RESUMEN
Increasing evidence suggests heterogeneity in the molecular pathogenesis of cystic fibrosis (CF). Mutations such as deletion of phenylalanine at position 508 (delta F508) within the cystic fibrosis transmembrane conductance regulator (CFTR), for example, appear to cause disease by abrogating normal biosynthetic processing, a mechanism which results in retention and degradation of the mutant protein within the endoplasmic reticulum. Other mutations, such as the relatively common glycine-->aspartic acid replacement at CFTR position 551 (G551D) appear to be normally processed, and therefore must cause disease through some other mechanism. Because delta F508 and G551D both occur within a predicted nucleotide binding domain (NBD) of the CFTR, we tested the influence of these mutations on nucleotide binding by the protein. We found that G551D and the corresponding mutation in the CFTR second nucleotide binding domain, G1349D, led to decreased nucleotide binding by CFTR NBDs, while the delta F508 mutation did not alter nucleotide binding. These results implicate defective ATP binding as contributing to the pathogenic mechanism of a relatively common mutation leading to CF, and suggest that structural integrity of a highly conserved region present in over 30 prokaryotic and eukaryotic nucleotide binding domains may be critical for normal nucleotide binding.
Asunto(s)
Adenosina Trifosfato/metabolismo , Fibrosis Quística/genética , Proteínas de la Membrana/genética , Mutación , Secuencia de Bases , Sitios de Unión , Canales de Cloruro/fisiología , Regulador de Conductancia de Transmembrana de Fibrosis Quística , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/fisiología , Datos de Secuencia MolecularRESUMEN
The absorption and circular dichroic spectra of three stable isoelectric forms of purified rhodopsin (in Emulphogene BC 720) with isoelectric points of 5.19, 5.58 and 6.14 were analysed over the accessible wavelength region of 190-800 nm. It was found from the spectral analysis of the 5.58 and 6.14 forms that the tertiary structure of these isoelectric forms was different, without any change being observed in the secondary structure of these proteins. The difference in structure was removed by denaturation with SDS. Specifically, the aromatic amino acid residues of the 6.14 isoelectric form were suggested to be in a more polar environment as compared to those for the 5.58 isoelectric form. The changes of structure in the microenvironment of retinal in these proteins were minor in comparison to the tertiary changes observed in the proteins. This indicated that the site of charge perturbation is not localized in the retinylidene microenvironment. Furthermore, the spectral features of these isoelectric forms were features common to the spectra of both purified unfocused rhodopsin and crude rhodopsin, suggesting that the isoelectric forms are not a consequence of purification. Spectral analysis of the 5.19 isoelectric form indicated that this form of rhodopsin had an increased tendency to aggregate after focusing. Based on the denaturation properties of these isoelectric forms, it was shown that the 5.19 and 5.58 isoelectric forms had different conformations. It was concluded from this study that preparations of 'purified' rhodopsin are a heterogeneous mixture of stable proteins with different tertiary structures and different isoelectric points.
Asunto(s)
Pigmentos Retinianos/análisis , Rodopsina/análisis , Animales , Bovinos , Dicroismo Circular , Focalización Isoeléctrica , Solubilidad , EspectrofotometríaRESUMEN
A conformationally defined retinoic acid analog (1) which contains a dimethylene bridge to maintain the 6-s-trans orientation for two terminal double bonds in the polyene chain was synthesized. A Reformatsky reaction was utilized to extend the polyene chain of the starting enone, which provided exclusively the 9Z-configuration for the intermediate aldehyde. A Horners-Emmons condensation with this aldehyde then produced retinoic acid analogs with both 9Z- and 9Z,13Z-configurations. An I2-catalyzed isomerization of the intermediate 9Z-aldehyde yielded the all-E-aldehyde, which was olefinated as above to yield the (all-E)- and (13Z)-retinoic acid analogs of 1. Each configurational isomer of 1 was evaluated for its ability to inhibit the binding of retinoic acid to CRABP (chick skin) and to inhibit the chemical induction of ornithine decarboxylase in mouse skin. In each assay (all-E)-1 was the most active isomer, and this activity was comparable to or better than that for (all-E)-retinoic acid. (all-E)-1 and (13Z)-1 were both shown to be equally effective as (13Z)-retinoic acid in suppressing the proliferation of human sebaceous cells in vitro. (all-E)-1 was further evaluated for its ability to prevent the induction of mouse skin papillomas and to induce signs of vitamin A toxicity in mice. The cancer chemopreventive activity of (all-E)-1 was comparable to that of (all-E)-retinoic acid, and the toxicity was comparable to or slightly better than that of the natural vitamin.
