Identification of a prealbumin variant in the serum of a Japanese patient with familial amyloidotic polyneuropathy

Serum prealbumin isolated from a Japanese patient with familial amyloidotic polyneuropathy (FAP) has been found to consist of a mixture of normal prealbumin and a prealbumin variant which contains a methionine for valine substitution at position 30. The prealbumin variant in the serum is identical to the prealbumin variant derived from amyloid fibrils of a Japanese FAP patient. FAP likely results from the deposition of abnormal serum prealbumin in various organs as amyloid fibrils.     

Immunocytochemical detection of the nonfibrillar stage of amyloid formation in the mouse myocardium

The indirect electron microscope immunoperoxidase method utilizing pure rabbit antibodies to protein of mouse amyloid fibrils was used for studying casein-induced amyloidosis in mice. At the early stages of amyloidosis there appeared deposits of fine granular material in the mouse myocardium. These deposits contained an antigen similar to the amyloid fibrils antigen, but the deposits had no fibrillar ultrastructure. The results testify to the presence of early nonfibrillar stage of amyloid formation.     

Techniques to study amyloid fibril formation in vitro

Amyloid fibrils are ordered aggregates of peptides or proteins that are fibrillar in structure and contribute to the complications of many diseases (e.g., type 2 diabetes mellitus, Alzheimer's disease, and primary systemic amyloidosis). These fibrils can also be prepared in vitro and there are three criteria that define a protein aggregate as an amyloid fibril: green birefringence upon staining with Congo Red, fibrillar morphology, and beta-sheet secondary structure. The purpose of this review is to describe the techniques used to study amyloid fibril formation in vitro, address common errors in the collection and interpretation of data, and open a discussion for a critical review of the criteria currently used to classify a protein aggregate as an amyloid fibril.     

Production and characterization of RNA aptamers specific for amyloid fibril epitopes

One of the most fascinating features of amyloid fibrils is their generic cross-beta architecture that can be formed from many different and completely unrelated proteins. Nonetheless, amyloid fibrils with diverse structural and phenotypic properties can form, both in vivo and in vitro, from the same protein sequence. Here, we have exploited the power of RNA selection techniques to isolate small, structured, single-stranded RNA molecules known as aptamers that were targeted specifically to amyloid-like fibrils formed in vitro from beta(2)-microglobulin (beta(2)m), the amyloid fibril protein associated with dialysis-related amyloidosis. The aptamers bind with high affinity (apparent K(D) approximately nm) to beta(2)m fibrils with diverse morphologies generated under different conditions in vitro, as well as to amyloid fibrils isolated from tissues of dialysis-related amyloidosis patients, demonstrating that they can detect conserved epitopes between different fibrillar species of beta(2)m. Interestingly, the aptamers also recognize some other, but not all, amyloid fibrils generated in vitro or isolated from ex vivo sources. Based on these observations, we have shown that although amyloid fibrils share many common structural properties, they also have features that are unique to individual fibril types.     

Pathogenesis, diagnosis and treatment of systemic amyloidosis

Amyloidosis is a disorder of protein folding in which normally soluble proteins are deposited as abnormal, insoluble fibrils that disrupt tissue structure and cause disease. Although about 20 different unrelated proteins can form amyloid fibrils in vivo, all such fibrils share a common cross-beta core structure. Some natural wild-type proteins are inherently amyloidogenic, form fibrils and cause amyloidosis in old age or if present for long periods at abnormally high concentration. Other amyloidogenic proteins are acquired or inherited variants, containing amino-acid substitutions that render them unstable so that they populate partly unfolded states under physiological conditions, and these intermediates then aggregate in the stable amyloid fold. In addition to the fibrils, amyloid deposits always contain the non-fibrillar pentraxin plasma protein, serum amyloid P component (SAP), because it undergoes specific calcium-dependent binding to amyloid fibrils. SAP contributes to amyloidogenesis, probably by stabilizing amyloid fibrils and retarding their clearance. Radiolabelled SAP is an extremely useful, safe, specific, non-invasive, quantitative tracer for scintigraphic imaging of systemic amyloid deposits. Its use has demonstrated that elimination of the supply of amyloid fibril precursor proteins leads to regression of amyloid deposits with clinical benefit. Current treatment of amyloidosis comprises careful maintenance of impaired organ function, replacement of end-stage organ failure by dialysis or transplantation, and vigorous efforts to control underlying conditions responsible for production of fibril precursors. New approaches under development include drugs for stabilization of the native fold of precursor proteins, inhibition of fibrillogenesis, reversion of the amyloid to the native fold, and dissociation of SAP to accelerate amyloid fibril clearance in vivo.     

