcriptional modulator of gene expression in 20-hydroxyecdysone and juvenile hormone signal transduction. The aim of this study was to analyze the molecular determinants responsible for the subcellular distribution of an FKBP39-yellow fluorescent protein (YFP) fusion construct (YFP-FKBP39). We found that YFP-FKBP39 was predominantly nucleolar. To identify the nuclear localization signal (NLS), a series of YFP-tagged FKBP39 deletion mutants were prepared and examined in vivo. The identified NLS signal is located in a basic domain. Detailed mutagenesis studies revealed that residues K188 and K191 are crucial for the nuclear targeting of FKBP39 and its nucleoplasmin-like (NPL) domain contains the sequence that controls the nucleolar-specific translocation of the protein. These results show that FKBP39 possesses a specific NLS in close proximity to a putative helix-turn-helix (HTH) motif and FKBP39 may bind DNA in vivo and in vitro.


Asunto(s)
Proteínas de Drosophila/análisis , Señales de Localización Nuclear/análisis , Proteínas de Unión a Tacrolimus/análisis , Animales , Células COS , Células Cultivadas , Chlorocebus aethiops , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/metabolismo , Señales de Localización Nuclear/metabolismo , Proteínas de Unión a Tacrolimus/genética , Proteínas de Unión a Tacrolimus/metabolismo
4.
Biochim Biophys Acta ; 1844(6): 1153-66, 2014 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-24704038

RESUMEN

The mosquito Aedes aegypti is the principal vector of dengue, one of the most devastating arthropod-borne viral infections in humans. The isoform specific A/B region, called the N-terminal domain (NTD), is hypervariable in sequence and length and is poorly conserved within the Ultraspiracle (Usp) family. The Usp protein together with ecdysteroid receptor (EcR) forms a heterodimeric complex. Up until now, there has been little data on the molecular properties of the isolated Usp-NTD. Here, we describe the biochemical and biophysical properties of the recombinant NTD of the Usp isoform B (aaUsp-NTD) from A. aegypti. These results, in combination with in silico bioinformatics approaches, indicate that aaUsp-NTD exhibits properties of an intrinsically disordered protein (IDP). We also present the first experimental evidence describing the dimerization propensity of the isolated NTD of Usp. These characteristics also appear for other members of the Usp family in different species, for example, in the Usp-NTD from Drosophila melanogaster and Bombyx mori. However, aaUsp-NTD exhibits the strongest homodimerization potential. We postulate that the unique dimerization of the NTD might be important for Usp function by providing an additional platform for interactions, in addition to the nuclear receptor superfamily dimerization via DNA binding domains and ligand binding domains that has already been extensively documented. Furthermore, the unique NTD-NTD interaction that was observed might contribute new insight into the dimerization propensities of nuclear receptors.


Asunto(s)
Aedes/química , Bombyx/química , Proteínas de Unión al ADN/química , Proteínas de Drosophila/química , Drosophila melanogaster/química , Proteínas Intrínsecamente Desordenadas/química , Receptores de Esteroides/química , Factores de Transcripción/química , Aedes/genética , Animales , Bombyx/genética , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Escherichia coli , Hidrodinámica , Proteínas Intrínsecamente Desordenadas/genética , Cinética , Pliegue de Proteína , Multimerización de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores de Esteroides/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Homología de Secuencia de Aminoácido , Factores de Transcripción/genética , Activación Transcripcional
5.
Front Mol Biosci ; 11: 1352508, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38606289