Asunto(s)
Anticarcinógenos/síntesis química , Tretinoina/síntesis química , Células 3T3 , Adulto , Animales , Anticarcinógenos/farmacología , Anticarcinógenos/toxicidad , Gatos , Femenino , Humanos , Ratones , Ornitina Descarboxilasa/biosíntesis , Estereoisomerismo , Relación Estructura-Actividad , Tretinoina/farmacología , Tretinoina/toxicidadRESUMEN
We recently synthesized several conformationally constrained retinoic acid (RA) analogues [8-(2'-cyclohexen-1'-ylidene)-3, 7-dimethyl-2,4,6-octatrienoic acids with different alkyl substituents at 2' (R1) and 3' (R2) positions on the cyclohexene ring] (Muccio et al. J. Med. Chem. 1996, 39, 3625) as cancer chemopreventive agents. UAB8 (R1 = Et; R2 = iPr), which contains sufficient steric bulk at the terminal end of the polyene chain to mimic the trimethylcyclohexenyl ring of RA, displayed biological properties similar to those of RA. To explore the efficacy of this retinoid in acute promyelocytic leukemia (APL) and juvenile myelomonocytic leukemia (JMML), we evaluated UAB8 isomers in in vitro assays which measure the capacity of retinoids to inhibit aberrant myeloid colony growth from blood or bone marrow cells obtained from human JMML patients and in assays measuring the potential of retinoids to differentiate NB4 cells (an APL cell line). Both (all-E)- and (13Z)-UAB8 were 2-fold more active than RA in the NB4 cell differentiation assay; however, only (all-E)-UAB8 had comparable activity to the natural retinoids in the JMML cell assays. These results were compared to the biological effectiveness of a new retinoid, UAB30 [8-(3', 4'-dihydro-1'(2'H)-naphthalen-1'-ylidene)-3,7-dimethyl-2,4, 6-octatrienoic acid], which had different nuclear receptor binding and transactivational properties than UAB8. Relative to (all-E)-RA and (all-E)-UAB8, (all-E)-UAB30 bound well to RARalpha but did not activate transcription-mediated RARalpha homodimers, even though it was effective in RARbeta- and RARgamma-mediated transactivational assays. In APL assays, this retinoid had much reduced activity and was only moderately effective in JMML assays and in cancer chemoprevention assays.
