The chemical environment of the extracellular matrix may influence the tissue-selective deposition observed there in gelsolin amyloid disease. Previously, we have identified the proteases that generate the amyloidogenic fragments from the full length gelsolin variants, and demonstrated that heparin is capable of accelerating gelsolin amyloidogenesis. Herein, we identify the structural features of heparin that promote the 8 kDa disease-associated gelsolin fragments (residues 173-243) generated at the cell surface to form amyloid. In conjunction with electron microscopy analyses, our kinetic studies demonstrate that heparin efficiently accelerates the formation of gelsolin amyloid by enabling intermolecular β-sheet formation. The use of heparin analogues reveal that sulfation is important in accelerating amyloidogenesis and that the extent of acceleration is proportional to the molecular weight of heparin. In addition, heparin accelerated aggregation at both early and late stages of amyloidogenesis. Dynamic light scattering coupled to size exclusion chromatography showed that heparin promotes the formation of soluble aggregates. Collectively, these data reveal that heparin templates fibril formation and affords solubility to the aggregating peptides through its sulfated structure. By extension, the biochemical results herein suggest that tissue-selective deposition characteristic of the gelsolin amyloidoses are likely influenced by the extracellular localization of distinct glycosaminoglycans.Amyloid diseases are characterized by the extracellular deposition of a given protein into a variety of structures, including cross β-sheet-rich fibrillar aggregates called amyloid. More than twenty non-homologous human proteins become amyloidogenic later in life. The clinically most important example is the amyloid β-peptide (Aβ) 1 , the deposits of which are associated with Alzheimer's disease. Many amyloid diseases exhibit distinct tissue-selective deposition of a given amyloidenic protein-the exact tissue targeted being dependent on the † We thank the Skaggs Institute of Chemical Biology, the Lita Annenberg Hazen Foundation and the National Institutes of Health (grants: AG18917, HL62244, HL52622 and GM038060) for financial support. * Corresponding author: tel: +1-858-784-9880; fax: +1-858-784-9899; email: jkelly@scripps.edu. Supporting Information Available: (1) Pre-treated D187N gelsolin173-243 fragments were analyzed to confirm a monomeric starting structure and the analytical ultracentrifugation, atomic force microscopy analyses of these samples are shown. (2) WT and D187Y gelsolin173-243 fragments were subjected to amyloid formation plate-reader assays as controls. The amyloid formation profiles of WT gelsolin173-243 are shown in the presence of various GAGs and heparin derivatives. The D187Y gelsolin173-243 only aggregated at acidic (pH 5) conditions. (3) Dextran sulfate and polyglutamic acid did not accelerate D187N gelsolin173-243 amyloidogenesis in the TfT fluorescence assays shown. (4) Full, unadjusted TE...