RESUMEN

Antibodies are proteins produced by our immune system that have been harnessed as biotherapeutics. The discovery of antibody-based therapeutics relies on analyzing large volumes of diverse sequences coming from phage display or animal immunizations. Identification of suitable therapeutic candidates is achieved by grouping the sequences by their similarity and subsequent selection of a diverse set of antibodies for further tests. Such groupings are typically created using sequence-similarity measures alone. Maximizing diversity in selected candidates is crucial to reducing the number of tests of molecules with near-identical properties. With the advances in structural modeling and machine learning, antibodies can now be grouped across other diversity dimensions, such as predicted paratopes or three-dimensional structures. Here we benchmarked antibody grouping methods using clonotype, sequence, paratope prediction, structure prediction, and embedding information. The results were benchmarked on two tasks: binder detection and epitope mapping. We demonstrate that on binder detection no method appears to outperform the others, while on epitope mapping, clonotype, paratope, and embedding clusterings are top performers. Most importantly, all the methods propose orthogonal groupings, offering more diverse pools of candidates when using multiple methods than any single method alone. To facilitate exploring the diversity of antibodies using different methods, we have created an online tool-CLAP-available at (clap.naturalantibody.com) that allows users to group, contrast, and visualize antibodies using the different grouping methods.

6.
Biol Chem ; 393(6): 457-71, 2012 May.
Artículo en Inglés | MEDLINE | ID: mdl-22628309

RESUMEN

Ecdysteroids coordinate essential biological processes in Drosophila through a complex of two nuclear receptors, the ecdysteroid receptor (EcR) and the ultraspiracle protein (Usp). Biochemical experiments have shown that, in contrast to Usp, the EcR molecule is characterized by high intramolecular plasticity. To investigate whether this plasticity is sufficient to form EcR complexes with nuclear receptors other than Usp, we studied the interaction of EcR with the DHR38 nuclear receptor. Previous in vitro experiments suggested that DHR38 can form complexes with Usp and thus disrupt Usp-EcR interaction with the specific hsp27pal response element. This article provides the experimental evidence that EcR is able to form complexes with DHR38 as well. The recombinant DNA-binding domains (DBDs) of EcR and DHR38 interact specifically on hsp27pal. However, the interaction between the receptors is not restricted to their isolated DBDs. We pre\xadsent data that indicate that the full-length EcR and DHR38 can also form specific complexes within the nuclei of living cells. This interaction is mediated by the hinge region of EcR, which was recently classified as an intrinsically disordered region. Our results indicate that DHR38 might modulate the activity of the Usp-EcR heterodimer by forming complexes with both of its components.


Asunto(s)
Proteínas de Drosophila/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Esteroides/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Supervivencia Celular , ADN/genética , ADN/metabolismo , Proteínas de Drosophila/química , Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Proteínas de Choque Térmico HSP27/genética , Humanos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas/genética , Unión Proteica , Estructura Terciaria de Proteína , Receptores Citoplasmáticos y Nucleares/química , Receptores de Esteroides/química , Especificidad por Sustrato
7.
Cells ; 10(3)2021 03 05.
Artículo en Inglés | MEDLINE | ID: mdl-33807814

RESUMEN

The superfamily of nuclear receptors (NRs), composed of ligand-activated transcription factors, is responsible for gene expression as a reaction to physiological and environmental changes. Transcriptional machinery may require phase separation to fulfil its role. Although NRs have a similar canonical structure, their C-terminal domains (F domains) are considered the least conserved and known regions. This article focuses on the peculiar molecular properties of the intrinsically disordered F domain of the ecdysteroid receptor from the Aedes aegypti mosquito (AaFEcR), the vector of the world's most devastating human diseases such as dengue and Zika. The His-Pro-rich segment of AaFEcR was recently shown to form the unique poly-proline helix II (PPII) in the presence of Cu2+. Here, using widefield microscopy of fluorescently labeled AaFEcR, Zn2+- and Cu2+-induced liquid-liquid phase separation (LLPS) was observed for the first time for the members of NRs. The perspectives of this finding on future research on the F domain are discussed, especially in relation to other NR members.