Fluorometric determination of amyloid fibrils in vitro using the fluorescent dye, thioflavin T1

We used a fluorometric method to examine amyloid fibrils, in vitro. These fibrils in the case of both murine senile and secondary amyloidosis were purified to apparent homogeneity from the water-suspended fraction of the liver of senescence-accelerated mouse, using sucrose density ultracentrifugation, and then the following assays were performed. In the absence of amyloid fibrils, thioflavine T fluoresced faintly at the excitation and emission maxima of 350 and 438 nm, respectively. In the presence of amyloid fibrils, thioflavine T fluoresced brightly at the excitation and emission maxima of 450 and 482 nm, respectively, and the fluorescence change was linear from 0 to 2.0 micrograms/ml amyloid fibrils. This fluorescence was maximal around pH 9.0. Fluorescence intensity in the presence of a constant amount of amyloid fibrils reached a plateau with increase in the thioflavine T concentration. Normal high density lipoproteins which contain apo A-II, the precursor of amyloid fibrils in murine senile amyloidosis, and acute phase high density lipoproteins which contain serum amyloid protein A, the precursor of amyloid fibrils in secondary amyloidosis, showed little fluorescence. The fluorescence was considerably diminished when structure of the amyloid fibrils was disrupted by guanidine-HCl treatment. This method will be useful for the determination of amyloid fibrils in vitro.     

Apolipoprotein E inhibits the depolymerization of beta 2-microglobulin-related amyloid fibrils at a neutral pH.

beta 2-Microglobulin-related (A beta 2M) amyloidosis is a common and serious complication in patients on long-term hemodialysis, and beta 2-microglobulin (beta 2-m) is a major structural component of A beta 2M amyloid fibrils. Fluorescence spectroscopic analysis with thioflavin T and electron microscopic study revealed that A beta 2M amyloid fibrils readily depolymerize into monomeric beta 2-m at a neutral to basic pH. Circular dichroism analysis revealed that soon after the initiation of the depolymerization reaction at pH 7.5, the characteristic spectrum of beta 2-m in A beta 2M amyloid fibrils changes to resemble that of monomeric beta 2-m at pH 7.5. Apolipoprotein E (apoE), a representative amyloid-associated protein, formed a stable complex with A beta 2M amyloid fibrils and inhibited the depolymerization of A beta 2M amyloid fibrils dose-dependently in a range of 0--10 microM. These results showed that apoE could enhance the deposition of amyloid fibrils in vivo, possibly by binding directly to the surface of the fibrils and stabilizing the conformation of beta 2-m in the fibrils.     

The prealbumin nature of the amyloid protein in familial amyloid polyneuropathy (FAP)-Swedish variety

Familial amyloid polyneuropathy (FAP) is a dominant hereditary type of amyloidosis affecting kinships originating in many countries. We have isolated a 15,000 dalton protein from the amyloid laden tissue of a patient of Swedish origin with familial amyloid polyneuropathy. By N-terminal sequence analysis it is homologous to the normal plasma protein, prealbumin. An antiserum prepared to the isolated protein confirms this by reacting identically with the amyloid protein and prealbumin. The normal plasma protein, prealbumin, is linked to a disease syndrome for the first time.     

