Methods to study the structure of misfolded protein states in systemic amyloidosis.

Systemic amyloidosis is defined as a protein misfolding disease in which the amyloid is not necessarily deposited within the same organ that produces the fibril precursor protein. There are different types of systemic amyloidosis, depending on the protein constructing the fibrils. This review will focus on recent advances made in the understanding of the structural basis of three major forms of systemic amyloidosis: systemic AA, AL and ATTR amyloidosis. The three diseases arise from the misfolding of serum amyloid A protein, immunoglobulin light chains or transthyretin. The presented advances in understanding were enabled by recent progress in the methodology available to study amyloid structures and protein misfolding, in particular concerning cryo-electron microscopy (cryo-EM) and nuclear magnetic resonance (NMR) spectroscopy. An important observation made with these techniques is that the structures of previously described in vitro formed amyloid fibrils did not correlate with the structures of amyloid fibrils extracted from diseased tissue, and that in vitro fibrils were typically more protease sensitive. It is thus possible that ex vivo fibrils were selected in vivo by their proteolytic stability.

Cryo-EM fibril structures from systemic AA amyloidosis reveal the species complementarity of pathological amyloids.

Systemic AA amyloidosis is a worldwide occurring protein misfolding disease of humans and animals. It arises from the formation of amyloid fibrils from the acute phase protein serum amyloid A. Here, we report the purification and electron cryo-microscopy analysis of amyloid fibrils from a mouse and a human patient with systemic AA amyloidosis. The obtained resolutions are 3.0 Å and 2.7 Å for the murine and human fibril, respectively. The two fibrils differ in fundamental properties, such as presence of right-hand or left-hand twisted cross-ß sheets and overall fold of the fibril proteins. Yet, both proteins adopt highly similar ß-arch conformations within the N-terminal ~21 residues. Our data demonstrate the importance of the fibril protein N-terminus for the stability of the analyzed amyloid fibril morphologies and suggest strategies of combating this disease by interfering with specific fibril polymorphs.

Proteomic analysis of highly prevalent amyloid A amyloidosis endemic to endangered island foxes.

Amyloid A (AA) amyloidosis is a debilitating, often fatal, systemic amyloid disease associated with chronic inflammation and persistently elevated serum amyloid A (SAA). Elevated SAA is necessary but not sufficient to cause disease and the risk factors for AA amyloidosis remain poorly understood. Here we identify an extraordinarily high prevalence of AA amyloidosis (34%) in a genetically isolated population of island foxes (Urocyon littoralis) with concurrent chronic inflammatory diseases. Amyloid deposits were most common in kidney (76%), spleen (58%), oral cavity (45%), and vasculature (44%) and were composed of unbranching, 10 nm in diameter fibrils. Peptide sequencing by mass spectrometry revealed that SAA peptides were dominant in amyloid-laden kidney, together with high levels of apolipoprotein E, apolipoprotein A-IV, fibrinogen-α chain, and complement C3 and C4 (false discovery rate ≤ 0.05). Reassembled peptide sequences showed island fox SAA as an 111 amino acid protein, most similar to dog and artic fox, with 5 unique amino acid variants among carnivores. SAA peptides extended to the last two C-terminal amino acids in 5 of 9 samples, indicating that near full length SAA was often present in amyloid aggregates. These studies define a remarkably prevalent AA amyloidosis in island foxes with widespread systemic amyloid deposition, a unique SAA sequence, and the co-occurrence of AA with apolipoproteins.

Transmission of systemic AA amyloidosis in animals.

Amyloidoses are a group of protein-misfolding disorders that are characterized by the deposition of amyloid fibrils in organs and/or tissues. In reactive amyloid A (AA) amyloidosis, serum AA (SAA) protein forms deposits in mice, domestic and wild animals, and humans that experience chronic inflammation. AA amyloid fibrils are abnormal ß-sheet-rich forms of the serum precursor SAA, with conformational changes that promote fibril formation. Extracellular deposition of amyloid fibrils causes disease in affected animals. Recent findings suggest that AA amyloidosis could be transmissible. Similar to the pathogenesis of transmissible prion diseases, amyloid fibrils induce a seeding-nucleation process that may lead to development of AA amyloidosis. We review studies of possible transmission in bovine, avian, mouse, and cheetah AA amyloidosis.

