A brief overview of amyloids and Alzheimer’s disease

Amyloid fibrils are self-assembled fibrous protein aggregates that are associated with a number of presently incurable diseases such as Alzheimer’s and Parkinson’s disease. Millions of people worldwide suffer from amyloid diseases. This review summarizes the unique cross-β structure of amyloid fibrils, morphological variations, the kinetics of amyloid fibril formation, and the cytotoxic effects of these fibrils and oligomers. Alzheimer’s disease is also explored as an example of an amyloid disease to show the various approaches to treat these amyloid diseases. Finally, this review investigates the nanotechnological and biological applications of amyloid fibrils; as well as a summary of the typical biological pathways involved in the disposal of amyloid fibrils and their precursors.     

Peptides for disrupting and degrading amyloids

Amyloid proteins can aggregate into insoluble fibrils and form amyloid deposits in the human brain, which is the hallmark of many neurodegenerative diseases. Promising strategies toward pathological amyloid proteins and deposition include investigating inhibitors that can disrupt amyloid aggregation or induce misfolding protein degradation. In this review, recent progress of peptide-based inhibitors, including amyloid sequence–derived inhibitors, designed peptides, and peptide mimics, is highlighted. Based on the increased understanding of peptide design and precise amyloid structures, these peptides exhibit advanced inhibitory activities against fibrous aggregation as well as enhanced druggability.     

The distribution of alpha 1-antichymotrypsin and amyloid production in the brain in Alzheimer's disease.

In this immunohistopathological study alpha 1-antichymotrypsin, which is barely demonstrable in the normal brain, was found in amyloid fibrils, endothelial cells and the cytoplasm of astroglial cells in brains from patients with Alzheimer's disease. Amyloid precursors stained with methenamine silver were arrayed mainly along the membranes, and amyloid fibrils, which stained densely with anti-alpha 1-antichymotrypsin, were in direct contact with the fibrous structures connecting with the membranes of vascular feet or astrocytic processes. From the above findings, alpha 1-antichymotrypsin seems to play a role in the production of amyloid fibrils in Alzheimer's disease.     

Glycation induces formation of amyloid cross-beta structure in albumin

Amyloid fibrils are components of proteinaceous plaques that are associated with conformational diseases such as Alzheimer's disease, transmissible spongiform encephalopathies, and familial amyloidosis. Amyloid polypeptides share a specific quarternary structure element known as cross-beta structure. Commonly, fibrillar aggregates are modified by advanced glycation end products (AGE). In addition, AGE formation itself induces protein aggregation. Both amyloid proteins and protein-AGE adducts bind multiligand receptors, such as receptor for AGE, CD36, and scavenger receptors A and B type I, and the serine protease tissue-type plasminogen activator (tPA). Based on these observations, we hypothesized that glycation induces refolding of globular proteins, accompanied by formation of cross-beta structure. Using transmission electron microscopy, we demonstrate here that glycated albumin condensates into fibrous or amorphous aggregates. These aggregates bind to amyloid-specific dyes Congo red and thioflavin T and to tPA. In contrast to globular albumin, glycated albumin contains amino acid residues in beta-sheet conformation, as measured with circular dichroism spectropolarimetry. Moreover, it displays cross-beta structure, as determined with x-ray fiber diffraction. We conclude that glycation induces refolding of initially globular albumin into amyloid fibrils comprising cross-beta structure. This would explain how glycated ligands and amyloid ligands can bind to the same multiligand "cross-beta structure" receptors and to tPA.     

Amyloid fibril formation from sequences of a natural beta-structured fibrous protein, the adenovirus fiber

Amyloid fibrils are fibrous beta-structures that derive from abnormal folding and assembly of peptides and proteins. Despite a wealth of structural studies on amyloids, the nature of the amyloid structure remains elusive; possible connections to natural, beta-structured fibrous motifs have been suggested. In this work we focus on understanding amyloid structure and formation from sequences of a natural, beta-structured fibrous protein. We show that short peptides (25 to 6 amino acids) corresponding to repetitive sequences from the adenovirus fiber shaft have an intrinsic capacity to form amyloid fibrils as judged by electron microscopy, Congo Red binding, infrared spectroscopy, and x-ray fiber diffraction. In the presence of the globular C-terminal domain of the protein that acts as a trimerization motif, the shaft sequences adopt a triple-stranded, beta-fibrous motif. We discuss the possible structure and arrangement of these sequences within the amyloid fibril, as compared with the one adopted within the native structure. A 6-amino acid peptide, corresponding to the last beta-strand of the shaft, was found to be sufficient to form amyloid fibrils. Structural analysis of these amyloid fibrils suggests that perpendicular stacking of beta-strand repeat units is an underlying common feature of amyloid formation.     