Light Chain Amyloid Fibrils Cause Metabolic Dysfunction in Human Cardiomyocytes

Light chain (AL) amyloidosis is the most common form of systemic amyloid disease, and cardiomyopathy is a dire consequence, resulting in an extremely poor prognosis. AL is characterized by the production of monoclonal free light chains that deposit as amyloid fibrils principally in the heart, liver, and kidneys causing organ dysfunction. We have studied the effects of amyloid fibrils, produced from recombinant λ6 light chain variable domains, on metabolic activity of human cardiomyocytes. The data indicate that fibrils at 0.1 μM, but not monomer, significantly decrease the enzymatic activity of cellular NAD(P)H-dependent oxidoreductase, without causing significant cell death. The presence of amyloid fibrils did not affect ATP levels; however, oxygen consumption was increased and reactive oxygen species were detected. Confocal fluorescence microscopy showed that fibrils bound to and remained at the cell surface with little fibril internalization. These data indicate that AL amyloid fibrils severely impair cardiomyocyte metabolism in a dose dependent manner. These data suggest that effective therapeutic intervention for these patients should include methods for removing potentially toxic amyloid fibrils.     

Review: immunoglobulin light chain amyloidosis--the archetype of structural and pathogenic variability

AL amyloidosis is caused by deposition in target tissue of amyloid fibrils constituted by monoclonal immunoglobulin light chains. The amyloidogenic plasma cells derive from a transformed memory B cell that can be identified by anti-idiotype monoclonal antibodies. Comparison of the primary structures of amyloidogenic and nonamyloidogenic light chains does not show any common structural motif in the amyloidogenic variants but reveals peculiar replacements which can destabilize the folding state. Reduced folding stability now appears to be a unifying property of amyloidogenic light chains. The tendency of these proteins to populate a partially unfolded intermediate state is a key event in the self-association that progresses to the formation of oligomers and fibrils. The mechanism of organ damage caused by AL amyloid deposition is not known, but clinical findings suggest that the process of amyloid fibril formation itself exerts tissue toxic effects independently of the amount of amyloid deposited. Since the disease is caused by the neoplastic expansion of the plasma cell population synthesizing the amyloidogenic light chains, the clone represents the prime therapeutic target of conventional chemotherapy and experimental immunotherapy. In common with other types of amyloidosis the therapeutic strategy can take advantage of drugs able to improve the reabsorption of the amyloid deposits or able to bind and stabilize the light chain in the native-like folded state.     

Conformational stability of amyloid fibrils of beta2-microglobulin probed by guanidine-hydrochloride-induced unfolding

Although the stability of globular proteins has been studied extensively, that of amyloid fibrils is scarcely characterized. Beta2-microglobulin (beta2-m) is a major component of the amyloid fibrils observed in patients with dialysis-related amyloidosis. We studied the effects of guanidine hydrochloride on the amyloid fibrils of beta2-m, revealing a cooperative unfolding transition similar to that of the native state. The stability of amyloid fibrils increased on the addition of ammonium sulfate, consistent with a role of hydrophobic interactions. The results indicate that the analysis of unfolding transition is useful to obtain insight into the structural stability of amyloid fibrils.     

Normal transthyretin and synthetic transthyretin fragments form amyloid-like fibrils in vitro

In two general forms of amyloidosis, senile systemic amyloidosis and several familial amyloidoses the amyloid fibrils are built up by transthyretin and fragments of the molecule. It has previously been demonstrated that other amyloid fibril proteins e.g. atrial natriuretic factor and islet amyloid polypeptide form amyloid-like fibrils in vitro. In this study we used normal transthyretin and synthetic polypeptides corresponding to segments of the transthyretin molecule. We show that normal transthyretin and two of our tested polypeptides, which corresponded to the beta-strands A and G, easily form amyloid-like fibrils in vitro.     

