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.     

Immunoprecipitation of Amyloid Fibrils by the Use of an Antibody that Recognizes a Generic Epitope Common to Amyloid Fibrils

Amyloid fibrils are associated with many maladies, including Alzheimer’s disease (AD). The isolation of amyloids from natural materials is very challenging because the extreme structural stability of amyloid fibrils makes it difficult to apply conventional protein science protocols to their purification. A protocol to isolate and detect amyloids is desired for the diagnosis of amyloid diseases and for the identification of new functional amyloids. Our aim was to develop a protocol to purify amyloid from organisms, based on the particular characteristics of the amyloid fold, such as its resistance to proteolysis and its capacity to be recognized by specific conformational antibodies. We used a two-step strategy with proteolytic digestion as the first step followed by immunoprecipitation using the amyloid conformational antibody LOC. We tested the efficacy of this method using as models amyloid fibrils produced in vitro, tissue extracts from C. elegans that overexpress Aβ peptide, and cerebrospinal fluid (CSF) from patients diagnosed with AD. We were able to immunoprecipitate Aβ_1–40 amyloid fibrils, produced in vitro and then added to complex biological extracts, but not α-synuclein and gelsolin fibrils. This method was useful for isolating amyloid fibrils from tissue homogenates from a C. elegans AD model, especially from aged worms. Although we were able to capture picogram quantities of Aβ_1–40 amyloid fibrils produced in vitro when added to complex biological solutions, we could not detect any Aβ amyloid aggregates in CSF from AD patients. Our results show that although immunoprecipitation using the LOC antibody is useful for isolating Aβ_1–40 amyloid fibrils, it fails to capture fibrils of other amyloidogenic proteins, such as α-synuclein and gelsolin. Additional research might be needed to improve the affinity of these amyloid conformational antibodies for an array of amyloid fibrils without compromising their selectivity before application of this protocol to the isolation of amyloids.     

Scrapie-associated fibrils (SAF) purification method yields amyloid proteins from systemic and cerebral amyloidosis

We identified fibrils from non-transmissible systemic and cerebral amyloidosis using the purification method of scrapie-associated fibrils (SAF). The fibrils possessed the same nature of congophilia, filamentous structures and molecular weights as amyloid fibrils, and were resistant to Proteinase K digestion. This SAF method makes for a rapid extraction from amyloid-laden tissues. The method, therefore, may purify nontransmissible amyloids alone or together with SAF proteins.     

Plasticity of amyloid fibrils

In experiments designed to characterize the basis of amyloid fibril stability through mutational analysis of the Abeta (1-40) molecule, fibrils exhibit consistent, significant structural malleability. In these results, and in other properties, amyloid fibrils appear to more resemble plastic materials generated from synthetic polymers than globular proteins. Thus, like synthetic polymers and plastics, amyloid fibrils exhibit both polymorphism, the ability of one polypeptide to form aggregates of different morphologies, and isomorphism, the ability of different polypeptides to grow into a fibrillar amyloid morphology. This view links amyloid with the prehistorical and 20th century use of proteins as starting materials to make films, fibers, and plastics, and with the classic protein fiber stretching experiments of the Astbury group. Viewing amyloids from the point of view of the polymer chemist may shed new light on a number of issues, such as the role of protofibrils in the mechanism of amyloid formation, the biological potency of fibrils, and the prospects for discovering inhibitors of amyloid fibril formation.     

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.     

Cell Adhesion on Amyloid Fibrils Lacking Integrin Recognition Motif

Amyloids are highly ordered, cross-β-sheet-rich protein/peptide aggregates associated with both human diseases and native functions. Given the well established ability of amyloids in interacting with cell membranes, we hypothesize that amyloids can serve as universal cell-adhesive substrates. Here, we show that, similar to the extracellular matrix protein collagen, amyloids of various proteins/peptides support attachment and spreading of cells via robust stimulation of integrin expression and formation of integrin-based focal adhesions. Additionally, amyloid fibrils are also capable of immobilizing non-adherent red blood cells through charge-based interactions. Together, our results indicate that both active and passive mechanisms contribute to adhesion on amyloid fibrils. The present data may delineate the functional aspect of cell adhesion on amyloids by various organisms and its involvement in human diseases. Our results also raise the exciting possibility that cell adhesivity might be a generic property of amyloids.     