Atypical AA amyloid deposits in bovine AA amyloidosis.

In bovine amyloid protein A (AA) amyloidosis, amyloid deposits are typically observed in the kidney and spleen at necropsy. To determine the distribution of amyloid deposits in cows affected with AA amyloidosis, we examined organs known to be sites of amyloid deposits that are also processed for human consumption in 14 cows: 11 with typical clinical symptoms (typical amyloidosis) and three with no typical clinical symptoms (atypical amyloidosis). We found unusually high amounts of amyloid deposits in the tongue and other organs in all 14 cows regardless of the presence or absence of clinical amyloidosis symptoms. Cows with typical amyloidosis had heavier amyloid deposits in the spleen and renal glomeruli than cows with atypical amyloidosis. From clinical symptoms and histological examinations, we found that cows with typical and atypical amyloidosis can be classified into two groups, class I and class II, according to the presence or absence of heavy amyloid deposits in the spleen and renal glomeruli. However, no significant differences were observed between the amyloid fibrils of class I and class II amyloidosis by electron microscopy and Western blot analysis.

Identification of regions responsible for heparin-induced amyloidogenesis of human serum amyloid A using its fragment peptides.

Human serum amyloid A (SAA) is a precursor protein of amyloid fibrils. Although several studies have been performed, a detailed understanding of the molecular mechanism for SAA fibrillation remains elusive. Glycosaminoglycans such as heparin are suggested to serve as scaffolds in amyloid fibril formation in some cases. In the present study, amyloidogenic properties of synthetic fragment peptides corresponding to the N-terminal (residues 1-27), central (residues 43-63), and C-terminal (residues 77-104) regions of SAA molecule induced by heparin were examined using fluorescence, circular dichroism (CD), and electron microscopy. Fluorescence and CD measurements demonstrated that SAA (1-27) peptide is evidently involved in heparin-induced amyloidogenesis. Correspondingly, relatively minor changes in fluorescence and a quite different pattern in the CD spectrum were observed in SAA (43-63) peptide. In contrast, SAA (77-104) peptide did not show any changes induced by heparin. Transmission electron microscopy indicated that SAA (1-27) peptide forms short and straight fibrils, whereas SAA (43-63) peptide forms much longer and seemingly elastic fibrils. These results suggest that the N-terminal region plays a crucial role as a rigid core and the central region facilitates the elongation of fibrils in heparin-induced amyloidogenesis of SAA molecule.

In vitro polymerization of a functional Escherichia coli amyloid protein.

Amyloid formation is characterized by the conversion of soluble proteins into biochemically and structurally distinct fibers. Although amyloid formation is traditionally associated with diseases such as Alzheimer disease, a number of biologically functional amyloids have recently been described. Curli are amyloid fibers produced by Escherichia coli that contribute to biofilm formation and other important physiological processes. We characterized the polymerization properties of the major curli subunit protein CsgA. CsgA polymerizes into an amyloid fiber in a sigmoidal kinetic fashion with a distinct lag, growth, and stationary phase. Adding sonicated preformed CsgA fibers to the polymerization reaction can significantly shorten the duration of the lag phase. We also demonstrate that the conversion of soluble CsgA into an insoluble fiber involves the transient formation of an intermediate similar to that characterized for several disease-associated amyloids. The CsgA core amyloid domain can be divided into five repeating units that share sequence and structural hallmarks. We show that peptides representing three of these repeating units are amyloidogenic in vitro. Although the defining characteristics of CsgA polymerization appear conserved with disease-associated amyloids, these proteins evolved in diverse systems and for different purposes. Therefore, amyloidogenesis appears to be an innate protein folding pathway that can be capitalized on to fulfill normal physiological tasks.

Direct observation of Abeta amyloid fibril growth and inhibition.