Clinical aspects of systemic amyloid diseases

Amyloidosis is a protein misfolding disorder in which soluble proteins aggregate as insoluble amyloid fibrils. Protein aggregates and amyloid fibrils cause functional and structural organ damage respectively. To date, at least 24 different proteins have been recognized as causative agents of amyloid diseases, localized or systemic. The two most common forms of systemic amyloidosis are light-chain (AL) amyloidosis and reactive AA amyloidosis due to chronic inflammatory diseases. beta(2)-microglobulin amyloidosis is a common complication associated with long-term hemodialysis. Hereditary systemic amyloidoses are a group of autosomal dominant disorders caused by mutations in the genes of several plasma proteins. Heterogeneity in clinical presentation, pattern of amyloid-related organ toxicity and rate of disease progression is observed among systemic amyloidoses. In particular, beta(2)-microglobulin presents unique clinical features compared to the other systemic forms. The phenotypic features of hereditary systemic amyloidoses may instead overlap those of the two more common forms of acquired amyloidoses mentioned above and therefore a correct diagnosis can not rely only on clinical grounds. Unequivocal identification of the deposited protein is essential in order to avoid misdiagnosis and inappropriate treatment. Amyloid deposits can be reabsorbed and organ dysfunction reversed if the concentration of the amyloidogenic protein is reduced or zeroed. At present, the most effective approach to treatment of the systemic amyloidoses involves shutting down, or substantially reducing the synthesis of the amyloid precursor, or, as in the case of beta(2)-microglobulin, promoting its clearance.     

Induction of protein conformational change in mouse senile amyloidosis

Aggregated amyloid fibrils can induce further polymerization of precursor proteins in vitro, thus providing a possible basis for propagation or transmission in the pathogenesis of amyloidoses. Previously, we postulated that the transmission of amyloid fibrils induces conformational changes of endogenous amyloid protein in mouse senile amyloidosis (Xing, Y., Nakamura, A., Chiba, T., Kogishi, K., Matsushita, T., Fu, L., Guo Z., Hosokawa, M., Mori, M., and Higuchi, K. (2001) Lab. Invest. 81, 493-499). To further characterize this transmissibility, we injected amyloid fibrils (AApoAII(C)) of amyloidogenic C type apolipoprotein A-II (APOAIIC) intravenously into 2-month-old SAMR1 mice, which have B type apolipoprotein A-II (APOAIIB), and develop few if any amyloid deposits spontaneously. 10 months after amyloid injection, deposits were detected in the tongue, stomach, intestine, lungs, heart, liver, and kidneys. The intensity of deposition increased thereafter, whereas no amyloid was detected in distilled water-injected SAMR1 mice, even after 20 months. The deposited amyloid was composed of endogenous APOAIIB with a different amyloid fibril conformation. The injection of these amyloid fibrils of APOAIIB (AApoAII(B)) induced earlier and more severe amyloidosis in SAMR1 mice than the injection of AApoAII(C) amyloid fibrils. Thus, AApoAII(C) from amyloidogenic mice could induce a conformational change of less amyloidogenic APOAIIB to a different amyloid fibril structure, which could also induce amyloidosis in the less amyloidogenic strain. These results provide important insights into the pathogenesis of amyloid diseases.     

Investigation into the role of macrophages in the formation and degradation of beta2-microglobulin amyloid fibrils

Dialysis related amyloidosis is a serious complication of long-term hemodialysis in which beta(2)-microglobulin (beta(2)m) forms amyloid fibrils that deposit predominantly in cartilaginous tissues. How these fibrils form in vivo, however, is poorly understood. Here we perform a systematic investigation into the role of macrophages in the formation and degradation of beta(2)m amyloid fibrils, building on observations that macrophages are found in association with beta(2)m amyloid deposits in vivo and that these cells contain intra-lysosomal beta(2)m amyloid. In live cell imaging experiments we demonstrate that macrophages internalize monomeric beta(2)m, whereupon it is sorted to lysosomes. At lysosomal pH beta(2)m self-associates in vitro to form amyloid-like fibrils with an array of morphologies as visualized by atomic force microscopy. Cleavage of the monomeric protein by both macrophages and lysosomal proteases isolated from these cells results in the rapid degradation of the monomeric protein, preventing amyloid formation. Incubation of macrophages with preformed fibrils revealed that macrophages internalize amyloid-like fibrils formed extracellularly, but in marked contrast with the monomeric protein, the fibrils were not degraded within macrophage lysosomes. Correspondingly beta(2)m fibrils were highly resistant to degradation by high concentrations of lysosomal proteases isolated from macrophages. Despite their enormous degradative capacity, therefore, macrophage lysosomes cannot ameliorate dialysis-related amyloidosis by degrading pre-existing amyloid fibrils, but lysosomal proteases may play a protective role by eliminating amyloid precursors before beta(2)m fibrils can accumulate in what may represent an otherwise fibrillogenic environment.     

