RESUMEN
GLP-1 analogs suffer from the main disadvantage of a short in vivo half-life. Lithocholic acid (LCA), one of the four main bile acids in the human body, possesses a high albumin binding rate. We therefore envisioned that a LCA-based peptide delivery system could extend the half-life of GLP-1 analogs by facilitating the noncovalent binding of peptides to human serum albumin. On the basis of our previously identified Xenopus GLP-1 analogs (1-3), a series of LCA-modified Xenopus GLP-1 conjugates were designed (4a-4r), and the bioactivity studies of these conjugates were performed to identify compounds with balanced in vitro receptor activation potency and plasma stability. 4c, 4i, and 4r were selected, and their LCA side chains were optimized to further increase their stability, affording 5a-5c. Compound 5b showed a more increased albumin affinity and prolonged in vitro stability than that of 4i and liraglutide. In db/ db mice, 5b exhibited comparable hypoglycemic and insulinotropic activity to liraglutide and semaglutide. Importantly, the enhanced albumin affinity of 5b resulted in a prolonged in vivo antidiabetic duration. Finally, chronic treatment investigations of 5b demonstrated the therapeutic effects of 5b on HbA1c, body weight, blood glucose, and pancreatic endocrine deficiencies on db/ db mice. Our studies revealed 5b as a promising antidiabetic candidate. Furthermore, our study suggests the derivatization of Xenopus GLP-1 analogs with LCA represents an effective strategy to develop potent long-acting GLP-1 receptor agonists for the treatment of type 2 diabetes.
Asunto(s)
Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Péptido 1 Similar al Glucagón/farmacología , Hipoglucemiantes/farmacología , Animales , Glucemia/efectos de los fármacos , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Tipo 2/sangre , Evaluación Preclínica de Medicamentos , Estabilidad de Medicamentos , Péptido 1 Similar al Glucagón/análogos & derivados , Péptido 1 Similar al Glucagón/química , Péptidos Similares al Glucagón/farmacología , Péptidos Similares al Glucagón/uso terapéutico , Células HEK293 , Semivida , Humanos , Hipoglucemiantes/química , Hipoglucemiantes/uso terapéutico , Liraglutida/farmacología , Liraglutida/uso terapéutico , Ácido Litocólico/química , Masculino , Ratones , Ratones Endogámicos C57BL , Ratas , Ratas Sprague-Dawley , Albúmina Sérica Humana/metabolismo , Resultado del Tratamiento , Proteínas de Xenopus/química , Proteínas de Xenopus/farmacologíaRESUMEN
Cadmium is a highly toxic environmental pollutant that can cause many adverse effects including cancer, neurological disease and kidney damage. Aquatic amphibians are particularly susceptible to this toxicant as it was shown to cause developmental abnormalities and genotoxic effects. In mammalian cells, the accumulation of heme oxygenase-1 (HO-1), which catalyzes the breakdown of heme into CO, free iron and biliverdin, was reported to protect cells against potentially lethal concentrations of CdCl2. In the present study, CdCl2 treatment of A6 kidney epithelial cells, derived from the frog, Xenopus laevis, induced the accumulation of HO-1, heat shock protein 70 (HSP70) and HSP30 as well as an increase in the production of aggregated protein and aggresome-like structures. Treatment of cells with inhibitors of HO-1 enzyme activity, tin protoporphyrin (SnPP) and zinc protoporphyrin (ZnPP), enhanced CdCl2-induced actin cytoskeletal disorganization and the accumulation of HO-1, HSP70, aggregated protein and aggresome-like structures. Treatment of cells with hemin and baicalein, which were previously shown to provide cytoprotection against various stresses, induced HO-1 accumulation in a concentration-dependent manner. Also, treatment of cells with hemin and baicalein suppressed CdCl2-induced actin dysregulation and the accumulation of aggregated protein and aggresome-like structures. This cytoprotective effect was inhibited by SnPP. These results suggest that HO-1-mediated protection against CdCl2 toxicity includes the maintenance of actin cytoskeletal and microtubular structure and the suppression of aggregated protein and aggresome-like structures.