The dye SYPRO orange binds to amylin amyloid fibrils but not pre‐fibrillar intermediates

Amyloid deposition underlies a broad range of diseases including multiple neurodegenerative diseases, systemic amyloidosis and type‐2 diabetes. Amyloid sensitive dyes, particularly thioflavin‐T, are widely used to detect ex‐vivo amyloid deposits, to monitor amyloid formation in vitro and to follow the kinetics of amyloid self‐assembly. We show that the dye SYPRO‐orange binds to amyloid fibrils formed by human amylin, the polypeptide responsible for islet amyloid formation in type‐2 diabetes. No fluorescence enhancement is observed in the presence of pre‐fibrillar species or in the presence of non‐amyloidogenic rat amylin. The kinetics of human amylin amyloid formation can be monitored by SYPRO‐orange fluorescence and match the time course determined with thioflavin‐T assays. Thus, SYPRO‐orange offers an alternative to thioflavin‐T assays of amylin amyloid formation. The implications for the interpretation of SYPRO‐orange‐based assays of protein stability and protein‐ligand interactions are discussed.     

Inhibition of amyloid formation

Amyloid is aggregated protein in the form of insoluble fibrils. Amyloid deposition in human tissue-amyloidosis-is associated with a number of diseases including all common dementias and type II diabetes. Considerable progress has been made to understand the mechanisms leading to amyloid formation. It is, however, not yet clear by which mechanisms amyloid and protein aggregates formed on the path to amyloid are cytotoxic. Strategies to prevent protein aggregation and amyloid formation are nevertheless, in many cases, promising and even successful. This review covers research on intervention of amyloidosis and highlights several examples of how inhibition of protein aggregation and amyloid formation has been achieved in practice. For instance, rational design can provide drugs that stabilize a native folded state of a protein, protein engineering can provide new binding proteins that sequester monomeric peptides from aggregation, small molecules and peptides can be designed to block aggregation or direct it into non-cytotoxic paths, and monoclonal antibodies have been developed for therapies towards neurodegenerative diseases based on inhibition of amyloid formation and clearance.     

Functional amyloid formation within mammalian tissue

Amyloid is a generally insoluble, fibrous cross-β sheet protein aggregate. The process of amyloidogenesis is associated with a variety of neurodegenerative diseases including Alzheimer, Parkinson, and Huntington disease. We report the discovery of an unprecedented functional mammalian amyloid structure generated by the protein Pmel17. This discovery demonstrates that amyloid is a fundamental nonpathological protein fold utilized by organisms from bacteria to humans. We have found that Pmel17 amyloid templates and accelerates the covalent polymerization of reactive small molecules into melanin—a critically important biopolymer that protects against a broad range of cytotoxic insults including UV and oxidative damage. Pmel17 amyloid also appears to play a role in mitigating the toxicity associated with melanin formation by sequestering and minimizing diffusion of highly reactive, toxic melanin precursors out of the melanosome. Intracellular Pmel17 amyloidogenesis is carefully orchestrated by the secretory pathway, utilizing membrane sequestration and proteolytic steps to protect the cell from amyloid and amyloidogenic intermediates that can be toxic. While functional and pathological amyloid share similar structural features, critical differences in packaging and kinetics of assembly enable the usage of Pmel17 amyloid for normal function. The discovery of native Pmel17 amyloid in mammals provides key insight into the molecular basis of both melanin formation and amyloid pathology, and demonstrates that native amyloid (amyloidin) may be an ancient, evolutionarily conserved protein quaternary structure underpinning diverse pathways contributing to normal cell and tissue physiology.     

Consensus features in amyloid fibrils: sheet-sheet recognition via a (polar or nonpolar) zipper structure

Amyloid fibrils characterized as highly intractable thread-like species are associated with many neurodegenerative diseases. Although neither the mechanism of amyloid formation nor the origin of amyloid toxicity is currently completely understood, the detailed three-dimensional atomic structures of the yeast protein Sup35 and Abeta amyloid protein determined by recent experiments provide the first and important step towards the comprehension of the pathogenesis and aggregation mechanisms of amyloid diseases. By analyzing these two amyloid peptides which have available crystal structures and other amyloid sequences with proposed structures using computational simulations, we delineate three common features in amyloid organizations and amyloid structures. These could contribute to an improved understanding of the molecular mechanism of amyloid formation, the nature of the aggregation driving forces that stabilize these structures and the development of potential therapeutic agents against amyloid diseases.     