The Functional Curli Amyloid Is Not Based on In-register Parallel ��-Sheet Structure

The extracellular curli proteins of Enterobacteriaceae form fibrous structures that are involved in biofilm formation and adhesion to host cells. These curli fibrils are considered a functional amyloid because they are not a consequence of misfolding, but they have many of the properties of protein amyloid. We confirm that fibrils formed by CsgA and CsgB, the primary curli proteins of Escherichia coli, possess many of the hallmarks typical of amyloid. Moreover we demonstrate that curli fibrils possess the cross-β structure that distinguishes protein amyloid. However, solid state NMR experiments indicate that curli structure is not based on an in-register parallel β-sheet architecture, which is common to many human disease-associated amyloids and the yeast prion amyloids. Solid state NMR and electron microscopy data are consistent with a β-helix-like structure but are not sufficient to establish such a structure definitively.Interest in amyloid is largely because of its association with many late onset human diseases, including Alzheimer disease (Aβ),² Parkinson disease (α-synuclein), type II diabetes (amylin), and the transmissible spongiform encephalopathies (PrP). In each case a particular endogenous protein becomes incorporated into large aggregates known as amyloid, which was originally defined by pathologists as a tissue deposit staining like starch (1). However, the term amyloid has come to mean a filamentous protein aggregate with cross-β secondary structure (cross-β means that the β-strands that form β-sheets in the amyloid fibrils run approximately perpendicular to the long axis of the fibril with interstrand hydrogen bonds that run approximately parallel to the long axis) and protease resistance. Morphologically amyloid fibrils may vary in length from tens of nanometers to micrometers and have diameters in the range of 3–10 nm, although lateral association can produce much larger apparent diameters.Proteins from a variety of organisms can form amyloid both in vitro and in vivo, and the propensity to form amyloid may be a common property of many proteins (2). In addition to disease-associated amyloids, there are several confirmed cases of functional amyloid (for a review, see Ref. 3). For example, hydrophobins are amyloid-like proteins that coat the surface of fungal cells, and amyloid fibrils coating fish eggs protect them from dehydration (4, 5). The [Het-s] prion of Podospora anserina is involved in heterokaryon incompatibility, a recognition of non-self reaction believed to be important as a defense against fungal virus infection (6).Curli are extracellular filamentous structures of Enterobacteriaceae (7) that are integral to biofilm formation and are the major protein component of the extracellular matrix of these organisms (8). Curli of Escherichia coli are composed of the secreted proteins CsgA and CsgB. The latter is believed to prime the polymerization of the former and anchor the fibrils to the outer membrane (9). Both CsgA and CsgB fibrils are β-sheet-rich and, like amyloids, stain with the dye Congo red (10, 11).Because amyloid fibrils are non-crystalline and insoluble, solution NMR and x-ray crystallography are not directly applicable in structural studies. Solid state NMR and electron spin resonance have both been useful in obtaining constraints on amyloid structures and, in some cases, determining detailed structural information. The disease-associated amyloids formed by Aβ, amylin, α-synuclein, and tau along with the infectious amyloids of several yeast prions each have in-register parallel β-sheet structure (12–19).Here we confirm that the fibrils formed in vitro by CsgA and CsgB proteins are amyloids and explore their structure using solid state NMR and electron microscopy. Our results indicate that, unlike the pathogenic amyloids of humans and yeast, CsgA and CsgB amyloids are not in-register parallel β-sheet structures. Solid state NMR and electron microscopy data are consistent with a β-helix-like structure but do not establish such a structure definitively.     