Asunto(s)
Cadmio/toxicidad , Contaminantes Ambientales/toxicidad , Proteínas del Choque Térmico HSP30/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Hemo-Oxigenasa 1/metabolismo , Riñón/efectos de los fármacos , Agregación Patológica de Proteínas/inducido químicamente , Animales , Antioxidantes/química , Antioxidantes/metabolismo , Línea Celular , Suplementos Dietéticos , Inhibidores Enzimáticos/farmacología , Flavanonas/antagonistas & inhibidores , Flavanonas/metabolismo , Hemo-Oxigenasa 1/antagonistas & inhibidores , Hemo-Oxigenasa 1/química , Hemina/antagonistas & inhibidores , Hemina/metabolismo , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/metabolismo , Riñón/citología , Riñón/metabolismo , Riñón/patología , Metaloporfirinas/farmacología , Microscopía Confocal , Agregación Patológica de Proteínas/patología , Agregación Patológica de Proteínas/prevención & control , Protoporfirinas/farmacología , Proteínas de Xenopus/agonistas , Proteínas de Xenopus/antagonistas & inhibidores , Proteínas de Xenopus/química , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMEN
Allostery is essential to neuronal receptor function, but its transient nature poses a challenge for characterization. The N-terminal domains (NTDs) distinct from ligand binding domains are a major locus for allosteric regulation of NMDA receptors (NMDARs), where different modulatory binding sites have been observed. The inhibitor ifenprodil, and related phenylethanoamine compounds specifically targeting GluN1/GluN2B NMDARs have neuroprotective activity. However, whether they use differential structural pathways than the endogenous inhibitor Zn2+ for regulation is unknown. We applied genetically encoded unnatural amino acids (Uaas) and monitored the functional changes in living cells with photo-cross-linkers specifically incorporated at the ifenprodil binding interface between GluN1 and GluN2B subunits. We report constraining the NTD domain movement, by a light induced crosslinking bond that introduces minimal perturbation to the ligand binding, specifically impedes the transduction of ifenprodil but not Zn2+ inhibition. Subtle distance changes reveal interfacial flexibility and NTD rearrangements in the presence of modulators. Our results present a much richer dynamic picture of allostery than conventional approaches targeting the same interface, and highlight key residues that determine functional and subtype specificity of NMDARs. The light-sensitive mutant neuronal receptors provide complementary tools to the photo-switchable ligands for opto-neuropharmacology.
Asunto(s)
Adenosina Trifosfato/metabolismo , Aminoácidos/genética , Reactivos de Enlaces Cruzados/farmacología , Receptores de N-Metil-D-Aspartato/metabolismo , Zinc/metabolismo , Regulación Alostérica , Aminoácidos/farmacología , Animales , Sitios de Unión , Ligandos , Modelos Moleculares , Mutación , Piperidinas/farmacología , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Receptores de Glutamato/química , Receptores de Glutamato/genética , Receptores de Glutamato/metabolismo , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/genética , Proteínas de Xenopus/química , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMEN
BACKGROUND: The venoms of predators have been an excellent source of diverse highly specific peptides targeting ion channels. Here we describe the first known peptide antagonist of the nociceptor ion channel transient receptor potential ankyrin 1 (TRPA1). RESULTS: We constructed a recombinant cDNA library encoding â¼100 diverse GPI-anchored peptide toxins (t-toxins) derived from spider venoms and screened this library by coexpression in Xenopus oocytes with TRPA1. This screen resulted in identification of protoxin-I (ProTx-I), a 35-residue peptide from the venom of the Peruvian green-velvet tarantula, Thrixopelma pruriens, as the first known high-affinity peptide TRPA1 antagonist. ProTx-I was previously identified as an antagonist of voltage-gated sodium (NaV) channels. We constructed a t-toxin library of ProTx-I alanine-scanning mutants and screened this library against NaV1.2 and TRPA1. This revealed distinct partially overlapping surfaces of ProTx-I by which it binds to these two ion channels. Importantly, this mutagenesis yielded two novel ProTx-I variants that are only active against either TRPA1or NaV1.2. By testing its activity against chimeric channels, we identified the extracellular loops of the TRPA1 S1-S4 gating domain as the ProTx-I binding site. CONCLUSIONS: These studies establish our approach, which we term "toxineering," as a generally applicable method for isolation of novel ion channel modifiers and design of ion channel modifiers with altered specificity. They also suggest that ProTx-I will be a valuable pharmacological reagent for addressing biophysical mechanisms of TRPA1 gating and the physiology of TRPA1 function in nociceptors, as well as for potential clinical application in the context of pain and inflammation.
