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.     

Amphoterin includes a sequence motif which is homologous to the Alzheimer's beta-amyloid peptide (Abeta), forms amyloid fibrils in vitro, and binds avidly to Abeta

Many of the proteins associated with amyloidoses have been found to share structural and sequence similarities, which are believed to be responsible for their capability to form amyloid fibrils. Interestingly, some proteins seem to be able to form amyloid-like fibrils although they are not associated with amyloidoses. This indicates that the ability to form amyloid fibrils may be a general property of a greater number of proteins not associated with these diseases. In the present work, we have searched for amyloidogenic consensus sequences in two current protein/peptide databases and show that many proteins share structures which can be predicted to form amyloid. One of these potentially amyloidogenic proteins is amphoterin (also known as HMG-1), involved in neuronal development and a ligand for the receptor for advanced glycation end products (RAGE). It contains an amyloidogenic peptide fragment which is highly homologous to the Alzheimer's amyloid beta-peptide. If enzymatically released from the native protein, it forms amyloid-like fibrils which are visible in electron microscopy, exhibit apple green birefringence under polarized light after Congo red staining, and increases thioflavin T fluorescence. This fragment also shows high affinity to Abeta as a free peptide or while part of the native protein. Our results support the hypothesis that the potential to form amyloid is a common characteristic of a number of proteins, independent of their relation to amyloidoses, and that this potential can be predicted based on the physicochemical properties of these proteins.     

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.     

Alzheimer beta-amyloid peptides: structures of amyloid fibrils and alternate aggregation products

The amyloidoses are a heterogeneous group of diseases, which are characterized by the local or systemic deposition of amyloid. At the root of these diseases are changes in protein conformation where normal innocuous proteins transform into insoluble amyloid fibrils and deposit in tissues. The amyloid fibrils of Alzheimer's disease are composed of the Abeta peptide and deposit in the form of senile plaques. Neurodegeneration surrounds the amyloid deposits, indicating that neurotoxic substances are produced during the deposition process. Whether the neurotoxic species is the amyloid fibril or a fibril precursor is a current area of active research. This review focuses on advancements made in elucidating the molecular structures of the Abeta amyloid fibril and alternate aggregation products of the Abeta peptide formed during fibrillogenesis.     

Amyloid Fibrils Trigger the Release of Neutrophil Extracellular Traps (NETs), Causing Fibril Fragmentation by NET-associated Elastase

The accumulation of amyloid fibrils is a feature of amyloid diseases, where cell toxicity is due to soluble oligomeric species that precede fibril formation or are formed by fibril fragmentation, but the mechanism(s) of fragmentation is still unclear. Neutrophil-derived elastase and histones were found in amyloid deposits from patients with different systemic amyloidoses. Neutrophil extracellular traps (NETs) are key players in a death mechanism in which neutrophils release DNA traps decorated with proteins such as elastase and histones to entangle pathogens. Here, we asked whether NETs are triggered by amyloid fibrils, reasoning that because proteases are present in NETs, protease digestion of amyloid may generate soluble, cytotoxic species. We show that amyloid fibrils from three different sources (α-synuclein, Sup35, and transthyretin) induced NADPH oxidase-dependent NETs in vitro from human neutrophils. Surprisingly, NET-associated elastase digested amyloid fibrils into short species that were cytotoxic for BHK-21 and HepG2 cells. In tissue sections from patients with primary amyloidosis, we also observed the co-localization of NETs with amyloid deposits as well as with oligomers, which are probably derived from elastase-induced fibril degradation (amyloidolysis). These data reveal that release of NETs, so far described to be elicited by pathogens, can also be triggered by amyloid fibrils. Moreover, the involvement of NETs in amyloidoses might be crucial for the production of toxic species derived from fibril fragmentation.     

Conformation-dependent scFv antibodies specifically recognize the oligomers assembled from various amyloids and show colocalization of amyloid fibrils with oligomers in patients with amyloidoses

Increasing evidence indicates that amyloid aggregates, including oligomers, protofibrils or fibrils, are pivotal toxins in the pathogenesis of many amyloidoses such as Alzheimer's disease (AD), Parkinson's disease, Huntington's disease, prion-related diseases, type 2 diabetes and hereditary renal amyloidosis. Various oligomers assembled from different amyloid proteins share common structures and epitopes. Here we present data indicating that two oligomer-specific single chain variable fragment (scFv) antibodies isolated from a na?ve human scFv library could conformation-dependently recognize oligomers assembled from α-synuclein, amylin, insulin, Aβ1-40, prion peptide 106-126 and lysozyme, and fibrils from lysozyme. Further investigation showed that both scFvs inhibited the fibrillization of α-synuclein, amylin, insulin, Aβ1-40 and prion peptide 106-126, and disaggregated their preformed fibrils. However, they both promoted the aggregation of lysozyme. Nevertheless, the two scFv antibodies could attenuate the cytotoxicity of all amyloids tested. Moreover, the scFvs recognized the amyloid oligomers in all types of plaques, Lewy bodies and amylin deposits in the brain tissues of AD and PD patients and the pancreas of type 2 diabetes patients respectively, and showed that most amyloid fibril deposits were colocalized with oligomers in the tissues. Such conformation-dependent scFv antibodies may have potential application in the investigation of aggregate structures, the mechanisms of aggregation and cytotoxicity of various amyloids, and in the development of diagnostic and therapeutic reagents for many amyloidoses.     