Bacterial functional amyloids: Order from disorder

The discovery of intrinsic disorderness in proteins and peptide regions has given a new and useful insight into the working of biological systems. Due to enormous plasticity and heterogeneity, intrinsically disordered proteins or regions in proteins can perform myriad of functions. The flexibility in disordered proteins allows them to undergo conformation transition to form homopolymers of proteins called amyloids. Amyloids are highly structured protein aggregates associated with many neurodegenerative diseases. However, amyloids have gained much appreciation in recent years due to their functional roles. A functional amyloid fiber called curli is assembled on the bacterial cell surface as a part of the extracellular matrix during biofilm formation. The extracellular matrix that encases cells in a biofilm protects the cells and provides resistance against many environmental stresses. Several of the Csg (curli specific genes) proteins that are required for curli amyloid assembly are predicted to be intrinsically disordered. Therefore, curli amyloid formation is highly orchestrated so that these intrinsically disordered proteins do not inappropriately aggregate at the wrong time or place. The curli proteins are compartmentalized and there are chaperone-like proteins that prevent inappropriate aggregation and allow the controlled assembly of curli amyloids. Here we review the biogenesis of curli amyloids and the role that intrinsically disordered proteins play in the process.     

Switching in amyloid structure within individual fibrils: Implication for strain adaptation, species barrier and strain classification

Amyloid fibrils are highly ordered crystal-like structures. It is generally assumed that individual amyloid fibrils consist of conformationally uniform cross-β-sheet structures that enable the amyloids to replicate their individual conformations via a template-dependent mechanism. Recent studies revealed that amyloids are capable of accommodating a global conformational switch from one amyloid strain to another within individual fibrils. The current review highlights the high adaptation potential of amyloid structures and discusses the implication of these findings for several emerging issues including prion strain adaptation (i.e. gradual change in strain structure). It also proposes that the catalytic activity of an amyloid structure should be separated from its templating effect, and raises the question of strain classification according to their promiscuous or species-specific nature.     

Diversity, biogenesis and function of microbial amyloids

Amyloid is a distinct β-sheet-rich fold that many proteins can acquire. Frequently associated with neurodegenerative diseases in humans, including Alzheimer's, Parkinson's and Huntington's diseases, amyloids are traditionally considered the product of protein misfolding. However, the amyloid fold is now recognized as a ubiquitous part of normal cellular biology. Functional amyloids have been identified in nearly all facets of cellular life, with microbial functional amyloids leading the way. Unlike disease-associated amyloids, functional amyloids are assembled by dedicated, directed pathways and ultimately perform a physiological function that benefits the organism. The evolved amyloid assembly and disassembly pathways of microbes have provided novel insights into how cells have harnessed the amyloid assembly process for productive means. An understanding of functional amyloid biogenesis promises to provide a fresh perspective on the molecular events that underlie disease-associated amyloidogenesis. Here, we review functional microbial amyloids with an emphasis on curli fibers and their role in promoting biofilm formation and other community behaviors.     

Amyloid-Like Adhesins Produced by Floc-Forming and Filamentous Bacteria in Activated Sludge

Amyloid proteins (fimbriae or other microbial surface-associated structures) are expressed by many types of bacteria, not yet identified, in biofilms from various habitats, where they likely are of key importance to biofilm formation and biofilm properties. As these amyloids are potentially of great importance to the floc properties in activated sludge wastewater treatment plants (WWTP), the abundance of amyloid adhesins in activated sludge flocs from different WWTP and the identity of bacteria producing these were investigated. Amyloid adhesins were quantified using a combination of conformationally specific antibodies targeting amyloid fibrils, propidium iodide to target all fixed bacterial cells, confocal laser scanning microscopy, and digital image analysis. The biovolume fraction containing amyloid adhesins ranged from 10 to 40% in activated sludge from 10 different WWTP. The identity of bacteria producing amyloid adhesins was determined using fluorescence in situ hybridization with oligonucleotide probes in combination with antibodies or thioflavin T staining. Among the microcolony-forming bacteria, amyloids were primarily detected among Alpha- and Betaproteobacteria and Actinobacteria. A more detailed analysis revealed that many denitrifiers (from Thauera, Azoarcus, Zoogloea, and Aquaspirillum-related organisms) and Actinobacteria-related polyphosphate-accumulating organisms most likely produced amyloid adhesins, whereas nitrifiers did not. Many filamentous bacteria also expressed amyloid adhesins, including several Alphaproteobacteria (e.g., Meganema perideroedes), some Betaproteobacteria (e.g., Aquaspirillum-related filaments), Gammaproteobacteria (Thiothrix), Bacteroidetes, Chloroflexi (e.g., Eikelboom type 1851), and some foam-forming Actinobacteria (e.g., Gordonia amarae). The results show that amyloid adhesins were an abundant component of activated sludge extracellular polymeric substances and seem to have unexpected, divers functions.     