Asunto(s)
Péptidos/farmacología , Venenos de Araña/química , Canales de Potencial de Receptor Transitorio/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Sitios de Unión , Evaluación Preclínica de Medicamentos/métodos , Femenino , Biblioteca de Genes , Humanos , Datos de Secuencia Molecular , Canal de Sodio Activado por Voltaje NAV1.2/metabolismo , Oocitos , Péptidos/genética , Estructura Terciaria de Proteína , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacología , Proteínas de Xenopus/antagonistas & inhibidores , Proteínas de Xenopus/química , Proteínas de Xenopus/metabolismoRESUMEN
Foxl2 is a transcription factor that plays a crucial role in the regulation of the early development of the female gonad in mammals and fish. However, little is known regarding its role in ovarian differentiation in amphibians. In this study, we isolated a Foxl2 cDNA from the ovary of the frog Rana rugosa and Xenopuslaevis and examined its expression during gonadal sex differentiation in R. rugosa. Alignment of known Foxl2 sequences from vertebrates showed high identity of the Foxl2 open reading frame and protein sequences, in particular the forkhead domain and C-terminal region, with other vertebrate sequences. Among different adult tissues, Foxl2 was expressed at its highest level in the ovary. Real-time RT-PCR analysis showed that Foxl2 expression was sexually dimorphic during gonadal sex differentiation in R. rugosa. In addition, Foxl2, which was detected immunochemically in somatic cells surrounding oocytes in the ovary, promoted R. rugosaCYP19 transcription in luciferase promoter assays conducted in A6 cells. We also found by FISH analysis that Foxl2 was an autosomal gene. Altogether, these results suggest that Foxl2 probably plays a very important role in ovarian differentiation of R. rugosa by possibly regulating CYP19 expression. The factor that up-regulates Foxl2 expression in female gonads still remains to be identified.
Asunto(s)
Clonación Molecular , Factores de Transcripción Forkhead/metabolismo , Regulación de la Expresión Génica/fisiología , Ranidae/metabolismo , Proteínas de Xenopus/metabolismo , Secuencia de Aminoácidos , Animales , Aromatasa/genética , Aromatasa/metabolismo , Bufonidae , Línea Celular , ADN Complementario/genética , ADN Complementario/metabolismo , Femenino , Proteína Forkhead Box L2 , Factores de Transcripción Forkhead/química , Factores de Transcripción Forkhead/genética , Hibridación Fluorescente in Situ , Larva , Masculino , Datos de Secuencia Molecular , Ovario/metabolismo , Filogenia , Regiones Promotoras Genéticas , ARN Mensajero/metabolismo , Ranidae/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Diferenciación Sexual , Proteínas de Xenopus/química , Proteínas de Xenopus/genética , Xenopus laevisRESUMEN
The early stages of vertebrate development depend heavily on control of maternally transcribed mRNAs that are stored for long periods in complexes termed messenger ribonucleoprotein particles (mRNPs) and utilized selectively following maturation and fertilization. The transcription factor Yin Yang 1 (YY1) is associated with cytoplasmic mRNPs in vertebrate oocytes; however, the mechanism by which any of the mRNP proteins associate with mRNA in the oocyte is unknown. Here we demonstrate the mechanism by which YY1 associates with mRNPs depends on its direct RNA binding activity. High affinity binding for U-rich single-stranded RNA and A:U RNA duplexes was observed in the nanomolar range, similar to the affinity for the cognate double-stranded DNA-binding element. Similar RNA binding affinity was observed with endogenous YY1 isolated from native mRNP complexes. In vivo expression experiments reveal epitope-tagged YY1 assembled into high molecular mass mRNPs, and assembly was blocked by microinjection of high affinity RNA substrate competitor. These findings present the first clues to how mRNPs assemble during early development.
Asunto(s)
ARN/química , Ribonucleoproteínas/química , Proteínas de Xenopus/fisiología , Factor de Transcripción YY1/fisiología , Animales , Sitios de Unión , Unión Competitiva , Epítopos/química , Cinética , Modelos Biológicos , Modelos Genéticos , Oocitos/metabolismo , Unión Proteica , Proteínas Recombinantes/química , Especificidad por Sustrato , Proteínas de Xenopus/química , Xenopus laevis/metabolismo , Factor de Transcripción YY1/químicaRESUMEN
To contribute to the study of the calcium-signaling mechanism of egg, we cloned and characterized a 26 kDa Ca(2+)-binding protein from Xenopus laevis eggs, a homologue of Rana catesbeiana dicalcin (renamed from p26olf) that was isolated from the olfactory epithelium. The primary structure of Xenopus dicalcin shows approximately 61% identity to that of Rana dicalcin and consists of two S100-like regions aligned in tandem, as seen in Rana dicalcin. Genomic Southern blot analysis indicated that Xenopus dicalcin is a unique orthologue of Rana dicalcin. Northern blot analysis showed that Xenopus dicalcin mRNA is expressed in Xenopus eggs and also in other tissues. These results indicated that Xenopus dicalcin is a novel S100-like Ca(2+)-binding protein in Xenopus eggs.