Asunto(s)
Antineoplásicos , Ácidos Grasos Insaturados , Leucemia Mielomonocítica Crónica/prevención & control , Leucemia Promielocítica Aguda/prevención & control , Naftalenos , Tretinoina/análogos & derivados , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Línea Celular , Pollos , Niño , Ácidos Grasos Insaturados/síntesis química , Ácidos Grasos Insaturados/química , Ácidos Grasos Insaturados/metabolismo , Ácidos Grasos Insaturados/farmacología , Células HL-60 , Humanos , Técnicas In Vitro , Ratones , Conformación Molecular , Naftalenos/síntesis química , Naftalenos/química , Naftalenos/metabolismo , Naftalenos/farmacología , Papiloma/prevención & control , Ensayo de Unión Radioligante , Receptores de Ácido Retinoico/metabolismo , Piel/metabolismo , Neoplasias Cutáneas/prevención & control , Estereoisomerismo , Transcripción Genética/efectos de los fármacos , Tretinoina/química , Tretinoina/metabolismo , Tretinoina/farmacología , Ensayo de Tumor de Célula MadreRESUMEN
We recently demonstrated that conformationally defined 6-s-trans-retinoic acid (RA) analogs were effective in the prevention of skin papillomas (Vaezi et al. J. Med. Chem. 1994, 37, 4499-4507) and selective agonists for nuclear receptor binding and activation (Alam et al. J. Med. Chem. 1995, 38, 2302-2310). In order to probe important structure-activity relationships, we evaluated a homologous series of four 6-s-trans-retinoids that are 8-(2'-cyclohexen-1'-ylidene)-3,7-dimethyl-2,4,6-octatrienoic acids with different substituents at 2' (R2) and 3' (R1) positions on the cyclohexene ring. UAB1 (R1 = R2 = H), UAB4 (R1 = R2 = Me), UAB7 (R1 = Me, R2 = iPr), and UAB8 (R1 = Et, R2 = iPr) contain alkyl R groups that mimic, to different extents, portions of the trimethylcyclohexenyl ring of RA. Both 9Z- and all-E-isomers of these retinoids were evaluated in binding assays for cellular retinoic acid-binding proteins (CRABP-I and CRABP-II), a nuclear retinoic acid receptor (RAR alpha), and a nuclear retinoid X receptor (RXR alpha). The all-E-isomers of UAB retinoids bound tightly to CRABPs and RAR alpha, the binding affinity of the all-E-isomer increased systematically from UAB1 to UAB8, and binding for the latter was comparable to that of all-E-RA. In contrast to RA, the (9Z)-UAB retinoids were at least 200-fold less active than the all-E-isomers in binding to RAR alpha. The (9Z)-UAB isomers exhibited increasingly stronger binding to RXR alpha, and (9Z)-UAB8 was nearly as effective as (9Z)-RA in binding affinity. The retinoids were also evaluated in gene expression assays mediated by RAR alpha and RXR alpha homodimers or RAR alpha/RXR alpha heterodimers. Consistent with the binding affinities, the (all-E)-UAB retinoids activated gene transciption mediated by RAR alpha homodimers or RAR alpha/RXR alpha heterodimers, while the (9Z)-UAB isomers activated only the RXR alpha homodimer-mediated transcription. The all-E- and 9Z-isomers of the UAB retinoids were further evaluated for their capacity to prevent the induction of mouse skin papillomas. When compared to RA, only the (all-E)-UAB retinoids containing bulky R1 and R2 groups were effective in this chemoprevention assay. (9Z)-RA displayed equal capacity as RA to prevent papillomas, while the 9Z-isomers of the UAB retinoids were much less effective. Taken together, these studies demonstrate that the cyclohexenyl ring substituents of 6-s-trans-UAB retinoids are important for their biological activities and that the chemopreventive effect of the all-E-isomers of these retinoids correlates well with their capacity to bind to RARs and activate RAR/RXR-mediated transcription.