Amyloid fibril formation is a phenomenon common to many proteins and peptides, including amyloid beta (Abeta) peptide associated with Alzheimer's disease. To clarify the mechanism of fibril formation and to create inhibitors, real-time monitoring of fibril growth is essential. Here, seed-dependent amyloid fibril growth of Abeta(1-40) was visualized in real-time at the single fibril level using total internal reflection fluorescence microscopy (TIRFM) combined with the binding of thioflavin T, an amyloid-specific fluorescence dye. The clear image and remarkable length of the fibrils enabled an exact analysis of the rate of growth of individual fibrils, indicating that the fibril growth was a highly cooperative process extending the fibril ends at a constant rate. It has been known that Abeta amyloid formation is a stereospecific reaction and the stability is affected by l/d-amino acid replacement. Focusing on these aspects, we designed several analogues of Abeta(25-35), a cytotoxic fragment of Abeta(1-40), consisting of l and d-amino acid residues, and examined their inhibitory effects by TIRFM. Some chimeric Abeta(25-35) peptides inhibited the fibril growth of Abeta(25-35) strongly, although they could not inhibit the growth of Abeta(1-40). The results suggest that a more rational design of stereospecific inhibitors, combined with real-time monitoring of fibril growth, will be useful to invent a potent inhibitor preventing the amyloid fibril growth of Abeta(1-40) and other proteins.

AA protein in experimental murine AA amyloid fibrils: a high resolution ultrastructural and immunohistochemical study comparing aldehyde-fixed and cryofixed tissues.

In a previous study, the fibrils of experimental murine AA amyloid were found to be microfibril-like structures with the AA protein (in the form of 1 nm wide flexible filaments) on their exterior surface. In this study, we have re-examined the AA amyloid fibrils with advanced methods of cryofixation and freeze substitution which are known to retain ultrastructural detail as close as possible to the living state. The observations were compared to those obtained with conventional methods of aldehyde fixation. Cryofixation and freeze substitution confirmed the microfibril-like nature of the inner part of the AA amyloid fibril. The AA protein was present on the exterior surface in the form of 3 nm wide 'helical rods' formed by the tight coiling of the 1 nm wide AA protein flaments. The 'helical rods' were arranged parallel to the axis of the fibril and to one another with a uniform center-to-center distance of 5 nm. This arrangement was fully preserved in amyloid fibrils after cryofixation and freeze substitution, but was present in only some areas of formaldehyde fixed mouse spleen AA amyloid This conformation and orientation of AA protein is likely to be that in its native state, given the ability of these advanced methods of biological preservation to retain structures close to that of the living state. This information shoulld be of considerable value in comparing the structure of amyloid fibrils observed in situ with those isolated from tissue or generated in vitro.

The protofilament substructure of amyloid fibrils.

Tissue deposition of normally soluble proteins, or their fragments, as insoluble amyloid fibrils causes the usually fatal, acquired and hereditary systemic amyloidoses and is associated with the pathology of Alzheimer's disease, type 2 diabetes and the transmissible spongiform encephalopathies. Although each type of amyloidosis is characterised by a specific amyloid fibril protein, the deposits share pathognomonic histochemical properties and the structural morphology of all amyloid fibrils is very similar. We have previously demonstrated that transthyretin amyloid fibrils contain four constituent protofilaments packed in a square array. Here, we have used cross-correlation techniques to average electron microscopy images of multiple cross-sections in order to reconstruct the sub-structure of ex vivo amyloid fibrils composed of amyloid A protein, monoclonal immunoglobulin lambda light chain, Leu60Arg variant apolipoprotein AI, and Asp67His variant lysozyme, as well as synthetic fibrils derived from a ten-residue peptide corresponding to the A-strand of transthyretin. All the fibrils had an electron-lucent core but the packing arrangement comprised five or six protofilaments rather than four. The structural similarity that defines amyloid fibres thus exists principally at the level of beta-sheet folding of the polypeptides within the protofilament, while the different types vary in the supramolecular assembly of their protofilaments.

Amyloid goiter: report of two cases and review of the literature.