Seeding-dependent propagation and maturation of amyloid fibril conformation

Recent studies of amyloid fibrils have focused on the presence of multiple amyloid forms even with one protein and their propagation by seeding, leading to conformational memory. To establish the structural basis of these critical features of amyloid fibrils, we used the amyloidogenic fragment Ser20-Lys41 (K3) of beta2-microglobulin, a protein responsible for dialysis-related amyloidosis. In 20% (v/v) 2,2,2-trifluoroethanol and 10 mM HCl (pH approximately 2), K3 peptide formed two types of amyloid-like fibrils, f218 and f210, differing in the amount of beta-sheet as measured by circular dichroism spectroscopy and Fourier transform infrared spectroscopy. Atomic force microscopy showed that the fibril with a larger amount of beta-sheet (f210) is thinner and longer. Both fibrils were reproduced by seeding, showing the template-dependent propagation of a fibril's conformation. However, upon repeated self-seeding, f218 fibrils were gradually transformed into f210 fibrils, revealing the conformational maturation. The observed maturation can be explained fully by a competitive propagation of two fibrils. The maturation of amyloid fibrils might play a role during the development of amyloidosis.     

Current perspectives on cardiac amyloidosis

Amyloidosis represents a group of diseases in which proteins undergo misfolding to form insoluble fibrils with subsequent tissue deposition. While almost all deposited amyloid fibers share a common nonbranched morphology, the affected end organs, clinical presentation, treatment strategies, and prognosis vary greatly among this group of diseases and are largely dependent on the specific amyloid precursor protein. To date, at least 27 precursor proteins have been identified to result in either local tissue or systemic amyloidosis, with nine of them manifesting in cardiac deposition and resulting in a syndrome termed "cardiac amyloidosis" or "amyloid cardiomyopathy." Although cardiac amyloidosis has been traditionally considered to be a rare disorder, as clinical appreciation and understanding continues to grow, so too has the prevalence, suggesting that this disease may be greatly underdiagnosed. The most common form of cardiac amyloidosis is associated with circulating amyloidogenic monoclonal immunoglobulin light chain proteins. Other major cardiac amyloidoses result from a misfolding of products of mutated or wild-type transthyretin protein. While the various cardiac amyloidoses share a common functional consequence, namely, an infiltrative cardiomyopathy with restrictive pathophysiology leading to progressive heart failure, the underlying pathophysiology and clinical syndrome varies with each precursor protein. Herein, we aim to provide an up-to-date overview of cardiac amyloidosis from nomenclature to molecular mechanisms and treatment options, with a particular focus on amyloidogenic immunoglobulin light chain protein cardiac amyloidosis.     

Endocytosed β2-Microglobulin Amyloid Fibrils Induce Necrosis and Apoptosis of Rabbit Synovial Fibroblasts by Disrupting Endosomal/Lysosomal Membranes: A Novel Mechanism on the Cytotoxicity of Amyloid Fibrils

Dialysis-related amyloidosis is a major complication in long-term hemodialysis patients. In dialysis-related amyloidosis, β2-microglobulin (β2-m) amyloid fibrils deposit in the osteoarticular tissue, leading to carpal tunnel syndrome and destructive arthropathy with cystic bone lesions, but the mechanism by which these amyloid fibrils destruct bone and joint tissue is not fully understood. In this study, we assessed the cytotoxic effect of β2-m amyloid fibrils on the cultured rabbit synovial fibroblasts. Under light microscopy, the cells treated with amyloid fibrils exhibited both necrotic and apoptotic changes, while the cells treated with β2-m monomers and vehicle buffer exhibited no morphological changes. As compared to β2-m monomers and vehicle buffer, β2-m amyloid fibrils significantly reduced cellular viability as measured by the lactate dehydrogenase release assay and the 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay and significantly increased the percentage of apoptotic cells as measured by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method. β2-m amyloid fibrils added to the medium adhered to cell surfaces, but did not disrupt artificial plasma membranes as measured by the liposome dye release assay. Interestingly, when the cells were incubated with amyloid fibrils for several hours, many endosomes/lysosomes filled with amyloid fibrils were observed under confocal laser microscopy and electron microscopy, Moreover, some endosomal/lysosomal membranes were disrupted by intravesicular fibrils, leading to the leakage of the fibrils into the cytosol and adjacent to mitochondria. Inhibition of actin-dependent endocytosis by cytochalasin D attenuated the toxicity of amyloid fibrils. These results suggest that endocytosed β2-m amyloid fibrils induce necrosis and apoptosis by disrupting endosomal/lysosomal membranes, and this novel mechanism on the cytotoxicity of amyloid fibrils is described.     