Identification of beta-aggregate sites in protein chain

Studying amyloid diseases (amyloidoses) has been especially actual and has attracted the attention of researchers all over the world in connection with the appearance of epidemic prion diseases such as Bovine Spongiform Encephalopathie in cattle and Creutzfeldt-Jakob in human. Amyloidoses are caused by transition of a "healthy" protein molecule or peptide from a native conformation to a very stable pathologic form: molecules in a pathologic conformation aggregate, that results in the amyloid fibrils which can grow infinitely. Investigation of molecular mechanisms of these diseases and the ability to find protein regions responsible for the appearance of the given diseases is a fundamental problem. Theoretical and experimental studies of amyloid fibril formation are considered in this review.     

Amyloid fibrils

Amyloid refers to the abnormal fibrous, extracellular, proteinaceous deposits found in organs and tissues. Amyloid is insoluble and is structurally dominated by β?sheet structure. Unlike other fibrous proteins it does not commonly have a structural, supportive or motility role but is associated with the pathology seen in a range of diseases known as the amyloidoses. These diseases include Alzheimer’s, the spongiform encephalopathies and type II diabetes, all of which are progressive disorders with associated high morbidity and mortality. Not surprisingly, research into the physicochemical properties of amyloid and its formation is currently intensely pursued. In this work we will highlight the key scientific findings and discuss how the stability of amyloid fibrils impacts on bionanotechnology.     

Imaging Amyloid Fibrils within Cells Using a Se-Labelling Strategy

The process of aggregation leading to amyloid formation by peptides and proteins is associated with diseases ranging from systemic amyloidoses to neurodegenerative disorders such as Alzheimer's disease. A key question in understanding the link between amyloid formation and its pathological consequences is the ultrastructural localisation and morphological form of amyloid species within the cellular environment. The acquisition of such information has proven to be challenging, but we report here a novel approach that enables amyloid fibrils to be visualised directly within a cell. First, fibrils are assembled from selenium analogues of the sulfur-containing cysteine peptides, and then, atomic number contrast transmission electron microscopy is used to detect the selenium doped species selectively within the carbon-rich background of the cell. We demonstrate the power of this approach by imaging human monocyte-derived macrophage cells that have been exposed to fibrils from an amyloidogenic fragment of the disease-associated protein transthyretin. The ready incorporation of seleno-cysteine and methionine instead of their natural sulfur-containing analogues, a feature that is already commonly used in X-ray diffraction studies of proteins, suggests that this method can be used as a general strategy to image specific peptides and proteins within the cellular environment using electron microscopy.     

Direct visualisation of the beta-sheet structure of synthetic Alzheimer's amyloid

Amyloid fibrils are a major pathological feature of Alzheimer's disease as well as other amyloidoses including the prion diseases. They are an unusual phenomenon, being made up of different, normally soluble proteins which undergo a profound conformational change and assemble to form very stable, insoluble fibrils which accumulate in the extracellular spaces. In Alzheimer's disease the amyloid fibrils are composed of the A beta protein. Knowledge of the structure of amyloid is essential for understanding the abnormal assembly and deposition of these fibrils and could lead to the rational design of therapeutic agents for their prevention or disaggregation. Here we reveal the core structure of an Alzheimer's amyloid fibril by direct visualisation using cryo-electron microscopy. Synthetic amyloid fibrils composed of A beta residues 11 to 25 and 1 to 42 were examined. The A beta (11-25) fibrils are clearly composed of beta-sheet structure that is observable as striations across the fibres. The beta-strands run perpendicular to the fibre axis and the projections show that the fibres are composed of beta-sheets with the strands in direct register. This observation has implications not only for the further understanding of amyloid, but also for the development of cryo-electron microscopy for direct visualisation of secondary structure.     

Study of the cytotoxicity of protein X amyloid fibrils

Amyloid oligomers, protofibrils, and fibrils of various amyloidogenic proteins are known to induce cell death. Tetracycline prevents the formation of fibrils of Aβ peptide and other amyloidogenic proteins and decomposes mature fibrils. It was previously shown that sarcomeric cytoskeletal proteins of the titin family (protein X, protein C, and protein H) in vitro form amyloid fibrils and tetracycline decomposes them. In this work, the concentration and time dependence of the survival of polymorphonuclear leukocytes in the presence of protein X amyloid fibrils is demonstrated. It is also shown that the survival rate increases as fibrils are decomposed by tetracycline. The antibiotic itself is found to be nontoxic. The results obtained show that this approach can be used to evaluate the efficiency of drugs that prevent or rectify amyloidoses.     

