PubMed · 6197490
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K Hashimoto. 1984. Progress on cutaneous amyloidoses.. https://doi.org/10.1111/1523-1747.ep12258661
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The unfolding and refolding properties of human lysozyme and two amyloidogenic variants (Ile56Thr and Asp67His) have been studied by stopped-flow fluorescence and hydrogen exchange pulse labeling coupled with mass spectrometry. The unfolding of each protein in 5.4 M guanidine hydrochloride (GuHCl) is well described as a two-state process, but the rates of unfolding of the Ile56Thr variant and the Asp67His variant in 5.4 M GuHCl are ca. 30 and 160 times greater, respectively, than that of the wild type. The refolding of all three proteins in 0.54 M GuHCl at pH 5.0 proceeds through persistent intermediates, revealed by multistep kinetics in fluorescence experiments and by the detection of well-defined populations in quenched-flow hydrogen exchange experiments. These findings are consistent with a predominant mechanism for refolding of human lysozyme in which one of the structural domains (the alpha-domain) is formed in two distinct steps and is followed by the folding of the other domain (the beta-domain) prior to the assembly of the two domains to form the native structure. The refolding kinetics of the Asp67His variant are closely similar to those of the wild-type protein, consistent with the location of this mutation in an outer loop of the beta-domain which gains native structure only toward the end of the refolding process. By contrast, the Ile56Thr mutation is located at the base of the beta-domain and is involved in the domain interface. The refolding of the alpha-domain is unaffected by this substitution, but the latter has the effect of dramatically slowing the folding of the beta-domain and the final assembly of the native structure. These studies suggest that the amyloidogenic nature of the lysozyme variants arises from a decrease in the stability of the native fold relative to partially folded intermediates. The origin of this instability is different in the two variants, being caused in one case primarily by a reduction in the folding rate and in the other by an increase in the unfolding rate. In both cases this results in a low population of soluble partially folded species that can aggregate in a slow and controlled manner to form amyloid fibrils.
Amyloid aggregates have been recognized to be a pathological hallmark of several fatal diseases, including Alzheimer's disease, the prion-related diseases, and type II diabetes. Pancreatic amyloidosis is characterized by the deposition of amyloid consisting of islet amyloid polypeptide (IAPP). We followed the steps preceding IAPP insolubilization and amyloid formation in vitro using a variety of biochemical methods, including a filtration assay, far and near-UV circular dichroism (CD) spectropolarimetry, 1-anilino-8-naphthalenesulfonic acid (ANS) binding, and atomic force (AFM) and electron (EM) microscopy. IAPP insolubilization and amyloid formation followed kinetics that were consistent with the nucleation-dependent polymerization mechanism. Nucleation of IAPP amyloid formation with traces of preformed fibrils induced a rapid conformational transition into beta-sheets that subsequently aggregated into insoluble amyloid fibrils. Transition proceeded via a molten globule-like conformeric state with large contents of secondary structure, fluctuating tertiary and quaternary aromatic interactions, and strongly solvent-exposed hydrophobic patches. In the temperature denaturation pathway at 5 microM peptide, we found that this state was mostly populated at about 45 degrees C, and either aggregated rapidly into amyloid by prolonged exposure to this temperature, or melted into denaturated but still structured IAPP, when heated further to 65 degrees C. The state at 45 degrees C was also found to be populated at 4.25 M GdnHCl at 25 degrees C during GdnHCl-induced equilibrium denaturation, and was stable in solution for several hours before aggregating into amyloid fibrils. Our studies suggested that this amyloidogenic state was a self-associated form of an aggregation-prone, partially folded state of IAPP. We propose that this partially folded population and its self-associated forms are in a concentration-dependent equilibrium with a non-amyloidogenic IAPP conformer and may act as early, soluble precursors of beta-sheet and amyloid formation. Our findings on the molecular mechanism of IAPP amyloid formation in vitro should assist in gaining insight into the pathogenesis and inhibition of pancreatic amyloidosis and other amyloid-related diseases.
Environmental manipulations during early development can induce permanent alterations in hypothalamic-pituitary-adrenal (HPA) and behavioral responses to stressors. However, little is known about the impact of early life experiences on appetitive responses. The present investigation assessed the effects of brief handling/separation or protracted separation from the dams, on feeding and anxiety responses during development. During the first 3 weeks post-partum, Sprague-Dawley rat pups were exposed daily to either brief (15 min) handling/isolation (H), a more protracted (3 h) period of maternal separation (MS), or were not handled (NH). When tested on the elevated plus-maze (at 5-6 weeks) H groups displayed less anxiety than NH gender-matched controls. Surprisingly, so did the MS females. At weaning (Day 22), the MS rats weighed significantly less than both the H and NH animals; the difference between the H and MS was more robust and persisted throughout the experiment (D 62). The H animals of both genders, and the females of the MS group, consumed more of the palatable 'snack' than their NH counterparts. The feeding suppressant response to the various satiety peptides (bombesin, cholecystokinin, and amylin) was not affected by the early life experience, with exception of cholecystokinin (CCK) effects, which were more pronounced in H and MS males. These results suggest that early life events may contribute to anxiety and/or ingestive disorders such as anorexia nervosa, bulimia and obesity.