OBJECTIVE: To describe two recent cases of amyloid goiter that occurred in two patients with a history of diffuse enlargement of the thyroid gland, progressing over several years' time. METHODS: We reviewed the medical histories as well as the histologic, immunohistochemical, and electron microscopic findings in these patients. RESULTS: Assessment of the clinical records of a 59-year-old man and a 64-year-old woman revealed histories that included chronic psoriasiform arthritis and asthma in conjunction with chronic obstructive pneumonia and bronchiectasis. In both cases, total thyroidectomy was performed. On histologic examination of both thyroid glands, the appearance was characterized by moderate to severe distortion of the normal thyroid architecture by amyloid in a perifollicular distribution and focally abundant interfollicular adipose tissue. In both cases, the amyloid stained intensely positive with Congo red, which bleached after treatment with potassium permanganate. Immunohistochemical staining patterns were consistent with AA amyloid, and electron microscopy showed nonbranching 9-nm fibrils consistent with amyloid. CONCLUSION: The diagnosis of amyloid goiter should be suspected in patients with a diffusely enlarging thyroid gland and an appropriate clinical history.

Transgenic mouse model of AA amyloidosis.

AA amyloidosis can be induced in mice experimentally through injection of certain chemical or biological compounds. However, the usefulness of this approach is limited by its dependence on exogenous inflammatory agents that stimulate cytokines to increase the synthesis of precursor serum amyloid A (SAA) protein and the transitory nature of the pathological fibrillar deposits. We now report that transgenic mice carrying the human interleukin 6 gene under the control of the metallothionein-I promoter had markedly increased concentrations of SAA and developed amyloid in the spleen, liver, and kidneys by 3 months of age. At the time of death about 6 months later, organs obtained from these animals had extensive amyloid deposits. This disease process was apparent radiographically using small-animal computer axial tomography and magnetic resonance imaging equipment. The AA nature of the amyloid was evidenced immunohistochemically and was unequivocally established by sequence analysis of protein extracted from the fibrils. The availability of this unique in vivo experimental model of AA amyloidosis provides the means to assess the therapeutic efficacy of agents designed to reduce or prevent the fibrillar deposits found in AA and other types of amyloid-associated disease.

A high resolution ultrastructural comparison of isolated and in situ murine AA amyloid fibrils.

There is an inconsistency between the ultrastructural organization of AA amyloid fibrils that have been isolated, which are composed of a slowly twisting set of two or more protofibrils, and those seen in situ, which are tubular entities with a tight helical substructure. In this study, the ultrastructure of fibrils isolated from experimental murine AA amyloid were observed at high resolution and compared with those seen in situ in the hope of clarifying the reason for this inconsistency. The fibrils in situ were composed of a microfibril-like 8-9 nm wide core covered by a layer of heparan sulfate proteoglycan (HSPG) to which 1 nm wide filaments, immunohistochemically identified as AA protein, were externally associated. Following isolation with the standard distilled water washing procedure, the HSPG layer and AA protein filaments detached from their core and dispersed into the water. The remaining denuded, variously loosened cores lost their typical appearance. In distilled water the detached 1 nm wide AA protein filaments became quite conspicuous and coiled themselves into 3 nm wide tight helices which in turn assembled into the characteristic slowly twisting sets of two parallel protofibrils similar to that previously reported as "isolated amyloid fibrils". The results emphasize that great caution must be taken in extrapolating amyloid fibril structure from isolated preparations to in situ tissue conditions.

Adenoviral expression of murine serum amyloid A proteins to study amyloid fibrillogenesis.

Serum amyloid A (SAA) proteins are one of the most inducible acute-phase reactants and are precursors of secondary amyloidosis. In the mouse, SAA1 and SAA2 are induced in approximately equal quantities in response to amyloid induction models. These two isotypes differ in only 9 of 103 amino acid residues; however, only SAA2 is selectively deposited into amyloid fibrils. SAA expression in the CE/J mouse species is an exception in that gene duplication did not occur and the CE/J variant is a hybrid molecule sharing features of SAA1 and SAA2. However, even though it is more closely related to SAA2 it is not deposited as amyloid fibrils. We have developed an adenoviral vector system to overexpress SAA proteins in cell culture to determine the ability of these proteins to form amyloid fibrils, and to study the structural features in relation to amyloid formation. Both the SAA2 and CE/J SAA proteins were synthesized in large quantities and purified to homogeneity. Electron microscopic analysis of the SAA proteins revealed that the SAA2 protein was capable of forming amyloid fibrils, whereas the CE/J SAA was incapable. Radiolabelled SAAs were associated with normal or acute-phase high-density lipoproteins (HDLs); we examined them for their clearance from the circulation. In normal mice, SAA2 had a half-life of 70 min and CE/J SAA had a half-life of 120 min; however, in amyloid mice 50% of the SAA2 cleared in 55 min, compared with 135 min for the CE/J protein. When the SAA proteins were associated with acute-phase HDLs, SAA2 clearance was decreased to 60 min in normal mice compared with 30 min in amyloidogenic mice. Both normal and acute-phase HDLs were capable of depositing SAA2 into preformed amyloid fibrils, whereas the CE/J protein did not become associated with amyloid fibrils. This established approach opens the doors for large-scale SAA production and for the examination of specific amino acids involved in the fibrillogenic capability of the SAA2 molecule in vitro and in vivo.