Asunto(s)
Anticarcinógenos , Núcleo Celular/metabolismo , Receptores de Ácido Retinoico/metabolismo , Retinoides/química , Transcripción Genética/efectos de los fármacos , Animales , Ratones , Modelos Moleculares , Conformación Molecular , Estructura Molecular , Papiloma/prevención & control , Receptores X Retinoide , Retinoides/metabolismo , Retinoides/uso terapéutico , Neoplasias Cutáneas/prevención & control , Estereoisomerismo , Relación Estructura-Actividad , Termodinámica , Factores de Transcripción/metabolismoRESUMEN
PURPOSE: The release of N-acetyl-proline-glycine-proline (PGP), a chemoattractant resulting from direct alkaline hydrolysis of corneal proteins, is believed to be the initial trigger for neutrophil invasion into the alkali-injured cornea. The purpose of this study is twofold: (1) to compare the activity of N-acetyl-PGP with the bioactivities of other similar synthetic peptides in an effort to uncover information about this chemoattractant molecule, and (2) to test these peptide analogs as potential antagonists of N-acetyl-PGP. METHODS: The polarization assay was used to measure the potential chemotactic response of human neutrophils to peptides. Bioactivity was expressed as the peptide concentration required to produce 50% neutrophil polarization (EC50). Antagonist activity was expressed as the peptide concentration required to produce 50% inhibition (ID50) of polarization activated by N-acetyl-PGP. RESULTS: Peptide bioactivities (EC50) were ranked as follows: APGPR (0.34 mM) > N-acetyl-PGP (0.5 mM) > N-(PGP)4-PGLG (3 mM) = t-Boc-PGP (3 mM) > N-acetyl-PG (3.4 mM) > N-methyl-PGP (15 mM) = PGP (15 mM) > peptides without detectable activity (t-Boc-PGP-OMe, N-acetyl-P, PG, PGG, GP, GG and gly-pro-hyp). Peptides with no detectable bioactivity were tested as potential antagonists of neutrophil polarization induced by N-acetyl-PGP. Gly-Pro-Hyp inhibited N-acetyl-PGP activation of polarization at 20 mM (ID50). No other synthetic peptide demonstrated a capacity for inhibition. CONCLUSIONS: The minimum requirement to elicit bioactivity was the presence of PGP alone or derivatives of PG in which the N-terminal proline is blocked. Using this approach, active and inactive mimetic peptides of N-acetyl-PGP were produced. The most active peptide, APGPR, was equal to or slightly greater than N-acetyl-PGP, suggesting that more potent analogs might be designed. Gly-pro-hyp was the only inactive peptide analog to inhibit the chemoattractant.
Asunto(s)
Factores Quimiotácticos/farmacología , Quimiotaxis de Leucocito/fisiología , Activación Neutrófila/efectos de los fármacos , Neutrófilos/fisiología , Oligopéptidos/farmacología , Prolina/análogos & derivados , Factores Quimiotácticos/síntesis química , Humanos , Oligopéptidos/síntesis química , Prolina/síntesis química , Prolina/farmacología , Relación Estructura-ActividadRESUMEN
Bovine rhodopsin was purified from n-octylglucoside-solubilized retinas by high-performance size-exclusion chromatography. In one chromatographic step, six protein fractions were separated with baseline resolution. The major fraction was identified as monomeric rhodopsin by absorption spectroscopy. Amino acid analysis of this fraction further supported the assignment. A comparison of the elution profiles of rhodopsin purified by this method with that purified by Concanavalin A-Sepharose 4B affinity chromatography suggested that rhodopsin from high-performance chromatography was slightly purer than the conventionally purified rhodopsin.
Asunto(s)
Pigmentos Retinianos/aislamiento & purificación , Rodopsina/aislamiento & purificación , Aminoácidos/análisis , Animales , Bovinos , Cromatografía de Afinidad/métodos , Cromatografía Líquida de Alta Presión/métodos , Concanavalina A , Peso Molecular , Células Fotorreceptoras/análisis , Retina/análisis , Sefarosa , Espectrofotometría UltravioletaRESUMEN
The absorption and circular dichroic (CD) spectra of purple membrane films in which the plane of the membranes is oriented perpendicular to the incident beam are compared with the solution spectra. This enables one to relate structural features of the purple membrane to a coordinate system as defined by a normal to the membrane plane and two mutually perpendicular in-plane axes. The film and solution absorption spectra were similar except for a relative depression in the 200 - 225-nm region of the film spectrum. However, the CD spectra showed significant differences in the visible region, where the biphasic band in the solution spectrum was replaced by a single positive band at 555 nm in the film spectrum and in the far ultraviolet region, where the 208-nm band was deleted from the film spectra of the native and regenerated membranes. Moreover, a small shoulder occurred at 208 nm in the film spectrum of the bleached membrane. The near ultraviolet spectra also showed differences, whereas the 317-nm band remained essentially the same for both spectra. Based on excitonic interpretations of the visible and far ultraviolet spectra the following conclusions were reached: (a) a relatively strong in-plane monomeric interaction occurs between te retinyl chromophore and apoprotein; (b) the helical axes of the native and regenerated membrane proteins are oriented primarily normal to the membrane plane; and (c) the helical axes of the bleached membrane proteins are tilted more in-plane than the axes of the native or regenerated membrane. Additional conclusions were that an interaction occurs between an in-plane magnetic dipole moment of the retinyl chromophore and probably an in-plane electric dipole moment of a nearby aromatic amino acid(s), and that although the membrane is anisotropic with respect to coupling between electric and magnetic moments of the aromatic amino acids, the transition dipole moments of the aromatic amino acids are not preferentially oriented in either direction.