Asunto(s)
Iones/metabolismo , Mosquitos Vectores/patogenicidad , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Esteroides/metabolismo , Aedes , Animales , Humanos
8.
Int J Biol Macromol ; 163: 108-119, 2020 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-32615218

RESUMEN

The FKBP39 from Drosophila melanogaster is a multifunctional regulatory immunophilin. It contains two globular domains linked by a highly charged disordered region. The N-terminal domain shows homology to the nucleoplasmin core domain, and the C-terminal domain is characteristic for the family of the FKBP immunophilin ligand binding domain. The specific partially disordered structure of the protein inspired us to investigate whether FKBP39 can drive spontaneous liquid-liquid phase separation (LLPS). Preliminary analyses using CatGranule and Pi-Pi contact predictors suggested a propensity for LLPS. Microscopy observations revealed that FKBP39 can self-concentrate to form liquid condensates. We also found that FKBP39 can lead to LLPS in the presence of RNA and peptides containing Arg-rich linear motifs derived from selected nuclear and nucleolar proteins. These heterotypic interactions have a stronger propensity for driving LLPS when compared to the interactions mediated by self-associating FKBP39 molecules. To investigate whether FKBP39 can drive LLPS in the cellular environment, we analysed it in fusion with YFP in COS-7 cells. The specific distribution and diffusion kinetics of FKBP39 examined by FRAP experiments provided evidence that immunophilin is an important driver of phase separation. The ability of FKBP39 to go into heterotypic interaction may be fundamental for ribosome subunits assembly.


Asunto(s)
Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/química , Inmunofilinas/química , Inmunofilinas/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas de Unión a Tacrolimus/química , Proteínas de Unión a Tacrolimus/metabolismo , Animales , Arginina/metabolismo , Células COS , Chlorocebus aethiops , Simulación por Computador , Drosophila melanogaster/metabolismo , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/metabolismo , Espectrometría de Masas , Microscopía Confocal , Microscopía Fluorescente , Agregado de Proteínas , Unión Proteica , ARN/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Ribosomas/metabolismo , Cloruro de Sodio/química , Espectrofotometría
9.
Biochim Biophys Acta ; 1779(6-7): 390-401, 2008.
Artículo en Inglés | MEDLINE | ID: mdl-18515113

RESUMEN

The juvenile hormone binding protein (JHBP) plays a key role in the protection and transport of the hormone to target tissues. In this report the sequence of the jhbp promoter comprising about 2000 bp is characterized. Using a minimized false positive algorithm, six putative regulatory elements, Hunchback, Heat shock factor binding element, Ultrabithorax, Broad-Complex Z3, Elf-1 and Chorion factor 1/ultraspiracle (CF1/Usp) were found in the distal promoter of the jhbp gene. Proteins from nuclear extract of Galleria mellonella fat body form four specific complexes with probe containing TATA box, five complexes with Inr probe and one protein complex with DPE probe. EMSA and footprinting analyses showed that one of the three CF1/Usp elements (starting at -1053) has an exceptionally high affinity to Usp protein. An unknown, high-affinity Usp/EcRDBD-binding element (TCAACA-AAC-TGTTCA), distinct from 20-hydroxyecdysone response elements, was identified in the jhbp gene promoter, based on a footprinting assay. Deletions of jhbp promoter in the regions containing the CF1/Usp elements enhance the transcriptional activity of luciferase reporter gene in the Trichoplusia ni High Five cell line. Obtained data suggest that jhbp promoter is TATA- and Inr-driven, CF1/Usp elements exhibit inhibitory effect on jhbp expression, and an interaction between Usp and DNA relies on recognition of the consensus sequence (GGGTCA) and on ionic interactions of several phosphate groups outside from this element.


Asunto(s)
Proteínas Portadoras/genética , Proteínas de Unión al ADN/metabolismo , Genes de Insecto , Proteínas de Insectos/genética , Hormonas Juveniles/metabolismo , Mariposas Nocturnas/genética , Mariposas Nocturnas/metabolismo , Receptores de Esteroides/metabolismo , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Sitios de Unión/genética , ADN/genética , ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Drosophila , Manduca/genética , Manduca/metabolismo , Datos de Secuencia Molecular , Complejos Multiproteicos , Regiones Promotoras Genéticas , Receptores de Esteroides/genética , Elementos Reguladores de la Transcripción , Especificidad de la Especie , TATA Box , Factores de Transcripción/genética
10.
Nucleic Acids Res ; 35(8): 2705-18, 2007.
Artículo en Inglés | MEDLINE | ID: mdl-17426125