Freezing of a fish antifreeze protein results in amyloid fibril formation

Amyloid is associated with a number of diseases including Alzheimer's, Huntington's, Parkinson's, and the spongiform encephalopathies. Amyloid fibrils have been formed in vitro from both disease and nondisease related proteins, but the latter requires extremes of pH, heat, or the presence of a chaotropic agent. We show, using fluorescence spectroscopy, electron microscopy, and solid-state NMR spectroscopy, that the alpha-helical type I antifreeze protein from the winter flounder forms amyloid fibrils at pH 4 and 7 upon freezing and thawing. Our results demonstrate that the freezing of some proteins may accelerate the formation of amyloid fibrils.     

The amyloid state of proteins in human diseases

Amyloid fibers and oligomers are associated with a great variety of human diseases including Alzheimer's disease and the prion conditions. Here we attempt to connect recent discoveries on the molecular properties of proteins in the amyloid state with observations about pathological tissues and disease states. We summarize studies of structure and nucleation of amyloid and relate these to observations on amyloid polymorphism, prion strains, coaggregation of pathogenic proteins in tissues, and mechanisms of toxicity and transmissibility. Molecular studies have also led to numerous strategies for biological and chemical interventions against amyloid diseases.     

Discovery and characterization of a mammalian amyloid disaggregation activity

The formation of amyloid, a cross‐β‐sheet fibrillar aggregate, is associated with a variety of aging‐associated degenerative diseases. Herein, we report the existence of a mammalian amyloid disaggregase activity that is present in all tissues and cell types tested. Homogenates from mammalian tissues and cell lines are able to disaggregate amyloid fibrils composed of amyloid β (Aβ)_1–40 or the 8 kDa plasma gelsolin fragment. The mammalian disaggregase activity is sensitive to proteinase K digestion and can be uncoupled from proteolysis activity using a protease inhibitor cocktail. Amyloid disaggregation and proteolysis activities are remarkably resistant to changes in temperature and pH. Identification and manipulation of the proteins responsible for the amyloid disaggregation/degradation activities offers the possibility of ameliorating aggregation‐associated diseases.     

Structural insights into functional and pathological amyloid

Amyloid is traditionally viewed as a consequence of protein misfolding and aggregation and is most notorious for its association with debilitating and chronic human diseases. However, a growing list of examples of "functional amyloid" challenges this bad reputation and indicates that many organisms can employ the biophysical properties of amyloid for their benefit. Because of developments in the structural studies of amyloid, a clearer picture is emerging about what defines amyloid structure and the properties that unite functional and pathological amyloids. Here, we review various amyloids and place them within the framework of the latest structural models.     

The distribution of α1-antichymotrypsin and amyloid production in the brain in Alzheimer’s disease

In this immunohistopathological study α₁-antichymotrypsin, which is barely demonstrable in the normal brain, was found in amyloid fibrils, endothelial cells and the cytoplasm of astroglial cells in brains from patients with Alzheimer’s disease. Amyloid precursors stained with methenamine silver were arrayed mainly along the membranes, and amyloid fibrils, which stained densely with anti-α₁-antichymotrypsin, were in direct contact with the fibrous structures connecting with the membranes of vascular feet or astrocytic processes. From the above findings, α₁-antichymotrypsin seems to play a role in the production of amyloid fibrils in Alzheimer’s disease.     

Structural polymorphism of Alzheimer Aβ and other amyloid fibrils

Deposits of amyloid fibrils characterize a diverse group of human diseases that includes Alzheimer’s disease, Creutzfeldt-Jakob disease and type II diabetes. Amyloid fibrils formed from different polypeptides contain a common cross-β spine. Nevertheless, amyloid fibrils formed from the same polypeptide can occur in a range of structurally different morphologies. The heterogeneity of amyloid fibrils reflects different types of polymorphism: (i) variations in the protofilament number, (ii) variations in the protofilament arrangement and (iii) different polypeptide conformations. Amyloid fibril polymorphism implies that fibril formation can lead, for the same polypeptide sequence, to many different patterns of inter- or intra-residue interactions. This property differs significantly from native, monomeric protein folding reactions that produce, for one protein sequence, only one ordered conformation and only one set of inter-residue interactions.     