Asunto(s)
Bacteriorodopsinas , Carotenoides , Dicroismo Circular , Halobacterium , Cinética , Conformación Proteica , EspectrofotometríaRESUMEN
Bacteriorhodopsin, an integral membrane protein of purple membranes, was solubilized with n-octylglucoside and isolated as intact monomeric micelles by high-performance size-exclusion chromatography. It was shown that separation was obtained between these micelles and either those containing bacterio-opsin or retinal as well as bacterio-opsin in the aggregated state. Estimates of the apparent molecular weights and Stokes radii were obtained by comparison with water-soluble proteins with known properties. Thermal denaturation of the native protein micelle induced the formation of a denatured species, which was similar to that found from denaturation at 4 degrees C.
Asunto(s)
Bacteriorodopsinas/aislamiento & purificación , Carotenoides/aislamiento & purificación , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Detergentes , Glucósidos , Halobacterium/análisis , Micelas , Peso Molecular , Desnaturalización Proteica , Espectrofotometría , Espectrofotometría Ultravioleta , TemperaturaRESUMEN
The thermal denaturation of bacteriorhodopsin in the purple membrane of Halobacterium halobium has been studied by differential scanning calorimetry (DSC) and temperature-dependent spectroscopy in the pH range from 5 to 11. Monitoring of protein fluorescence and absorbance in the near-UV and visible regions indicates that changes primarily occur in tertiary structure with denaturation. Far-UV circular dichroism shows only small changes in the secondary structure, unlike most globular water-soluble proteins of comparable molecular weight. The DSC transition can best be described as a two-state denaturation of the trimer. Thermodynamic analysis of the calorimetric transition reveals some similarity between the unfolding of bacteriorhodopsin and water-soluble proteins. Specifically, a pH dependence of the midpoint temperature of denaturation is seen as well as a temperature-dependent enthalpy of denaturation. Proteolysis experiments on denatured purple membrane suggest that bacteriorhodopsin may be partially extruded from the membrane as it denatures. Exposure of buried hydrophobic residues to the aqueous environment upon denaturation is consistent with the observed temperature-dependent enthalpy.
Asunto(s)
Bacteriorodopsinas/metabolismo , Halobacterium/metabolismo , Rastreo Diferencial de Calorimetría , Concentración de Iones de Hidrógeno , Peso Molecular , Desnaturalización Proteica , Espectrofotometría , TermodinámicaRESUMEN
The complete 1H nuclear magnetic resonance assignments have been made for the common mono-, di-, and trihydroxy 5 beta-cholanoic acids; lithocholic acid, chenodeoxycholic acid, ursodeoxycholic acid, deoxycholic acid, cholic acid, and the unsubstituted parent compound, 5 beta-cholanoic acid, by heteronuclear-correlated two-dimensional NMR. The known 13C chemical shifts of these compounds were used to make the proton resonance assignments, and consistency of the carbon and proton assignments was verified by expected changes due to substituent effects. This has led to clarification of previously published 13C NMR resonance assignments. Addition of the 3 alpha, 7 alpha, and 12 alpha hydroxyl substituent effects derived from the mono- and dihydroxycholanoic acids yielded predicted values for proton chemical shifts of the trihydroxy-substituted 5 beta-cholanoic acid, cholic acid, that agreed well with experimental values. It is suggested that the individual substituent effects can be used to predict proton chemical shifts for hydroxycholanic acids containing other combinations of 3 alpha, 7 alpha, 7 beta, and 12 alpha hydroxyl groups.