RESUMEN

The heterodimer of the ecdysone receptor (EcR) and ultraspiracle (Usp), members of the nuclear receptors superfamily, is considered as the functional receptor for ecdysteroids initiating molting and metamorphosis in insects. Here we report the 1.95 A structure of the complex formed by the DNA-binding domains (DBDs) the EcR and the Usp, bound to the natural pseudopalindromic response element. Comparison of the structure with that obtained previously, using an idealized response element, shows how the EcRDBD, which has been previously reported to possess extraordinary flexibility, accommodates DNA-induced structural changes. Part of the C-terminal extension (CTE) of the EcRDBD folds into an alpha-helix whose location in the minor groove does not match any of the locations previously observed for nuclear receptors. Mutational analyses suggest that the alpha-helix is a component of EcR-box, a novel element indispensable for DNA-binding and located within the nuclear receptor CTE. This element seems to be a general feature of all known EcRs.


Asunto(s)
Proteínas de Unión al ADN/química , Modelos Moleculares , Receptores de Esteroides/química , Elementos de Respuesta , Factores de Transcripción/química , Sustitución de Aminoácidos , Animales , Sitios de Unión , Cristalografía por Rayos X , ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Choque Térmico/genética , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores de Esteroides/genética , Factores de Transcripción/genética
11.
J Steroid Biochem Mol Biol ; 186: 42-55, 2019 02.
Artículo en Inglés | MEDLINE | ID: mdl-30243841

RESUMEN

The dominant vector of dengue and Zika diseases is a female Aedes aegypti mosquito. Its reproduction is controlled by the formation of an active heterodimer complex of the 20-hydroxyecdysone receptor (EcR) and Ultraspiracle protein (Usp). Although EcR exhibits a structural and functional organization typical of nuclear receptors (NRs), the EcR C-terminus has an additional F domain (AaFEcR) that is rarely present in the NRs superfamily. The presence of F domains is evolutionarily not well conserved in the NRs. The structure-function relationship of EcR F domains in arthropods is unclear and enigmatic. To date, there have been no data concerning the structure and function of AaFEcR. Our results showed that AaFEcR belongs to a family of intrinsically disordered proteins (IDPs) and possesses putative pre-molten globule (PMG) characteristics. Unexpectedly, additional amino acid composition in silico analyses revealed the presence of short unique repeated Pro-His clusters forming an HGPHPHPHG motif, which is similar to those responsible for Zn2+ and Cu2+ binding in histidine-proline-rich glycoproteins (HPRGs). Using SEC, SV-AUC and ESI-TOF MS, we showed that the intrinsically disordered AaFEcR is able to bind metal ions and form complexes with these ions. Our studies provide new insight into the structural organization and activities of the F domains of NRs. This unique for the F domains of NRs ion-binding propensity demonstrated by the AaFEcR domain may be a part of the ecdysteroid receptor's mechanism for regulating the expression of genes encoding oxidative stress-protecting proteins.


Asunto(s)
Aedes/metabolismo , Proteínas de Insectos/metabolismo , Proteínas Intrínsecamente Desordenadas/metabolismo , Metales/metabolismo , Receptores de Esteroides/metabolismo , Aedes/química , Animales , Proteínas de Insectos/química , Proteínas Intrínsecamente Desordenadas/química , Metales/química , Unión Proteica , Dominios Proteicos , Receptores de Esteroides/química
12.
Mol Cell Endocrinol ; 294(1-2): 45-51, 2008 Nov 06.
Artículo en Inglés | MEDLINE | ID: mdl-18771703

RESUMEN

The heterodimer of the ecdysone receptor (EcR) and ultraspiracle (Usp), members of the nuclear receptor superfamily, is considered to be functional receptor for the ecdysteroids that coordinate essential biological processes in insects. In this work we have applied a bimolecular fluorescence complementation (BiFC) method to directly analyze the formation of the EcR/Usp complex. The BiFC experiments were carried out in mammalian cells which are routinely used for heterologous studies of the EcR/Usp complex, including experiments on EcR-based artificial molecular gene switches. BiFC analysis, supported by flow cytometry, revealed that EcR-Usp interaction is nuclei-restricted. If expressed separately, Usp and EcR are able to form nuclear complexes in the absence of the cognate dimerization partner. We have observed that Muristerone A that is widely used for the induction of ecdysteroid-dependent transcription in mammalian cells, does not significantly change the number of EcR/Usp and EcR/EcR complexes, and it does not influence their subcellular localization.


