RESUMEN
CF2H-Pseudoprolines obtained from difluoroacetaldehyde hemiacetal and serine are stable proline surrogates. The consequence of the incorporation of the CF2H group is an important decrease of the trans to cis amide bond isomerization energy and a remarkable stabilisation of the cis conformer by an hydrogen bond.
Asunto(s)
Péptidos/química , Prolina/análogos & derivados , Tiazoles/química , Tolueno/análogos & derivados , Enlace de Hidrógeno , Metilación , Conformación Molecular , Prolina/química , Estereoisomerismo , Tolueno/químicaRESUMEN
Protein-protein interactions are crucial for signal transductions required for cell differentiation and proliferation. Their modulation is therefore key to the development of therapeutic alternatives, particularly in the context of cancer. According to literature data, the polyproline-rich nuclear receptor coactivators PNRC and PNRC2 interact with estrogen receptor (ERα) through their PxxP SH3-binding motifs. In a search to identify the molecular features governing this interaction, we explored using electronic circular dichroism (ECD) spectroscopy and molecular dynamics (MD) calculations, the capacity of a range of putative biologically active peptides derived from these proteins and containing this PxxP motif(s) to form polyproline II (PPII) domains. An additional more exhaustive structural study on a lead PPII peptide was also performed using 2D nuclear magnetic resonance (NMR) spectroscopy. With the exception of one of all the investigated peptides (PNRC-D), binding assays failed to detect any affinity for Grb2 SH3 domains, suggesting that PPII motifs issued from Grb2 antagonists have a binding mode distinct from those derived from Grb2 agonists. Instead, the peptides revealed a competitive binding ability against a synthetic peptide (ERα17p) with a putative PPII-cognate domain located within a coregulator recruitment region of ERα (AF-2 site). Our work, which constitutes the first structure-related interaction study concerning PNRC and PNRC2, supports not only the existence of PxxP-induced PPII sequences in these coregulators, but also confirms the presence of a PPII recognition site in the AF-2 of the steroid receptor ERα, a region important for transcription regulation.
Asunto(s)
Receptor alfa de Estrógeno/química , Proteínas Nucleares , Coactivadores de Receptor Nuclear/química , Péptidos/química , Prolina/química , Receptores Citoplasmáticos y Nucleares , Transactivadores , Factores de Transcripción , Dominios Homologos src/fisiología , Secuencias de Aminoácidos/fisiología , Secuencia de Aminoácidos , Dicroismo Circular , Receptor alfa de Estrógeno/fisiología , Proteína Adaptadora GRB2/química , Proteína Adaptadora GRB2/fisiología , Humanos , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Proteínas Nucleares/genética , Proteínas Nucleares/fisiología , Unión Proteica , Estructura Terciaria de Proteína , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/fisiología , Alineación de Secuencia , Transactivadores/genética , Transactivadores/fisiología , Factores de Transcripción/genética , Factores de Transcripción/fisiologíaRESUMEN
The pentose-phosphate pathway provides reductive power and nucleotide precursors to the cell through oxidative and nonoxidative branches, respectively. 6-Phosphogluconolactonase is the second enzyme of the oxidative branch and catalyzes the hydrolysis of 6-phosphogluconolactones, the products of glucose 6-phosphate oxidation by glucose-6-phosphate dehydrogenase. The role of 6-phosphogluconolactonase was still questionable, because 6-phosphogluconolactones were believed to undergo rapid spontaneous hydrolysis. In this work, nuclear magnetic resonance spectroscopy was used to characterize the chemical scheme and kinetic features of the oxidative branch. We show that 6-phosphogluconolactones have in fact a nonnegligible lifetime and are highly electrophilic compounds. The delta form (1-5) of the lactone is the only product of glucose 6-phosphate oxidation. Subsequently, it leads to the gamma form (1-4) by intramolecular rearrangement. However, only the delta form undergoes spontaneous hydrolysis, the gamma form being a "dead end" of this branch. The delta form is the only substrate for 6-phosphogluconolactonase. Therefore, 6-phosphogluconolactonase activity accelerates hydrolysis of the delta form, thus preventing its conversion into the gamma form. Furthermore, 6-phosphogluconolactonase guards against the accumulation of delta-6-phosphogluconolactone, which may be toxic through its reaction with endogenous cellular nucleophiles. Finally, the difference between activity of human, Trypanosoma brucei, and Plasmodium falciparum 6-phosphogluconolactonases is reported and discussed.
Asunto(s)
Hidrolasas de Éster Carboxílico/fisiología , Vía de Pentosa Fosfato , Glucosa-6-Fosfato/metabolismo , Humanos , Cinética , Espectroscopía de Resonancia Magnética , Oxidación-Reducción , Especificidad por SustratoRESUMEN
Cystic fibrosis is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). This protein belongs to the large ATP-binding cassette (ABC) family of transporters. Most patients with cystic fibrosis bear a mutation in the nucleotide-binding domain 1 (NBD1) of CFTR, which plays a key role in the activation of the channel function of CFTR. Determination of the three dimensional structure of NBD1 is essential to better understand its structure-function relationship, and relate it to the biological features of CFTR. In this paper, we report the first preparation of recombinant His-tagged NBD1, as a soluble, stable and isolated domain. The method avoids the use of renaturing processes or fusion constructs. ATPase activity assays show that the recombinant domain is functional. Using tryptophan intrinsic fluorescence, we point out that the local conformation, in the region of the most frequent mutation DeltaF508, could differ from that of the nucleotide-binding subunit of histidine permease, the only available ABC structure. We have undertaken three dimensional structure determination of NBD1, and the first two dimensional 15N-1H NMR spectra demonstrate that the domain is folded. The method should be applicable to the structural studies of NBD2 or of other NBDs from different ABC proteins of major biological interest, such as multidrug resistance protein 1 or multidrug resistance associated protein 1.