Asunto(s)
Ácidos y Sales Biliares/análisis , Ácido Quenodesoxicólico/análisis , Colanos/análisis , Ácido Cólico , Ácidos Cólicos/análisis , Ácido Desoxicólico/análisis , Ácido Litocólico/análisis , Espectroscopía de Resonancia Magnética/métodos , Relación Estructura-Actividad , Ácido Ursodesoxicólico/análisisRESUMEN
Through the use of CD and DSC, the thermal unfolding of holo serum retinol binding protein containing a single, tightly bound retinol ligand was studied at pH 7.4. The DSC endotherm of the holoprotein ([retinol]/[protein] = 1) was asymmetric about the transition temperature of 78 degrees C. Using changes in ellipticity at 230 nm, the thermal unfolding curve was also asymmetric about the inflection point centered near 78 degrees C. van't Hoff enthalpies were determined by three means and compared to the calorimetric enthalpy (delta Hcal) of 200 kcal/mol. A van't Hoff enthalpy of 190 kcal/mol was determined from the dependence of transition temperature on the concentration of the ligand-bound protein. This value agreed well with the van't Hoff enthalpies found from fits of the DSC (delta HvH = 184 kcal/mol) and spectroscopic (delta HvH = 181 kcal/mol) curves to a two-state thermodynamic model that included ligand dissociation (NR in equilibrium with U+R, where NR is the native holoprotein, U is the unfolded apoprotein, and R is retinol). Poor agreement was obtained with a two-state model that ignored ligand dissociation (N in equilibrium with U). Furthermore, the NR in equilibrium with U+R model accounted for the asymmetry in both CD and DSC transitions and yielded a much improved fit of the data over the N in equilibrium with U model. From these considerations and simulations on other equilibrium models, it is suggested that the NR in equilibrium with U+R model is the simplest model that describes the thermal unfolding of this ligand-bound protein.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Proteínas de Unión al Retinol/química , Sitios de Unión , Rastreo Diferencial de Calorimetría , Dicroismo Circular , Calor , Humanos , Ligandos , Desnaturalización Proteica , Espectrofotometría Ultravioleta , Termodinámica , Vitamina A/metabolismoRESUMEN
The microsomal enzyme LRAT esterifies retinol and has been implicated in the hepatic storage of vitamin A. Previously, we showed that hepatic LRAT activity is negligible during vitamin A deficiency and that all-trans-retinoic acid (all-trans-RA) rapidly induces the activity of liver LRAT in retinoid-deficient rats. In the present studies, we have examined the ability of natural and synthetic retinoids to induce liver LRAT activity in retinoid-deficient rats. The natural retinoids retinol, all-trans-RA (100 microg), 9-cis-RA, or equal molar amounts of other retinoids were injected ip and LRAT specific activity was measured in liver homogenates 17-18 h later. In retinoid-deficient rats, liver LRAT activity was extremely low [0.13 +/- 0.03 pmol retinyl ester (RE)/min/mg liver protein, mean +/- SE]. The natural retinoids retinol and all-trans-RA strongly induced LRAT activity (12.71 +/- 1.09 and 13.10 +/- 1.55 pmol RE/min/mg, respectively), whereas 9-cis-RA induced a lower level of LRAT activity (3.96 +/- 1.88 pmol RE/min/mg, P < 0.001 vs all-trans-RA). The retinoic acid receptor (RAR)-selective analog (RAR pan-agonist) all-trans-UAB8 and the RAR-alpha-selective retinoid Am580 also strongly induced LRAT activity. In contrast, neither RXR-selective agonists nor retinoids having a retro structure were active. For retinoids with significant RAR-alpha binding activity there was a strong direct correlation between receptor binding in vitro and the ability to induce hepatic LRAT activity in vivo (r2 = 0.920). These data implicate the RARs in the induction of hepatic LRAT and suggest a predominant role for RAR-alpha-active ligands.