Asunto(s)
Núcleo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Drosophila melanogaster/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Esteroides/metabolismo , Factores de Transcripción/metabolismo , Animales , Células CHO , Células COS , Supervivencia Celular , Chlorocebus aethiops , Cricetinae , Cricetulus , Proteínas de Drosophila , Citometría de Flujo , Fluorescencia , Células HeLa , Humanos , Espacio Intracelular , Unión Proteica , Transporte de Proteínas
13.
Cell Signal ; 19(3): 490-503, 2007 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-17011166

RESUMEN

Ecdysteroids coordinate development, reproduction and other essential biological processes in insects and other arthropods through the receptor which is a heterodimer of two members of the nuclear receptors superfamily, the ecdysteroid receptor (EcR) and the Ultraspiracle (Usp). Although the transcriptionally active EcR/Usp heterocomplex is believed to be the only functional form of the receptor, there are data indicating that EcR may be involved in the mediation of the non-genomic effects outside of the nucleus. Since the nucleocytoplasmic shuttling could be a key element determining participation of the single nuclear receptor molecule both in the genomic and non-genomic functions we have analyzed nuclear import and export properties of the EcR and Usp from Drosophila melanogaster. We show for the first time that both receptors exhibit differential distribution of the nuclear localization and nuclear export signals (NLSs and NESs). In particular, the Usp which exhibits exclusively nuclear localization in all cell types analyzed, contains apparently only NLS activity within the DNA-binding domain. In contrast, the three known EcR isoforms (A, B1 and B2) are mosaics of elements which can potentially mediate their nucleocytoplasmic shuttling. We have found two active NESs in ligand binding domain and NLS activity within the DNA-binding domain of all isoforms. Simultaneously we demonstrate that B1 and A isoforms possess an additional NLS activity localized in AB regions. We speculate that this characteristic, along with the previously reported structural pliability of the EcR molecule, allows the single receptor to evoke many different genomic as well as non-genomic ecdysteroid-dependent responses.


Asunto(s)
Transporte Activo de Núcleo Celular , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Receptores de Esteroides/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Células CHO , Células COS , Chlorocebus aethiops , Cricetinae , Cricetulus , Proteínas de Unión al ADN/genética , Proteínas de Drosophila , Drosophila melanogaster , Células HeLa , Humanos , Proteínas Luminiscentes/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Señales de Exportación Nuclear/genética , Señales de Exportación Nuclear/fisiología , Señales de Localización Nuclear/metabolismo , Receptores de Esteroides/genética , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Fracciones Subcelulares , Factores de Transcripción/genética
14.
Nutrients ; 10(8)2018 Jul 25.
Artículo en Inglés | MEDLINE | ID: mdl-30044398

RESUMEN

This study investigated the efficacy of a plant-derived dietary supplement with respect to decreasing postprandial glucose and insulin peaks after the intake of real-world meals. Two randomized, double-blind, placebo-controlled, cross-over experiments were conducted on healthy subjects who received a supplement containing extracts of white mulberry, white bean, and green coffee or one containing the three extracts with added fibre before consuming high-GI/GL (glycaemic index/glycaemic load) meals. In study one, 32 subjects received an investigational product/placebo before a standardized meal at two visits. In study two, 150 subjects received an investigational product/placebo before five different standardized meals. Postprandial glucose and insulin concentrations were lower 20⁻35 min after meal intake among subjects taking the investigational product, and fewer episodes of postprandial reactive hypoglycaemia were noted. For example, after consuming breakfast cereal with milk, lower glucose peaks were observed for the investigational product (vs. placebo) after 20 min (100.2 ± 1.97 vs. 112.5 ± 3.12 mg/dL, respectively; p < 0.01); lower insulin peaks were noted at the same time point (45.9 ± 4.02 IU/mL vs. 68.2 ± 5.53 IU/mL, respectively, p < 0.01). The combined formulation decreases the adverse consequences of high-GI/GL meal consumption. It can be an effective dietary supplement for the management of metabolic syndrome and type 2 diabetes mellitus.