Islet amyloid and type 2 diabetes: from molecular misfolding to islet pathophysiology.

Islet amyloid polypeptide (IAPP, amylin) is secreted from pancreatic islet beta-cells and converted to amyloid deposits in type 2 diabetes. Conversion from soluble monomer, IAPP 1-37, to beta-sheet fibrils involves changes in the molecular conformation, cellular biochemistry and diabetes-related factors. In addition to the recognised amyloidogenic region, human IAPP (hIAPP) 20-29, the peptides human or rat IAPP 30-37 and 8-20, assume beta-conformation and form fibrils. These three amyloidogenic regions of hIAPP can be modelled as a folding intermediate with an intramolecular beta-sheet. A hypothesis is proposed for co-secretion of proIAPP with proinsulin in diabetes and formation of a 'nidus' adjacent to islet capillaries for subsequent accumulation of secreted IAPP to form the deposit. Although intracellular fibrils have been identified in experimental systems, extracellular deposition predominates in animal models and man. Extensive fibril accumulations replace islet cells. The molecular species of IAPP that is cytotoxic remains controversial. However, since fibrils form invaginations in cell membranes, small non-toxic IAPP fibrillar or amorphous accumulations could affect beta-cell stimulus-secretion coupling. The level of production of hIAPP is important but not a primary factor in islet amyloidosis; there is little evidence for inappropriate IAPP hypersecretion in type 2 diabetes and amyloid formation is generated in transgenic mice overexpressing the gene for human IAPP only against a background of obesity. Animal models of islet amyloidosis suggest that diabetes is induced by the deposits whereas in man, fibril formation appears to result from diabetes-associated islet dysfunction. Islet secretory failure results from progressive amyloidosis which provides a target for new therapeutic interventions.     

A common beta-sheet architecture underlies in vitro and in vivo beta2-microglobulin amyloid fibrils

Misfolding and aggregation of normally soluble proteins into amyloid fibrils and their deposition and accumulation underlies a variety of clinically significant diseases. Fibrillar aggregates with amyloid-like properties can also be generated in vitro from pure proteins and peptides, including those not known to be associated with amyloidosis. Whereas biophysical studies of amyloid-like fibrils formed in vitro have provided important insights into the molecular mechanisms of amyloid generation and the structural properties of the fibrils formed, amyloidogenic proteins are typically exposed to mild or more extreme denaturing conditions to induce rapid fibril formation in vitro. Whether the structure of the resulting assemblies is representative of their natural in vivo counterparts, thus, remains a fundamental unresolved issue. Here we show using Fourier transform infrared spectroscopy that amyloid-like fibrils formed in vitro from natively folded or unfolded beta(2)-microglobulin (the protein associated with dialysis-related amyloidosis) adopt an identical beta-sheet architecture. The same beta-strand signature is observed whether fibril formation in vitro occurs spontaneously or from seeded reactions. Comparison of these spectra with those of amyloid fibrils extracted from patients with dialysis-related amyloidosis revealed an identical amide I' absorbance maximum, suggestive of a characteristic and conserved amyloid fold. Our results endorse the relevance of biophysical studies for the investigation of the molecular mechanisms of beta(2)-microglobulin fibrillogenesis, knowledge about which may inform understanding of the pathobiology of this protein.