Conjugated polyelectrolytes: conformation-sensitive optical probes for detection of amyloid fibril formation

The in vivo deposition of amyloid fibrils is a hallmark of many devastating diseases known as the amyloidoses. Amyloid formation in vitro may also complicate production of proteins in the biotechnology industry. Simple, sensitive, and versatile tools that detect the fibrillar conformation of amyloidogenic proteins are thus of great importance. We have developed a negatively charged conjugated polyelectrolyte that displays different characteristic optical changes, detected visually or by absorption and emission, depending on whether the protein with which it forms a complex is in its native state or amyloid fibril conformation. This simple, rapid, and novel methodology was applied here to two amyloidogenic proteins, insulin and lysozyme, and its validity for detection of their fibrillar conformation was verified by currently used methods such as circular dichroism, transmission electron microscopy, and Congo red absorption.     

Amyloid cannot resist identification

The capacity to polymerize into amyloid fibrils is common to many proteins. While some proteins naturally form these fibrils to serve functional roles, amyloid is usually associated with pathogenic processes in which specific proteins aberrantly aggregate within cells or tissues. Though the contribution of amyloid fibrils to actual disease pathogenesis is not always clear, one possibility is that the titration of essential proteins from solution into aggregates contributes to the cellular degeneration common to many amyloid diseases. Using mammalian and yeast model systems, we recently showed that the common biophysical properties of amyloid aggregates—including strong resistance to dissolution—enable stringent purification and identification of both amyloid-forming and amyloid-associated proteins directly from cells. Strikingly, many proteins that were previously implicated in formation or clearance of intracellular aggregates, including several stress granule components, were found to co-aggregate with amyloid formed by a polyglutamine-expanded huntingtin fragment. This direct evaluation of proteins within aggregates can help identify new amyloid-forming proteins, as well as proteins that can indirectly contribute to disease mechanisms.     

Spontaneous generation of infectious nucleating amyloids in the transmissible and nontransmissible cerebral amyloidoses

The unconventional viruses of the transmissible subacute spongiform encephalopathies (kuru-CJD-GSS-FFI-scrapie-BSE) are nucleants spontaneously generated from host precursor proteins altered to β-pleated sheet configuration that polymerize into insoluble infectious amyloid fibrils. Thede novo conversion to infectious amyloids is facilitated or accelerated by many different point mutations causing amino acid changes, a stop codon, or octapeptide inserts that increase the likelihood of spontaneous conversion to infectious configuration by many orders of magnitude. Similar nucleating induction of configurational change to amyloid probably occurs in other amyloidoses of brain and in systemic amyloidoses. Thus, all amyloids, particularly so-called fibrillar amyloid enhancing factors, may be considered to be infectious scrapie-like agents. These events probably occur extracellularly, thus we are attempting to reproduce them in vitro, even from synthetic polypeptides.     

Multistep Changes in Amyloid Structure Induced by Cross-Seeding on a Rugged Energy Landscape

Amyloid fibrils are aberrant protein aggregates associated with various amyloidoses and neurodegenerative diseases. It is recently indicated that structural diversity of amyloid fibrils often results in different pathological phenotypes, including cytotoxicity and infectivity. The diverse structures are predicted to propagate by seed-dependent growth, which is one of the characteristic properties of amyloid fibrils. However, much remains unknown regarding how exactly the amyloid structures are inherited to subsequent generations by seeding reaction. Here, we investigated the behaviors of self- and cross-seeding of amyloid fibrils of human and bovine insulin in terms of thioflavin T fluorescence, morphology, secondary structure, and iodine staining. Insulin amyloid fibrils exhibited different structures, depending on species, each of which replicated in self-seeding. In contrast, gradual structural changes were observed in cross-seeding, and a new type of amyloid structure with distinct morphology and cytotoxicity was formed when human insulin was seeded with bovine insulin seeds. Remarkably, iodine staining tracked changes in amyloid structure sensitively, and singular value decomposition analysis of the ultraviolet-visible absorption spectra of the fibril-bound iodine has revealed the presence of one or more intermediate metastable states during the structural changes. From these findings, we propose a propagation scheme with multistep structural changes in cross-seeding between two heterologous proteins, which is accounted for as a consequence of the rugged energy landscape of amyloid formation.     