An ultrastructural study of the colocalization of biglycan and decorin with AA amyloid fibrils in human renal glomeruli.

An investigation was undertaken on paraformaldehyde-fixed, Lowicryl resin-embedded renal biopsies from patients with AA amyloidosis to study the association of two small chondroitin sulphate/dermatan sulphate proteoglycans, decorin and biglycan, with amyloid fibrils using an ultrastructural immunogold technique. Biglycan was present in glomerular endothelial cells in both normal kidney and in amyloidosis, but little biglycan or decorin was present in the normal mesangial matrix. By contrast, conspicuous amounts of both biglycan and decorin were seen to be associated with amyloid fibrils in the glomerular matrix in cases of renal AA amyloidosis. The results further emphasise the close association between amyloid and extracellular matrix components which are now considered to be an integral part of the amyloid fibrils.

In vitro amyloid fibril formation by synthetic peptides corresponding to the amino terminus of apoSAA isoforms from amyloid-susceptible and amyloid-resistant mice.

Specific proteins of the apolipoprotein serum amyloid (apoSAA) family that are synthesized in large quantities during the acute, early phase of inflammation can serve as the proteinaceous precursors for amyloid fibrils. To model fibrillogenesis in such inflammatory diseases, we have used electron microscopy and X-ray diffraction to examine the structures formed by synthetic peptides corresponding in sequence to the 11 amino-terminal amino acids of murine apoSAA1, apoSAAcej, and apoSAA2 and to the 15 amino-terminal amino acids of apoSAA2. This region is reported to be the major fibrillogenic determinant of apoSAA isoforms. Both in 1 mM Tris buffer and in 35% acetonitrile, 0.1% trifluoracetic acid (ACN/TFA), all of the peptides formed macromolecular assemblies consisting of twisted, approximately 40- to 60-A-thick ribbons, which varied in width from around 40-70 A (for 11-mer apoSAA2 in Tris) up to 900 A (for the other peptides). X-ray diffraction patterns recorded from lyophilized peptides, vapor-hydrated samples, and solubilized/dried samples showed hydrogen bonding and intersheet reflections typical of a beta-pleated sheet conformation. The coherent lengths measured from the breadths of the X-ray reflections indicated that with hydration the growth of the assemblies in the intersheet stacking direction was comparable to that in the hydrogen-bonding direction, and analysis of oriented samples showed that the beta-strands were oriented perpendicular to both the long axis and the face of the assemblies. These X-ray results are consistent with the ribbon- or plate-like morphology of the individual aggregates and emphasize the polymorphic nature of amyloidogenic peptides. Our findings demonstrate that X-ray diffraction measurements on vapor-hydrated or solubilized/dried versus lyophilized, amyloidogenic peptides are a good indicator of their fibrillogenic potential. For example, from the highest to the lowest potential, the peptides examined here were ranked as: Abeta1-28 > Abeta1-40 > apoSAA1 approximately apoSAAcej > apoSAA2 > Abeta17-42. Experiments in which the three different 11-mer apoSAA isoforms were solubilized in ACN/TFA and then combined as binary mixtures showed that the ribbon morphology was not affected but that the extent of hydrogen bonding in the assemblies was substantially reduced. Our observations on the in vitro assembly of apoSAA analogs emphasize that amyloid fibril formation and morphology depend on primary sequence, length of polypeptide chain, the presence of additional fibrillogenic polypeptides, and solvent conditions.

Effect of poly(vinylsulfonate) on murine AA amyloid: a high-resolution ultrastructural study.