Small molecule screening in context: Lipid-catalyzed amyloid formation

Islet Amyloid Polypeptide (IAPP) is a 37-residue hormone cosecreted with insulin by the β-cells of the pancreas. Amyloid fiber aggregation of IAPP has been correlated with the dysfunction and death of these cells in type II diabetics. The likely mechanisms by which IAPP gains toxic function include energy independent cell membrane penetration and induction of membrane depolarization. These processes have been correlated with solution biophysical observations of lipid bilayer catalyzed acceleration of amyloid formation. Although the relationship between amyloid formation and toxicity is poorly understood, the fact that conditions promoting one also favor the other suggests related membrane active structural states. Here, a novel high throughput screening protocol is described that capitalizes on this correlation to identify compounds that target membrane active species. Applied to a small library of 960 known bioactive compounds, we are able to report identification of 37 compounds of which 36 were not previously reported as active toward IAPP fiber formation. Several compounds tested in secondary cell viability assays also demonstrate cytoprotective effects. It is a general observation that peptide induced toxicity in several amyloid diseases (such as Alzhiemer’s and Parkinson’s) involves a membrane bound, preamyloid oligomeric species. Our data here suggest that a screening protocol based on lipid-catalyzed assembly will find mechanistically informative small molecule hits in this subclass of amyloid diseases.     

Towards revealing the structure of bacterial inclusion bodies

Protein aggregation is a widely observed phenomenon in human diseases, biopharmaceutical production, and biological research. Protein aggregates are generally classified as highly ordered, such as amyloid fibrils, or amorphous, such as bacterial inclusion bodies. Amyloid fibrils are elongated filaments with diameters of 6-12 nm, they are comprised of residue-specific cross-β structure, and display characteristic properties, such as binding with amyloid-specific dyes. Amyloid fibrils are associated with dozens of human pathological conditions, including Alzheimer disease and prion diseases. Distinguished from amyloid fibrils, bacterial inclusion bodies display apparent amorphous morphology. Inclusion bodies are formed during high-level recombinant protein production, and formation of inclusion bodies is a major concern in biotechnology. Despite of the distinctive morphological difference, bacterial inclusion bodies have been found to have some amyloid-like properties, suggesting that they might contain structures similar to amyloid-like fibrils. Recent structural data further support this hypothesis, and this review summarizes the latest progress towards revealing the structural details of bacterial inclusion bodies.     

Evaluating quinacrine as a potential amyloid imaging compound: studies on hen egg white lysozyme as model system

Amyloid plaque is associated with several neuronal and non-neuronal degenerative diseases. More than twenty human proteins can fold abnormally to form pathological deposits like amyloid plaque. Strategies for treating such diseases include therapies designed to decrease protein plaque formation or its complete clearance, but monitoring/clinical trials of these treatments are limited by the lack of effective methods to monitor amyloid deposits in the organs/tissues of living patients. The current study shows binding and staining ability of quinacrine to protein amyloid deposits, using Hen Egg White Lysozyme (HEWL) as model system and characterization of its binding interaction with HEWL, employing several biophysical techniques. Since quinacrine can pass the blood brain barrier, the current report suggests potential application of quinacrine for antemortem diagnostic of amyloid.     

Amino acid sequence determinants and molecular chaperones in amyloid fibril formation

Amyloid consists of cross-β-sheet fibrils and is associated with about 25 human diseases, including several neurodegenerative diseases, systemic and localized amyloidoses and type II diabetes mellitus. Amyloid-forming proteins differ in structures and sequences, and it is to a large extent unknown what makes them convert from their native conformations into amyloid. In this review, current understanding of amino acid sequence determinants and the effects of molecular chaperones on amyloid formation are discussed. Studies of the nonpolar, transmembrane surfactant protein C (SP-C) have revealed amino acid sequence features that determine its amyloid fibril formation, features that are also found in the amyloid β-peptide in Alzheimer’s disease and the prion protein. Moreover, a proprotein chaperone domain (CTC_Brichos) that prevents amyloid-like aggregation during proSP-C biosynthesis can prevent fibril formation also of other amyloidogenic proteins.     

Ca2+ binding protects against gelsolin amyloidosis

Amyloid diseases occur when native or mutant polypeptides misfold and aggregate to form deposits in the extracellular space. There are at least 20 proteins associated with amyloid diseases, including the well-known amyloid-beta peptide that is the causative agent for Alzheimer's disease (AD). This review describes familial amyloidosis of Finnish type (FAF), an amyloid disease caused by mutations in plasma gelsolin, a secreted protein that contains multiple Ca2+-binding domains. The FAF mutations result in a loss of the Ca2+-binding site in domain 2 of plasma gelsolin. The resulting decreased stability gives rise to susceptibility to the protease furin in the Golgi. Furin cleavage generates a secreted fragment that undergoes a second proteolytic event in the extracellular matrix to produce a peptide that self-assembles into amyloid plaques. Thus, Ca2+ binding in native plasma gelsolin protects against amyloid disease.