Asunto(s)
Aciltransferasas/metabolismo , Microsomas Hepáticos/enzimología , Receptores de Ácido Retinoico/metabolismo , Retinoides/farmacología , Animales , Femenino , Estructura Molecular , Unión Proteica , Ratas , Ratas Endogámicas , Receptores X Retinoide , Factores de Transcripción/metabolismo , Tretinoina/farmacología , Vitamina A/farmacología , Deficiencia de Vitamina A/metabolismoRESUMEN
Direct comparison of the absorption and circular dichroic spectra of dark- and light-adapted purple membrane from Halobacterium cutirubrum and Halobacterium halobium indicated no apparent species differences. In addition, sequential bleaching and regeneration of the purple membrane with concomitant monitoring of the absorption and circular dichroic spectra showed no species differences as well. Furthermore, perturbation of the structure of the purple membrane from either species with a detergent, Triton X-100, yielded similar spectral changes. It was concluded: (i) no apparent differences exist in the molecular organization and protein fine structure of the two purple membranes, (ii) if exciton interaction among the retinal chromophores is a reasonable possibility in the case of the purple membrane from Halobacterium halobium, it must be similarly so for the membrane from Halobacterium cutirubrum, (iii) the effects of light adaptation on the membrane structure of both species are essentially the same, and (iv) the underlying molecular mechanisms for the bleaching and regenerative processes must be similar, if not identical, for the purple membranes of the two species.
Asunto(s)
Bacteriorodopsinas , Carotenoides , Halobacterium/ultraestructura , Dicroismo Circular , Adaptación a la Oscuridad , Polietilenglicoles , Retinaldehído , Solubilidad , Especificidad de la Especie , Análisis EspectralRESUMEN
The majority of mutations which lead to clinical cystic fibrosis are located within the two predicted nucleotide binding domains of the cystic fibrosis gene product. We have used a prokaryotic expression system to synthesize and purify the first nucleotide binding domain (NBD-1, amino acids 426-588) with and without the most common mutation associated with the disease (the deletion of phenylalanine at position 508, delta F508). Both wild type and delta F508 NBD-1 bind ATP-agarose in a quantitatively comparable manner; this binding was inhibited by excess Na2ATP, trinitrophenol-ATP, or 8-azido-ATP. Irreversible NBD-1 labeling by an ATP analog was demonstrated using [32P]8-azido-ATP. This covalent labeling was inhibited by preincubation with Na2ATP, with half-maximal inhibition for Na2ATP occurring at approximately 5 mM for both the wild type and delta F508 nucleotide binding domain. These experiments are among the first to confirm the expectation that the cystic fibrosis transmembrane conductance regulator NBD-1 binds nucleotide. Since, under the conditions used in our study, NBD-1 without phenylalanine 508 displays very similar nucleotide binding characteristics to the wild type protein, our results support previous structural models which predict that the delta F508 mutation should not cause an alteration in ATP binding.
Asunto(s)
Fibrosis Quística/metabolismo , Proteínas de la Membrana/biosíntesis , Nucleótidos/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/química , Marcadores de Afinidad , Azidas/química , Secuencia de Bases , Regulador de Conductancia de Transmembrana de Fibrosis Quística , ADN/genética , Electroforesis en Gel de Poliacrilamida , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Mutación , Fenilalanina/genéticaRESUMEN
Recently antifreeze proteins (AFP) have been the subject of many structure-function relationship studies regarding their antifreeze activity. Attempts have been made to elucidate the structure-function relationship by various amino acid substitutions, but to our knowledge there has been no successful from first principles design of a polypeptide that would bind to designated ice planes along a specific direction. In this paper we show the results of our first attempt on an entirely de novo design of an alanine-lysine-rich antifreeze polypeptide. This 43 residue alanine-lysine peptide exhibits characteristic nonequilibrium freezing point depression and binds to the designated (210) planes of ice along the [122] vector. The structural and thermodynamic properties of this polypeptide were determined using circular dichroism spectroscopy and its nonequilibrium antifreeze properties were investigated using an ice-etching method and nanoliter osmometry.