Asunto(s)
Glucemia , Insulina/sangre , Comidas , Extractos Vegetales/farmacología , Periodo Posprandial , Adolescente , Adulto , Estudios Cruzados , Método Doble Ciego , Femenino , Humanos , Masculino , Persona de Mediana Edad , Extractos Vegetales/química , Adulto Joven
15.
J Steroid Biochem Mol Biol ; 183: 167-183, 2018 10.
Artículo en Inglés | MEDLINE | ID: mdl-29944921

RESUMEN

Nuclear receptors (NRs) are a family of ligand-dependent transcription factors activated by lipophilic compounds. NRs share a common structure comprising three domains: a variable N-terminal domain (NTD), a highly conserved globular DNA-binding domain and a ligand-binding domain. There are numerous papers describing the molecular details of the latter two globular domains. However, very little is known about the structure-function relationship of the NTD, especially as an intrinsically disordered fragment of NRs that may influence the molecular properties and, in turn, the function of globular domains. Here, we investigated whether and how an intrinsically disordered NTD consisting of 58 amino acid residues affects the functions of the globular domains of the Ultraspiracle protein from Helicoverpa armigera (HaUsp). The role of the NTD was examined for two well-known and easily testable NR functions, i.e., interactions with specific DNA sequences and dimerization. Electrophoretic mobility shift assays showed that the intrinsically disordered NTD influences the interaction of HaUsp with specific DNA sequences, apparently by destabilization of HaUsp-DNA complexes. On the other hand, multi-angle light scattering and sedimentation velocity analytical ultracentrifugation revealed that the NTD acts as a structural element that stabilizes HaUsp homodimers. Molecular models based on small-angle X-ray scattering indicate that the intrinsically disordered NTD may exert its effects on the tested HaUsp functions by forming an unexpected scorpion-like structure, in which the NTD bends towards the ligand-binding domain in each subunit of the HaUsp homodimer. This structure may be crucial for specific NTD-dependent regulation of the functions of globular domains in NRs.


Asunto(s)
ADN/química , Proteínas de Insectos/química , Proteínas Intrínsecamente Desordenadas/química , Dominios y Motivos de Interacción de Proteínas , Animales , ADN/metabolismo , Proteínas de Insectos/metabolismo , Proteínas Intrínsecamente Desordenadas/metabolismo , Mariposas Nocturnas , Conformación Proteica
16.
Sci Rep ; 7: 40405, 2017 01 11.
Artículo en Inglés | MEDLINE | ID: mdl-28074868

RESUMEN

Nucleoplasmins are a nuclear chaperone family defined by the presence of a highly conserved N-terminal core domain. X-ray crystallographic studies of isolated nucleoplasmin core domains revealed a ß-propeller structure consisting of a set of five monomers that together form a stable pentamer. Recent studies on isolated N-terminal domains from Drosophila 39-kDa FK506-binding protein (FKBP39) and from other chromatin-associated proteins showed analogous, nucleoplasmin-like (NPL) pentameric structures. Here, we report that the NPL domain of the full-length FKBP39 does not form pentameric complexes. Multi-angle light scattering (MALS) and sedimentation equilibrium ultracentrifugation (SE AUC) analyses of the molecular mass of the full-length protein indicated that FKBP39 forms homotetrameric complexes. Molecular models reconstructed from small-angle X-ray scattering (SAXS) revealed that the NPL domain forms a stable, tetrameric core and that FK506-binding domains are linked to it by intrinsically disordered, flexible chains that form tentacle-like segments. Analyses of full-length FKBP39 and its isolated NPL domain suggested that the distal regions of the polypeptide chain influence and determine the quaternary conformation of the nucleoplasmin-like protein. These results provide new insights regarding the conserved structure of nucleoplasmin core domains and provide a potential explanation for the importance of the tetrameric structural organization of full-length nucleoplasmins.