Structures for amyloid fibrils

Alzheimer's disease and Creutzfeldt-Jakob disease are the best-known examples of a group of diseases known as the amyloidoses. They are characterized by the extracellular deposition of toxic, insoluble amyloid fibrils. Knowledge of the structure of these fibrils is essential for understanding the process of pathology of the amyloidoses and for the rational design of drugs to inhibit or reverse amyloid formation. Structural models have been built using information from a wide variety of techniques, including X-ray diffraction, electron microscopy, solid state NMR and EPR. Recent advances have been made in understanding the architecture of the amyloid fibril. Here, we describe and compare postulated structural models for the mature amyloid fibril and discuss how the ordered structure of amyloid contributes to its stability.     

Promotion of formation of amyloid fibrils by aluminium adenosine triphosphate (AlATP)

The formation of amyloid fibrils is considered to be an important step in the aetiology of Alzheimer's disease and other amyloidoses. Fibril formation in vitro has been shown to depend on many different factors including modifications to the amino acid profile of fibrillogenic peptides and interactions with both large and small molecules of physiological significance. How these factors might contribute to amyloid fibril formation in vivo is not clear as very little is known about the promotion of fibril formation in undersaturated solutions of amyloidogenic peptides. We have used thioflavin T fluorescence and reverse phase high performance liquid chromatography to show that ATP, and in particular AlATP, promoted the formation of thioflavin T-reactive fibrils of beta amyloid and, an unrelated amyloidogenic peptide, amylin. Evidence is presented that induction of fibril formation followed the complexation of AIATP by one or more monomers of the respective peptide. However, the complex formed could not be identified directly and it is suggested that AlATP might be acting as a chaperone in the assembly of amyloid fibrils. The effect of AlATP was not mimicked by either AlADP or AlAMP. However, it was blocked by suramin, a P2 ATP receptor antagonist, and this has prompted us to speculate that the precursor proteins to beta amyloid and amylin may be substrates or receptors for ATP in vivo.     

Elongation of amyloid fibrils through lateral binding of monomers revealed by total internal reflection fluorescence microscopy

Amyloid fibrils are fibrillar aggregates of denatured proteins associated with a large number of amyloidoses. The formation of amyloid fibrils has been considered to occur by nucleation and elongation. Real-time imaging of the elongation as well as linear morphology of amyloid fibrils suggests that all elongation events occur at the growing ends of fibrils. On the other hand, we suggested that monomers also bind to the lateral sides of preformed fibrils during the seed-dependent elongation, diffuse to the growing ends, and finally make further conformation changes to the mature amyloid fibrils. To examine lateral binding during the elongation of fibrils, we used islet amyloid polypeptide (IAPP), which has been associated with type II diabetes, and prepared IAPP modified with the fluorescence dye, Alexa532. By monitoring the elongation process with amyloid specific thioflavin T and Alexa532 fluorescence, we obtained overlapping images of the two fluorescence probes, which indicated lateral binding. These results are similar to the surface diffusion-dependent growth of crystals, further supporting the similarities between amyloid fibrillation and the crystallization of substances.     

Sarcomeric proteins of the titin family form amyloids

It was shown for the first time that skeletal muscle sarcomeric proteins of the titin family (X-, C- and H-proteins) are able to form in vitro amyloid aggregates of different types: granular aggregates, protofibrils, helically twisted ribbons, linear fibrils, and bundles of linear fibrils. Their amyloid nature was confirmed by electron, polarization, and fluorescence microscopy and by spectral methods. As opposed to other amyloidogenic proteins, X-, C-, and H-proteins easily form amyloids under mild conditions close to physiological ones (pH, ionic strength, temperature). Like amyloid fibrils of Abeta-peptide and tau protein in Alzheimer's disease, amyloid aggregates formed by X-, C-, and H-proteins are destroyed by the antibiotic tetracycline. Thus, new proteins-precursors of amyloids and possible participants of amyloidoses in muscles were discovered. Further study of in vitro amyloidogenesis of these proteins would help to find approaches to controlling this process in organs and tissues.     

Cytotoxicity of amyloid fibrils of X-protein

It is known that amyloid oligomers, protofibrils, and fibrils induce cell death, and antibiotic tetracycline inhibits the fibrillization of beta amyloid peptides and other amyloidogenic proteins and disassembles their pre-formed fibrils. Earlier we have demonstrated that sarcomeric cytoskeletal proteins of the titin family (X-, C-, and H-proteins) are capable to form in vitro amyloid fibrils, and tetracycline effectively destroys these fibrils. Here we show that the viability of polymorphonuclear leukocytes in the presence of X-protein amyloids depends on the concentration of amyloid fibrils of X-protein and the time of incubation. In addition to the disaggregation of X-protein fibrils, tetracycline eliminated the cytotoxic effect of the protein. The antibiotic itself did not show a toxic effect, and the cell viability in its presence even increased. Our results evidence the potential of this approach for evaluating the effectiveness of drugs preventing or treating amyloidoses.