Asunto(s)
Proteínas de Unión al Calcio/genética , Clonación Molecular , Proteínas S100/genética , Proteínas de Xenopus/genética , Secuencia de Aminoácidos/genética , Animales , Autorradiografía , Secuencia de Bases/genética , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/metabolismo , Secuencia Conservada , ADN Complementario , Motivos EF Hand/genética , Femenino , Datos de Secuencia Molecular , Peso Molecular , Oocitos/metabolismo , ARN Mensajero/metabolismo , Proteínas S100/química , Proteínas S100/metabolismo , Homología de Secuencia de Aminoácido , Distribución Tisular , Xenopus , Proteínas de Xenopus/química , Proteínas de Xenopus/metabolismoRESUMEN
A recently developed implicit membrane model (IMM1) is supplemented with a Gouy-Chapman term describing counterion-screened electrostatic interactions of a solute with negatively charged membrane lipids. The new model is tested on peptides that bind to anionic membranes. Pentalysine binds just outside the plane of negative charge, whereas Lys-Phe peptides insert their aromatic rings into the hydrophobic core. Melittin and magainin 2 bind more strongly to anionic than to neutral membranes and in both cases insert their hydrophobic residues into the hydrocarbon core. The third domain of Antennapedia homeodomain (penetratin) binds as an alpha-helix in the headgroup region. Cardiotoxin II binds strongly to anionic membranes but marginally to neutral ones. In all cases, the location and configuration of the peptides are consistent with experimental data, and the effective energy changes upon binding compare favorably with experimental binding free energies. The model opens the way to exploring the effect of membrane charge on the location, conformation, and dynamics of a large variety of biologically active peptides on membranes.
Asunto(s)
Membrana Celular/metabolismo , Lípidos/química , Solventes/química , Animales , Aniones , Péptidos Catiónicos Antimicrobianos/química , Proteínas Cardiotóxicas de Elápidos/química , Simulación por Computador , Iones , Lisina/química , Magaininas , Meliteno/química , Modelos Químicos , Modelos Moleculares , Modelos Estadísticos , Modelos Teóricos , Distribución Normal , Péptidos/química , Fenilalanina/química , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Programas Informáticos , Electricidad Estática , Termodinámica , Proteínas de Xenopus/químicaRESUMEN
In order to investigate protein kinases expressed in the different developmental stages of Xenopus laevis, recently developed expression cloning was carried out. When two different expression libraries, Xenopus oocyte and Xenopus head (embryonic stage 28/30) cDNA libraries, were screened by kinase-specific monoclonal antibodies, cDNA clones for various known and novel protein serine/threonine kinases (Ser/Thr kinases) were isolated. In addition to well-characterized Ser/Thr kinases, one cDNA clone for a putative kinase was isolated from the Xenopus head library. The sequence of the open reading frame of the cDNA encoded a protein of 337 amino acid residues with a predicted molecular weight of 38,404. Since the deduced animo acid sequence of this protein was 75% identical to that of rat Ca(2+)/calmodulin-dependent protein kinase I (CaMKI), it was designated as CaMKIx. Although recombinant CaMKIx expressed in Escherichia coli showed no protein kinase activity against syntide-2, a synthetic peptide substrate, it was activated when phosphorylated by mouse Ca(2+)/calmodulin-dependent protein kinase kinase alpha (CaMKKalpha). Activated CaMKIx significantly phosphorylated various proteins including synapsin I, histones, and myelin basic protein. CaMKIx could not be detected in the early stages of embryogenesis, but was detected in late embryos of stages 37/38 and thereafter when examined by Western blotting using a specific antibody. This kinase was found to be highly expressed in adult brain and heart, and an upstream kinase that could activate CaMKIx was detected in these tissues. These results suggest that CaMKIx plays some critical role in the late stages of embryogenesis of Xenopus laevis.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/química , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Proteínas de Xenopus/química , Proteínas de Xenopus/genética , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/química , Secuencia de Bases , Biotinilación , Western Blotting , Quinasa de la Proteína Quinasa Dependiente de Calcio-Calmodulina , Proteína Quinasa Tipo 1 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/biosíntesis , Clonación Molecular , ADN Complementario/metabolismo , Electroforesis en Gel de Poliacrilamida , Escherichia coli/metabolismo , Biblioteca de Genes , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Péptidos/química , Fosforilación , Plásmidos/metabolismo , Proteínas Serina-Treonina Quinasas/química , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Factores de Tiempo , Proteínas de Xenopus/biosíntesis , Xenopus laevisRESUMEN