In experimental murine inflammation-associated amyloidosis (AA amyloidosis), an interaction between heparan sulfate and serum amyloid A (SAA), the AA precursor, has been demonstrated and is believed to play an important role in AA amyloidogenesis. Poly(vinylsulfonate) sodium salt (PVS) can arrest AA amyloid induction and cause established amyloid deposits to regress. PVS is thought to have this property by virtue of limited anionic structural similarities it has to heparan sulfate. In the present study, a comparison has been made of the in situ light microscopic and high-resolution ultrastructure of amyloid deposits before and after PVS treatment. As shown recently in situ, AA fibrils from untreated mice are composed of an outer layer of heparan sulfate proteoglycan and a 1- to 2-nm filament network of AA protein. This layer encloses a microfibril-like structure composed of chondroitin sulfate proteoglycan wound around a core of amyloid P component. After treatment with PVS, both the heparan sulfate proteoglycan and the AA filament network are lost from the fibrils, and the more central portion disintegrates into the chondroitin sulfate proteoglycan with associated amyloid P subunits. These findings add further support to the concept that heparan sulfate proteoglycan is important in amyloid fibril structure, and interference with its binding interactions with the amyloid filament protein provides a point of therapeutic attack.

A high resolution ultrastructural study of experimental murine AA amyloid.

An essential and distinguishing feature of all amyloids is the presence of fibrillar structures of approximately 10-nm width. The precise nature of the fibril is not yet clearly understood, particularly in situ, and the ultrastructure of isolated fibrils differs significantly from that of fibrils observed in situ. The fibrils are generally believed to be composed of a protein specific to each type of amyloid, but increasing evidence suggests additional associations with other components such as heparan sulfate proteoglycan (HSPG) and amyloid P component (AP). Experimental AA amyloidosis was induced in mice by amyloid enhancing factor and an inflammatory stimulus (subcutaneous AgNO3); fibrils were thereafter examined in detail. Particular attention was paid to ultrastructural characteristics known to represent particular molecular components of basement membranes such as HSPG and AP. Additionally, rabbit anti-mouse AA antisera was used with 5-nm and 1-nm gold particles to establish the location of the AA protein in-situ. Amyloid fibrils could be identified in their mature form as well as at apparent intermediate stages of formation. The fibril contained an apparent core which is composed of an assembly of 3.5-nm wide pentosomal particles having the characteristics of AP. Wound around the AP assembly in a helical fashion is a "double tracked" ribbon-like entity, 3 nm wide, having the morphologic characteristics of chondroitin sulfate proteoglycan (CSPG). Covering the surface of this structure is a second ribbon-like double track structure, but this one is wider (4.6 nm vs 3.0 nm) than the CSPG. These have the ultrastructural characteristics of HSPG. Routine fixation and tissue preparation techniques that usually remove HSPG from microfibrils did not do so with amyloid fibrils, suggesting an alteration in affinity between these components. The AA protein could be identified as a 1 - to 2-nm filament network on the most exterior surface of the fibril. The ultrastructure of AA amyloid fibrils in situ resembles that of connective tissue microfibrils, and, in addition to AA protein, is likely composed of HSPG, CSPG, and AP. Amyloid fibrils can be distinguished from microfibrils by the apparently stronger binding of HSPG to the surface of the amyloid fibril and the presence of the AA filaments. A model of the in situ organization of AA amyloid fibrils is proposed.

Arresting amyloidosis in vivo using small-molecule anionic sulphonates or sulphates: implications for Alzheimer's disease.

Amyloid is a term for extracellular protein fibril deposits that have characteristic tinctorial and structural properties. Heparan sulphate, or the heparan sulphate proteoglycan perlecan, has been identified in all amyloids and implicated in the earliest stages of inflammation-associated (AA) amyloid induction. Heparan sulphate interacts with the AA amyloid precursor and the beta-peptide of Alzheimer's amyloid, imparting characteristic secondary and tertiary amyloid structural features. These observations suggest that molecules that interfere with this interaction may prevent or arrest amyloidogenesis. We synthesized low-molecular-weight (135-1,000) anionic sulphonate or sulphate compounds. When administered orally, these compounds substantially reduced murine splenic AA amyloid progression. They also interfered with heparan sulphate-stimulated beta-peptide fibril aggregation in vitro.