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.     

FapA is an Intrinsically Disordered Chaperone for Pseudomonas Functional Amyloid FapC

Bacterial functional amyloids contribute to biofilm development by bacteria and provide protection from the immune system and prevent antibiotic treatment. Strategies to target amyloid formation and interrupt biofilm formation have attracted recent interest due to their antimicrobial potential. Functional amyloid in Pseudomonas (Fap) includes FapC as the major component of the fibril while FapB is a minor component suggested to function as a nucleator of FapC. The system also includes the small periplasmic protein FapA, which has been shown to regulate fibril composition and morphology. The interplay between these three components is central in Fap fibril biogenesis. Here we present a comprehensive biophysical and spectroscopy analysis of FapA, FapB and FapC and provide insight into their molecular interactions. We show that all three proteins are primarily disordered with some regions with structural propensities for α-helix and β-sheet. FapA inhibits FapC fibrillation by targeting the nucleation step, whereas for FapB the elongation step is modulated. Furthermore, FapA alters the morphology of FapC (more than FapB) fibrils. Complex formation is observed between FapA and FapC, but not between FapA and FapB, and likely involves the N-terminus of FapA. We conclude that FapA is an intrinsically disordered chaperone for FapC that guards against fibrillation within the periplasm. This new understanding of a natural protective mechanism of Pseudomonas against amyloid formations can serve as inspiration for strategies blocking biofilm formation in infections.     

Amyloid peptides derived from CsgA and FapC modify the viscoelastic properties of biofilm model matrices

The bacterial biofilm is a complex environment of cells, which secrete a matrix made of various components, mainly polysaccharides and proteins. An understanding of the precise role of these components in the stability and dynamics of biofilm architecture would be a great advantage for the improvement of anti-biofilm strategies. Here, artificial biofilm matrices made of polysaccharides and auto-assembled peptides were designed, and the influence of bacterial amyloid proteins on the mechanical properties of the biofilm matrix was studied. The model polysaccharides methylcellulose and alginate and peptides derived from the amyloid proteins curli and FapC found in biofilms of Enterobacteriaceae and Pseudomonas, respectively, were used. Rheological measurements showed that the amyloid peptides do not prevent the gelation of the polysaccharides but influence deformation of the matrices under shear stress and modify the gel elastic response. Hence the secretion of amyloids could be for the biofilm a way of adapting to environmental changes.     

Amyloid-Like Fibril Formation by Tachykinin Neuropeptides and Its Relevance to Amyloid β-Protein Aggregation and Toxicity

