RESUMEN
Human interleukin-1beta (IL1beta) was used as a presentation scaffold for the characterization of the reactive site loop (RSL) of the serpin alpha1-antitrypsin (A1AT), the physiological inhibitor of leukocyte elastase. A chimeric protein was generated by replacement of residues 50-53 of IL1beta, corresponding to an exposed reverse turn in IL1beta, with the 10-residue P5-P5' sequence EAIPMSIPPE from A1AT. The chimera (antitrypsin-interleukin, AT-IL) inhibits elastase specifically and also binds the IL1beta receptor. Multinuclear NMR characterization of AT-IL established that, with the exception of the inserted sequence, the structure of the IL1beta scaffold is preserved in the chimera. The structure of the inserted RSL was analyzed relative to that of the isolated 10-residue RSL peptide, which was shown to be essentially disordered in solution. The chimeric RSL was also found to be solvent exposed and conformationally mobile in comparison with the IL1beta scaffold, and there was no evidence of persisting interactions with the scaffold outside of the N- and C-terminal linkages. However, AT-IL exhibits sigificant differences in chemical shift and NOE patterns relative to the isolated RSL that are consistent with local features of non-random structure. The proximity of these features to the P1-P1' residues suggests that they may be responsible for the inhibitory activity of the chimera.
Asunto(s)
Interleucina-1/química , Serpinas/química , Secuencia de Aminoácidos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación Proteica , Proteínas Recombinantes de Fusión/química , Soluciones , TemperaturaRESUMEN
Cellular toxicity resulting from nucleation-dependent polymerization of amyloid beta-peptide (Abeta) is considered to be a major and possibly the primary component of Alzheimer's disease (AD). Inhibition of Abeta polymerization has thus been identified as a target for the development of therapeutic agents for the treatment of AD. The intrinsic affinity of Abeta for itself suggested that Abeta-specific interactions could be adapted to the development of compounds that would bind to Abeta and prevent it from polymerizing. Abeta-derived peptides of fifteen residues were found to be inhibitory of Abeta polymerization. The activity of these peptides was subsequently enhanced through modification of their amino termini with specific organic reagents. Additional series of compounds prepared to probe structural requirements for activity allowed reduction of the size of the inhibitors and optimization of the Abeta-derived peptide portion to afford a lead compound, cholyl-Leu-Val-Phe-Phe-Ala-OH (PPI-368), with potent polymerization inhibitory activity but limited biochemical stability. The corresponding all-D-amino acyl analogue peptide acid (PPI-433) and amide (PPI-457) retained inhibitory activity and were both stable in monkey cerebrospinal fluid for 24 h.
Asunto(s)
Péptidos beta-Amiloides/antagonistas & inhibidores , Péptidos beta-Amiloides/química , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/química , Péptidos/síntesis química , Péptidos/farmacología , Secuencia de Aminoácidos , Péptidos beta-Amiloides/líquido cefalorraquídeo , Péptidos beta-Amiloides/farmacocinética , Animales , Barrera Hematoencefálica , Encéfalo/metabolismo , Inyecciones Intravenosas , Macaca mulatta , Masculino , Datos de Secuencia Molecular , Oxidación-Reducción , Fragmentos de Péptidos/líquido cefalorraquídeo , Fragmentos de Péptidos/farmacocinética , Polímeros/química , Ratas , Ratas Sprague-Dawley , Eliminación de Secuencia , Relación Estructura-Actividad , Distribución TisularRESUMEN
Native state 1H NMR resonance assignments for 125 of the 129 residues of equine lysozyme have enabled measurement of the hydrogen exchange kinetics for over 60 backbone amide and three tryptophan indole hydrogen atoms in the native state. Native holo equine lysozyme hydrogen exchange protection factors are as large as 10(6), the most protected residues being located in elements of secondary structure. High exchange protection in the domain interface correlates with the binding of Ca2+ in this region. Equine lysozyme differs from most non-Ca2+ binding lysozymes in forming a highly populated partially folded state at low pH. The protein in this A-state at pH 2.0 has been found to bind 1-anilino-naphthalene-8-sulphonate with the enhancement of fluorescent intensity and blue shift in the spectral maximum characteristic of molten globules. NMR spectra indicate that the A-state is globally much less ordered than native equine lysozyme but does not contain significant regions of random coil structure. The amides most protected against hydrogen exchange in the A-state (protection factors up to 10(2) at 5 degrees C) correspond to residues of three of the four alpha-helices of the native state; the side-chains of these residues form a hydrophobic cluster that includes five aromatic residues. Circular dichroism and tryptophan fluorescence indicate that these residues are substantially more constrained than similar residues in "classical" molten globules. Taken together, the data suggest a model for the A-state of equine lysozyme in which a more ordered core is surrounded by a less ordered but still compact polypeptide chain.