PubMed · 12374886
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Giovanni Palladini, Laura Obici, Giampaolo Merlini. 2002-10-10. Hereditary amyloidosis.. https://doi.org/10.1056/nejm200210103471517
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The formation of amyloid fibrils by the human islet amyloid polypeptide is associated with type II diabetes. While it was previously suggested that the formed fibrils are toxic to pancreatic beta-cells due to membrane permeation activity, more recent studies suggested that protofibrillar assemblies have significantly higher potency in permeating lipid bilayers. Here, we specifically studied the membrane interaction activity of soluble and insoluble islet amyloid polypeptide assemblies at high temporal resolution. A colorimetric analysis using lipid/polydiacetylene (PDA) biomimetic vesicles clearly demonstrated the transient formation of soluble assemblies that strongly interact with the lipid vesicles. A peak in the level of membrane binding of the soluble fraction, as reflected by the colorimetric assay, was observed after incubation for approximately 1 h, followed by a decrease in the level of membrane interaction of the assemblies. The transient nature of the membrane-active assemblies was independently confirmed by a fluorescence quenching assay. Ultrastructural analysis using transmission electron microscopy provided morphological evidence of prefibrillar assemblies, supported the transient existence of membrane interacting soluble species, and facilitated observation of the non-membrane-active filaments in the solution. Taken together, our results provide experimental evidence for the formation of transient soluble prefibrillar assemblies which are highly membrane-active. The implications of these observations are discussed in light of designed fibrillization inhibitors.
Components of caveolae and lipid rafts are characterized by their buoyancy after detergent extraction. Using flotations in density gradients, we now show that non-raft membrane molecules are also associated with detergent-insoluble, buoyant assemblies. When Triton X-100 cellular extracts were spun to equilibrium in Nycodenz, only components of classical rafts floated. In contrast, with the zwitterionic detergent CHAPS, non-raft residents such as calnexin and APP also buoyed. When CHAPS extracts were spun in non-equilibrium (velocity) conditions, some raft components rapidly exited the input fractions while other raft markers and non-raft molecules remained relatively immobile. This pointed to size heterogeneities of CHAPS-insoluble complexes. Combined velocity/equilibrium gradients broadly divided CHAPS-insoluble membrane complexes into three size categories, which all contained cholesterol and the glycosphingolipid GM1. Large complexes were enriched in caveolin and ESA. Medium size complexes were enriched in PrP, whereas small complexes contained non-raft proteins, PrP, and some ESA. While Alzheimer's APP was primarily confined to small assemblies, a portion of its glycosylated form did buoy with large complexes. Large CHAPS-insoluble complexes resemble, but are not equal to, classical rafts. These findings extend considerably the range of detergent-insoluble membranal domains.
Beta2-microglobulin (beta2-m), a typical immunoglobulin domain made of seven beta-strands, is a major component of amyloid fibrils formed in dialysis-related amyloidosis. To understand the mechanism of amyloid fibril formation in the context of full-length protein, we prepared various mutants in which proline (Pro) was introduced to each of the seven beta-strands of beta2-m. The mutations affected the amyloidogenic potential of beta2-m to various degrees. In particular, the L23P, H51P, and V82P mutations significantly retarded fibril extension at pH 2.5. Among these, only L23P is included in the known "minimal" peptide sequence, which can form amyloid fibrils when isolated as a short peptide. This indicates that the residues in regions other than the minimal sequence, such as H51P and V82P, determine the amyloidogenic potential in the full-length protein. To further clarify the mutational effects, we measured their stability against guanidine hydrochloride of the native state at pH 8.0 and the amyloid fibrils at pH 2.5. The amyloidogenicity of mutants showed a significant correlation with the stability of the amyloid fibrils, and little correlation was observed with that of the native state. It has been proposed that the stability of the native state and the unfolding rate to the amyloidogenic precursor as well as the conformational preference of the denatured state determine the amyloidogenicity of the proteins. The present results reveal that, in addition, stability of the amyloid fibrils is a key factor determining the amyloidogenic potential of the proteins.