Familial amyloidosis of Finnish type (FAF) is a systemic amyloid disease associated with the deposition of proteolytic fragments of mutant (D187N/Y) plasma gelsolin. We report a mouse model of FAF featuring a muscle-specific promoter to drive D187N gelsolin synthesis. This model recapitulates the aberrant endoproteolytic cascade and the aging-associated extracellular amyloid deposition of FAF. Amyloidogenesis is observed only in tissues synthesizing human D187N gelsolin, despite the presence of full-length D187N gelsolin and its 68-kDa cleavage product in blood-demonstrating the importance of local synthesis in FAF. Loss of muscle strength was progressive in homozygous D187N gelsolin mice. The presence of misfolding-prone D187N gelsolin appears to exacerbate the age-associated decline in cellular protein homeostasis (proteostasis), reflected by the intracellular deposition of numerous proteins, a characteristic of the most common degenerative muscle disease of aging humans, sporadic inclusion body myositis.amyloid ͉ proteostasis ͉ sporadic inclusion body myositis ͉ FAF mouse P rotein homeostasis (proteostasis) refers to the cellular control of the synthesis, structure, trafficking, and degradation of proteins (1). An aging-associated decline in proteostasis capacity may contribute to the onset of many diseases associated with protein misfolding (2-4). Inheriting an aggregation-prone protein(s) further challenges proteostasis capacity upon aging (5). Such challenges can lead to loss-of-function disorders or aggregation-associated degenerative diseases (1-3, 5-9). Whether intracellular and/or extracellular aggregation leads to proteotoxicity in diseases associated with extracellular amyloid, such as Alzheimer's disease, remains unclear (3, 10).Familial amyloidosis of Finnish type (FAF) or gelsolin amyloidosis is thought to result from amyloidogenesis of a fragment of gelsolin causing aging-associated proteotoxicity (1,(11)(12)(13)(14). Plasma gelsolin is a 83-kDa 6-domain Ca 2ϩ -binding protein that is secreted from several cell types into the blood (Ͼ200 g/mL) (15), with muscle contributing significantly (16). A D187 mutation to N or Y (D187N/Y) in plasma gelsolin compromises Ca 2ϩ binding in domain 2 and thus folding (13). This enables aberrant cleavage by furin in the trans-Golgi, generating a 68-kDa fragment (C68) of gelsolin that is secreted (11, 17) (Fig. 1A). Only a fraction of D187N/Y plasma gelsolin in FAF is cleaved by furin; the remainder is secreted as full-length, functional plasma gelsolin. C68 can be further cleaved in the extracellular space by a type I matrix metalloprotease, like MT1-MMP (12), to generate the major (8-kDa) and minor (5-kDa) amyloidogenic fragments that deposit in blood vessel walls, skin, the autonomic nervous system, and the eye of humans, causing cranial and peripheral polyneuropathy, cutis laxa, and corneal lattice dystrophy (18). Deposition also occurs in skeletal and cardiac muscle, leading to cardiomyopathy and muscle weakness (14, 18). Given that human D187N/Y plasma gelso...

Amyloid diseases like Alzheimer's disease and familial amyloidosis of Finnish type (FAF) stem from endoproteolytic cleavage of a precursor protein to generate amyloidogenic peptides that accumulate as amyloid deposits in a tissue-specific manner. FAF patients deposit both 8 and 5 kDa peptides derived from mutant (D187Y/N) plasma gelsolin in the extracellular matrix (ECM). The first of two aberrant sequential proteolytic events is executed by furin to yield a 68 kDa (C68) secreted fragment. We now identify the metalloprotease MT1-matrix metalloprotease (MMP), an integral membrane protein active in the ECM, as a protease that processes C68 to the amyloidogenic peptides. We further demonstrate that ECM components are capable of accelerating gelsolin amyloidogenesis. Proteolysis by MT1-MMP-like proteases proximal to the unique chemical environment of the ECM offers an explanation for the tissue-specific deposition observed in FAF and provides critical insight into new therapeutic strategies.

The largest group of disease-causing mutations affecting calcium-binding epidermal growth factor-like (cbEGF) domain function in a wide variety of extracellular and transmembrane proteins is that which results in cysteine substitutions. Although known to introduce proteolytic susceptibility, the detailed structural consequences of cysteine substitutions in cbEGF domains are unknown. Here, we studied pathogenic mutations C1977Y and C1977R, which affect cbEGF30 of human fibrillin-1, in a recombinant three cbEGF domain fragment (cbEGF29 -31). Limited proteolysis, 1 H NMR, and calcium chelation studies have been used to probe the effect of each substitution on cbEGF30 and its flanking domains. Analysis of the wild-type fragment identified two high affinity and one low affinity calcium-binding sites. Each substitution caused the loss of high affinity calcium binding to cbEGF30, consistent with intradomain misfolding, but the calcium binding properties of cbEGF29 and cbEGF31 were surprisingly unaffected. Further analysis of mutant fragments showed that domain packing of cbEGF29 -30, but not cbEGF30 -31, was disrupted. These data demonstrate that C1977Y and C1977R have localized structural effects, confined to the N-terminal end of the mutant domain, which disrupt domain packing. Cysteine substitutions affecting other cbEGF disulfide bonds are likely to have different effects. This proposed structural heterogeneity may underlie the observed differences in stability and cellular trafficking of proteins containing such changes.