A Xenopus laevis egg cortical granule, calcium-dependent, galactosyl-specific lectin participates in forming the fertilization layer of the egg envelope and functions in establishing a block to polyspermy. We report the cDNA cloning of the lectin, expression of the cortical granule lectin gene during oogenesis and early development, and identification of a new family of lectins. The translated cDNA for the cortical granule lectin had a signal peptide, a structural sequence of 298 amino acids, a molecular weight of 32.7 K, contained consensus sequence sites for N-glycosylation and a fibrinogen domain. The lectin cDNA was expressed during early stages of oogenesis. Lectin glycoprotein levels were constant during development with 2/3 of the lectin associated with the extracellular perivitelline space and the egg/embryo fertilization envelope. Lectin mRNA levels were from 100- to 1000-fold greater in ovary than in other adult tissues. The lectin had no sequence homology to the previously identified lectin families. The lectin had 41-88% amino acid identity with nine translated cDNA sequences from an ascidian, lamprey, frog, mouse, and human. Based on the conserved carbohydrate binding and structural properties of these glycoproteins, we propose a new family of lectins, the eglectin family.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Lectinas/genética , Familia de Multigenes/genética , Proteínas de Xenopus/genética , Xenopus laevis/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario/genética , Etiquetas de Secuencia Expresada , Femenino , Perfilación de la Expresión Génica , Lectinas/química , Lectinas/metabolismo , Masculino , Datos de Secuencia Molecular , Oogénesis/genética , Filogenia , ARN Mensajero/análisis , ARN Mensajero/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Proteínas de Xenopus/química , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologíaRESUMEN
Here we report the purification, from Xenopus laevis oocyte nuclear extracts, of a new endoribonuclease, XendoU, that is involved in the processing of the intron-encoded box C/D U16 small nucleolar RNA (snoRNA) from its host pre-mRNA. Such an activity has never been reported before and has several uncommon features that make it quite a novel enzyme: it is poly(U)-specific, it requires Mn(2+) ions, and it produces molecules with 2'-3'-cyclic phosphate termini. Even if XendoU cleaves U-stretches, it displays some preferential cleavage on snoRNA precursor molecules. XendoU also participates in the biosynthesis of another intron-encoded snoRNA, U86, which is contained in the NOP56 gene of Xenopus laevis. A common feature of these snoRNAs is that their production is alternative to that of the mRNA, suggesting an important regulatory role for all the factors involved in the processing reaction.
Asunto(s)
Endorribonucleasas/genética , Intrones , ARN Nuclear Pequeño/genética , Proteínas de Xenopus/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Nucléolo Celular/genética , Núcleo Celular/enzimología , Cromatografía , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Clonación Molecular , ADN Complementario/genética , Durapatita , Embrión no Mamífero , Endorribonucleasas/química , Endorribonucleasas/aislamiento & purificación , Endorribonucleasas/metabolismo , Femenino , Datos de Secuencia Molecular , Oocitos/enzimología , Fragmentos de Péptidos/química , Proteínas de Xenopus/química , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMEN
The Armadillo family of catenin proteins function in multiple capacities including cadherin-mediated cell-cell adhesion and nuclear signaling. The newest catenin, p120(ctn), differs from the classical catenins and binds to the membrane-proximal domain of cadherins. Recently, a novel transcription factor Kaiso was found to interact with p120(ctn), suggesting that p120(ctn) also possesses a nuclear function. We isolated the Xenopus homolog of Kaiso, XKaiso, from a Xenopus stage 17 cDNA library. XKaiso contains an amino-terminal BTB/POZ domain and three carboxyl-terminal zinc fingers. The XKaiso transcript was present maternally and expressed throughout early embryonic development. XKaiso's spatial expression was defined via in situ hybridization and was found localized to the brain, eye, ear, branchial arches, and spinal cord. Co-immunoprecipitation of Xenopus p120(ctn) and XKaiso demonstrated their mutual association, whereas related experiments employing differentially epitope-tagged XKaiso constructs suggest that XKaiso additionally self-associates. Finally, reporter assays employing a chimera of XKaiso fused to the GAL4 DNA binding domain indicate that XKaiso is a transcriptional repressor. These data suggest that XKaiso functions throughout development and that its repressor functions may be most apparent in the context of neural tissues. The significance of the XKaiso-p120(ctn) interaction has yet to be determined, but it may include transducing information from cadherin-mediated cell-cell contacts to transcriptional processes within the nucleus.