Asunto(s)
Alanina/química , Lisina/química , Péptidos/química , Secuencia de Aminoácidos , Fenómenos Químicos , Química Física , Dicroismo Circular , Cristalización , Grabado por Congelación , Congelación , Conformación Molecular , Datos de Secuencia Molecular , Relación Estructura-Actividad , Termodinámica , Agua/químicaRESUMEN
Alkaline hydrolysis of corneal proteins in the alkali-injured eye releases N-acetyl-proline-glycine-proline (Ac-Pro-Gly-Pro-OH) among other peptides. It has been shown that this tripeptide is a neutrophil chemoattractant. Existing data suggest that the release of this peptide is the catalytic event for early neutrophil invasion of the cornea leading to corneal ulcers. In order to design inhibitors of this tripeptide chemoattractant that would block neutrophil invasion and diminish corneal ulcers, we studied the solution properties of this tripeptide by NMR spectroscopy and compared this peptide to Ac-Pro-Gly-OH (a weaker chemoattractant), and to Ac-Pro-OH (inactive). The NMR data were consistent with Ac-Pro-Gly-Pro-OH existing in solution as a mixture of four isomers with different cis and trans conformations about the two X-proline amide bonds. The isomer with two trans conformations (trans-trans) was the most dominant (41%) in aqueous solution. This was followed by the isomers with mixed cis and trans conformations (trans-cis, 26% and cis-trans, 20%). The isomer with two cis conformations (cis-cis) was the least favored (13%). The populations of these isomers were investigated in DMSO and they were similar to those reported in aqueous solutions except that the ordering of the trans-cis and cis-trans isomers were reversed. NMR NH temperature coefficients and nuclear Overhauser effect (NOE) measurements as well as CD spectroscopy were used to demonstrate that the four isomers exist primarily in an extended conformation with little hydrogen bonding. The available (NOE) information was used with molecular dynamics calculations to construct a dominant solution conformation for each isomer of the tripeptide. This information will serve as a model for the design of peptide and nonpeptide inhibitors of the chemoattractant.
Asunto(s)
Factores Quimiotácticos/química , Neutrófilos/fisiología , Oligopéptidos/química , Prolina/química , Álcalis/efectos adversos , Animales , Quimiotaxis de Leucocito , Córnea/química , Lesiones de la Cornea , Lesiones Oculares/inducido químicamente , Humanos , Resonancia Magnética Nuclear Biomolecular , EstereoisomerismoRESUMEN
(2E, 4E, 6E)-8-[3'-Ethyl-2'-(1-methylethyl)-2'-cyclohexen-1'-ylidene] -3, 7-dimethyl-2,4,6-octatrienoic acid (UAB-8) has potent activity in preventing papillomas on the skin of mice similar to that determined in a previous study for the homolog containing one less carbon atom. To evaluate the toxicological profile for UAB-8, relative to all-trans-retinoic acid (RA), female mice were dosed by oral gavage for 29 days with amounts of 0.05, 0.1, or 0.2 mmol/kg/day. For the two compounds, the effects on body weights were similar. Mice dosed with UAB-8, however, had a lower incidence of clinical signs of toxicity (alopecia, scaly skin, and limping). At necropsy, bone fractures, skin abnormalities, and splenomegaly were observed in some mice dosed with RA but not in any dosed with UAB-8. Lymph node hyperplasia was noted in some mice dosed with either dose of RA but only in those dosed with the highest dose of UAB-8. All dose levels of RA produced microscopic lesions in the bones of mice; only the highest dose of UAB-8 had this effect. RA and UAB-8 had similar effects on chondrogenesis in cultures of cells from mouse limb buds, an indication of comparable teratogenic effects. For mice dosed i.v. (10 mg/kg), there was a saturated phase of elimination of RA from plasma (Km = 0.61 microgram/ml and Vmax = 2572 micrograms/hr); no such phase was noted when UAB-8 was administered. UAB-8 had values for t1/2 alpha and t1/2 beta of 0.47 and 17.1 hr, respectively. Relative to RA, UAB-8 has a favorable toxicological profile and different pharmacokinetics.