Asunto(s)
Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Nucleoplasminas/metabolismo , Multimerización de Proteína , Proteínas de Unión a Tacrolimus/química , Proteínas de Unión a Tacrolimus/metabolismo , Animales , Área Bajo la Curva , Dicroismo Circular , Modelos Moleculares , Peso Molecular , Dominios Proteicos , Estructura Secundaria de Proteína , Transporte de Proteínas , Dispersión del Ángulo Pequeño , Soluciones , Fracciones Subcelulares/metabolismo , Difracción de Rayos X
17.
Mol Endocrinol ; 18(9): 2166-84, 2004 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-15192079

RESUMEN

Ecdysteroids coordinate molting and metamorphosis in insects via a heterodimer of two nuclear receptors, the ecdysone receptor (EcR) and the ultraspiracle (Usp) protein. Here we show how the DNA-recognition alpha-helix and the T box region of the EcR DNA-binding domain (EcRDBD) contribute to the specific interaction with the natural response element and to the stabilization of the EcRDBD molecule. The data indicate a remarkable mutational tolerance with respect to the DNA-binding function of the EcRDBD. This is particularly manifested in the heterocomplexes formed between the EcRDBD mutants and the wild-type Usp DNA-binding domain (UspDBD). Circular dichroism (CD) spectra and protein unfolding experiments indicate that, in contrast to the UspDBD, the EcRDBD is characterized by a lower alpha-helix content and a lower stability. As such, the EcRDBD appears to be an intrinsically unstructured protein-like molecule with a high degree of intramolecular plasticity. Because recently published crystal structures indicate that the ligand binding domain of the EcR is also characterized by the extreme adaptability, we suggest that plasticity of the EcR domains may be a key factor that allows a single EcR molecule to mediate diverse biological effects.


Asunto(s)
Receptores de Esteroides/química , Receptores de Esteroides/metabolismo , Elementos de Respuesta/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Análisis Mutacional de ADN , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Péptidos y Proteínas de Señalización Intracelular , Datos de Secuencia Molecular , Muda/genética , Mutación/genética , Regiones Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinasas/genética , Estructura Secundaria de Proteína/genética , Estructura Terciaria de Proteína/genética , Receptores de Esteroides/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
18.
PLoS One ; 9(1): e86052, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24465866

RESUMEN

The heterodimer of the ecdysone receptor (EcR) and ultraspiracle (Usp), members of the nuclear receptors superfamily, regulates gene expression associated with molting and metamorphosis in insects. The DNA binding domains (DBDs) of the Usp and EcR play an important role in their DNA-dependent heterodimerization. Analysis of the crystal structure of the UspDBD/EcRDBD heterocomplex from Drosophila melanogaster on the hsp27 gene response element, suggested an appreciable similarity between both DBDs. However, the chemical denaturation experiments showed a categorically lower stability for the EcRDBD in contrast to the UspDBD. The aim of our study was an elucidation of the molecular basis of this intriguing instability. Toward this end, we mapped the EcRDBD amino acid sequence positions which have an impact on the stability of the EcRDBD. The computational protein design and in vitro analyses of the EcRDBD mutants indicate that non-conserved residues within the α-helix 2, forming the EcRDBD hydrophobic core, represent a specific structural element that contributes to instability. In particular, the L58 appears to be a key residue which differentiates the hydrophobic cores of UspDBD and EcRDBD and is the main reason for the low stability of the EcRDBD. Our results might serve as a benchmark for further studies of the intricate nature of the EcR molecule.


Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Drosophila/química , Drosophila melanogaster , Simulación de Dinámica Molecular , Receptores de Esteroides/química , Factores de Transcripción/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Sitios de Unión , ADN/química , Interacciones Hidrofóbicas e Hidrofílicas , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Estabilidad Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Desplegamiento Proteico , Receptores de Esteroides/genética , Temperatura de Transición
19.
J Steroid Biochem Mol Biol ; 138: 162-73, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-23727127

RESUMEN

The 20-hydroxyecdysone receptor (EcR) is a transcription factor belonging to the nuclear receptor superfamily. Together with the ultraspiracle nuclear receptor (Usp) it coordinates critical biological processes in insects such as development and reproduction. EcR and its ligands are used in commercially available ecdysone-inducible expression systems and are considered to be artificial gene switches with potential therapeutic applications. However, the regulation of EcR action is still unclear, especially in mammals and as far as posttranslational modifications are concerned. Up until now, there has been no study on EcR sumoylation. Using bioinformatic predictors, a Ubc9 fusion-directed sumoylation system and mutagenesis experiments, we present EcR as a new target of SUMO1 and SUMO3 modification. Our research revealed that EcR undergoes isoform-specific multisumoylation. The pattern of modification remains unchanged in the presence of the ligand and the dimerization partner. The SUMO acceptor sites are located in the DNA-binding domain and the ligand-binding domain that both exhibit structural plasticity. We also demonstrated the existence of a sumoylation site in the F region and EcRA-A/B region, both revealing characteristics of intrinsically disordered regions. The consequences of modification and the resulting impact on conformation and function may be especially crucial for the disordered sequences in these two areas. The isoform-specificity of sumoylation may explain the differences in the transcriptional activity of EcR isoforms.


Asunto(s)
Proteínas de Drosophila/metabolismo , Receptores de Esteroides/metabolismo , Animales , Western Blotting , Línea Celular , Biología Computacional , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster , Humanos , Modelos Biológicos , Receptores de Esteroides/química , Receptores de Esteroides/genética , Sumoilación
20.
J Biomol Struct Dyn ; 30(4): 379-93, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22694217

RESUMEN

The ecdysone receptor (EcR) and the ultraspiracle protein (Usp) form the functional receptor for ecdysteroids that initiates metamorphosis in insects. The Usp and EcR DNA-binding domains (UspDBD and EcRDBD, respectively) form a heterodimer on the natural pseudopalindromic element from the hsp27 gene promoter. The conformational changes in the protein-DNA during the formation of the UspDBD-EcRDBD-hsp27 complex were analyzed. Recombined UspDBD and EcRDBD proteins were purified and fluorescein labeled (FL) using the intein method at the C-ends of both proteins. The changes in the distances from the respective C-ends of EcRDBD and/or UspDBD to the 5'- and/or 3'-end of the response element were measured using fluorescence resonance energy transfer (FRET) methodology. The binding of EcRDBD induced a strong conformational change in UspDBD and caused the C-terminal fragment of the UspDBD molecule to move away from both ends of the regulatory element. UspDBD also induced a significant conformational change in the EcRDBD molecule. The EcRDBD C-terminus moved away from the 5'-end of the regulatory element and moved close to the 3'-end. An analysis was also done on the effect that DHR38DBD, the Drosophila ortholog of the mammalian NGFI-B, had on the interaction of UspDBD and EcRDBD with hsp27. FRET analysis demonstrated that hsp27 bending was induced by DHR38DBD. Fluorescence data revealed that hsp27 had a shorter end-to-end distance both in the presence of EcRDBD as well as in the presence of EcRDBD together with DHR38DBD, with DNA bend angles of about 36.2° and 33.6°, respectively. A model of how DHR38DBD binds to hsp27 in the presence of EcRDBD is presented.


Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Drosophila/química , Drosophila/genética , Proteínas de Choque Térmico HSP27/química , Receptores de Esteroides/química , Elementos de Respuesta , Factores de Transcripción/química , Animales , Sitios de Unión , Clonación Molecular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico HSP27/metabolismo , Metamorfosis Biológica/genética , Modelos Moleculares , Conformación de Ácido Nucleico , Unión Proteica , Multimerización de Proteína , Estructura Terciaria de Proteína , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
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