Protein aggregation and amyloid formation are associated with both pathological conditions in humans such as Alzheimer's disease and native functions such as peptide hormone storage in the pituitary secretory granules in mammals. Here, we studied amyloid fibrils formation by three neuropeptides namely physalaemin, kassinin and substance P of tachykinin family using biophysical techniques including circular dichroism, thioflavin T, congo red binding and microscopy. All these neuropeptides under study have significant sequence similarity with Aβ(25-35) that is known to form neurotoxic amyloids. We found that all these peptides formed amyloid-like fibrils in vitro in the presence of heparin, and these amyloids were found to be nontoxic in neuronal cells. However, the extent of amyloid formation, structural transition, and morphology were different depending on the primary sequences of peptide. When Aβ(25-35) and Aβ40 were incubated with each of these neuropeptides in 1:1 ratio, a drastic increase in amyloid growths were observed compared to that of individual peptides suggesting that co-aggregation of Aβ and these neuropeptides. The electron micrographs of these co-aggregates were dissimilar when compared with individual peptide fibrils further supporting the possible incorporation of these neuropeptides in Aβ amyloid fibrils. Further, the fibrils of these neuropeptides can seed the fibrils formation of Aβ40 and reduced the toxicity of preformed Aβ fibrils. The present study of amyloid formation by tachykinin neuropeptides is not only providing an understanding of the mechanism of amyloid fibril formation in general, but also offering plausible explanation that why these neuropeptide might reduce the cytotoxicity associated with Alzheimer's disease related amyloids.     

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.     

Role of the C-terminal 28 residues of beta2-microglobulin in amyloid fibril formation

Beta2microglobulin (beta2m) is the major protein component of the fibrillar amyloid deposits isolated from patients diagnosed with dialysis-related amyloidosis (DRA). While investigating the molecular mechanism of amyloid fibril formation by beta2m, we found that the beta2m C-terminal peptide of 28 residues (cbeta2m) itself forms amyloid fibrils. When viewed by electron microscopy, cbeta2m aggregates appear as elongated unbranched fibers, the morphology typical for amyloids. Cbeta2m fibers stain with Congo red and show apple-green birefringence in polarized light, characteristic of amyloids. The observation that the beta2m C-terminal fragment readily forms amyloid fibrils implies that beta2m amyloid fibril formation proceeds via interactions of amyloid forming segments, which become exposed when the beta2m subunit is partially unfolded.     

The Role of Functional Amyloids in Bacterial Virulence

Amyloid fibrils are best known as a product of human and animal protein misfolding disorders, where amyloid formation is associated with cytotoxicity and disease. It is now evident that for some proteins, the amyloid state constitutes the native structure and serves a functional role. These functional amyloids are proving widespread in bacteria and fungi, fulfilling diverse functions as structural components in biofilms or spore coats, as toxins and surface-active fibers, as epigenetic material, peptide reservoirs or adhesins mediating binding to and internalization into host cells. In this review, we will focus on the role of functional amyloids in bacterial pathogenesis. The role of functional amyloids as virulence factor is diverse but mostly indirect. Nevertheless, functional amyloid pathways deserve consideration for the acute and long-term effects of the infectious disease process and may form valid antimicrobial targets.     

Study of Exosomes Shed New Light on Physiology of Amyloidogenesis

Accumulation of toxic amyloid oligomers, a key feature in the pathogenesis of amyloid-related diseases, results from an imbalance between generation and clearance of amyloidogenic proteins. Cell biology has brought to light the key roles of multivesicular endosomes (MVEs) and their intraluminal vesicles (ILVs), which can be secreted as exosomes, in amyloid generation and clearance. To better understand these roles, we have investigated a relevant physiological model of amyloid formation in pigment cells. These cells have tuned their endosomes to optimize the formation of functional amyloid fibrils from the premelanosome protein (PMEL) and to avoid potential accumulation of toxic species. The functional amyloids derived from PMEL reveal striking analogies with the generation of Aβ peptides. We have recently strengthened these analogies using extracellular vesicles as reporters of the endosomal processes that regulate PMEL melanogenesis. We have shown that in pigmented cells, apolipoprotein E (ApoE) is associated with ILVs and exosomes, and regulates the formation of PMEL amyloid fibrils in endosomes. This process secures the generation of amyloid fibrils by exploiting ILVs as amyloid-nucleating platforms. This physiological model of amyloidogenesis could shed new light on the roles of MVEs and exosomes in conditions with pathological amyloid metabolism, such as Alzheimer’s disease.