Neurodegenerative amyloidoses: Yeast model

More than 20 human diseases are associated with protein misfolding, which results in the appearance of amyloids, fibrillar aggregates of normally soluble proteins. Such diseases are termed amyloid diseases, or amyloidoses. Of these, only prion diseases are transmissible. Amyloids of the prion type are known for lower eukaryotes. While mammalian prions cause neurodegenerative diseases, prions of lower eukaryotes are associated with some nonchromosomally inherited phenotypic traits. The review summarizes the results of studying the prions of yeast Saccharomyces cerevisiae and data obtained using S. cerevisiae as a model to investigate some human amyloidoses such as Alzheimer’s, Parkinson’s, Huntington’s, and prion diseases.     

Amyloid by Design: Intrinsic Regulation of Microbial Amyloid Assembly

The term amyloid has historically been used to describe fibrillar aggregates formed as the result of protein misfolding and that are associated with a range of diseases broadly termed amyloidoses. The discovery of “functional amyloids” expanded the amyloid umbrella to encompass aggregates structurally similar to disease-associated amyloids but that engage in a variety of biologically useful tasks without incurring toxicity. The mechanisms by which functional amyloid systems ensure nontoxic assembly has provided insights into potential therapeutic strategies for treating amyloidoses. Some of the most-studied functional amyloids are ones produced by bacteria. Curli amyloids are extracellular fibers made by enteric bacteria that function to encase and protect bacterial communities during biofilm formation. Here we review recent studies highlighting microbial functional amyloid assembly systems that are tailored to enable the assembly of non-toxic amyloid aggregates.     

Mechanistic insights into accelerated α-synuclein aggregation mediated by human microbiome-associated functional amyloids

The gut microbiome has been shown to have key implications in the pathogenesis of Parkinson’s disease (PD). The Escherichia coli functional amyloid CsgA is known to accelerate α-synuclein aggregation in vitro and induce PD symptoms in mice. However, the mechanism governing CsgA-mediated acceleration of α-synuclein aggregation is unclear. Here, we show that CsgA can form stable homodimeric species that correlate with faster α-synuclein amyloid aggregation. Furthermore, we identify and characterize new CsgA homologs encoded by bacteria present in the human microbiome. These CsgA homologs display diverse aggregation kinetics, and they differ in their ability to modulate α-synuclein aggregation. Remarkably, we demonstrate that slowing down CsgA aggregation leads to an increased acceleration of α-synuclein aggregation, suggesting that the intrinsic amyloidogenicity of gut bacterial CsgA homologs affects their ability to accelerate α-synuclein aggregation. Finally, we identify a complex between CsgA and α-synuclein that functions as a platform to accelerate α-synuclein aggregation. Taken together, our work reveals complex interplay between bacterial amyloids and α-synuclein that better informs our understanding of PD causation.     

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.     

Catalytic amyloids: Is misfolding folding?

Originally regarded as a disease symptom, amyloids have shown a rich diversity of functions, including biologically beneficial ones. As such, the traditional view of polypeptide aggregation into amyloid-like structures being ‘misfolding’ should rather be viewed as ‘alternative folding.’ Various amyloid folds have been recently used to create highly efficient catalysts with specific catalytic efficiencies rivaling those of enzymes. Here we summarize recent developments and applications of catalytic amyloids, derived from both de novo and bioinspired designs, and discuss how progress in the last 2 years reflects on the field as a whole.     

Amyloid Formation by the Pro-Inflammatory S100A8/A9 Proteins in the Ageing Prostate

Background

The conversion of soluble peptides and proteins into polymeric amyloid structures is a hallmark of many age-related degenerative disorders, including Alzheimer''s disease, type II diabetes and a variety of systemic amyloidoses. We report here that amyloid formation is linked to another major age-related phenomenon − prostate tissue remodelling in middle-aged and elderly men.

Methodology/Principal Findings

By using multidisciplinary analysis of corpora amylacea inclusions in prostate glands of patients diagnosed with prostate cancer we have revealed that their major components are the amyloid forms of S100A8 and S100A9 proteins associated with numerous inflammatory conditions and types of cancer. In prostate protease rich environment the amyloids are stabilized by dystrophic calcification and lateral thickening. We have demonstrated that material closely resembling CA can be produced from S100A8/A9 in vitro under native and acidic conditions and shows the characters of amyloids. This process is facilitated by calcium or zinc, both of which are abundant in ex vivo inclusions. These observations were supported by computational analysis of the S100A8/A9 calcium-dependent aggregation propensity profiles. We found DNA and proteins from Escherichia coli in CA bodies, suggesting that their formation is likely to be associated with bacterial infection. CA inclusions were also accompanied by the activation of macrophages and by an increase in the concentration of S100A8/A9 in the surrounding tissues, indicating inflammatory reactions.

Conclusions/Significance

These findings, taken together, suggest a link between bacterial infection, inflammation and amyloid deposition of pro-inflammatory proteins S100A8/A9 in the prostate gland, such that a self-perpetuating cycle can be triggered and may increase the risk of malignancy in the ageing prostate. The results provide strong support for the prediction that the generic ability of polypeptide chains to convert into amyloids could lead to their involvement in an increasing number of otherwise apparently unrelated diseases, particularly those associated with ageing.     

Two distinct aggregation pathways in transthyretin misfolding and amyloid formation

Misfolding and amyloid formation of transthyretin (TTR) is implicated in numerous degenerative diseases. TTR misfolding is greatly accelerated under acidic conditions, and thus most of the mechanistic studies of TTR amyloid formation have been conducted at various acidic pH values (2–5). In this study, we report the effect of pH on TTR misfolding pathways and amyloid structures. Our combined solution and solid-state NMR studies revealed that TTR amyloid formation can proceed via at least two distinct misfolding pathways depending on the acidic conditions. Under mildly acidic conditions (pH 4.4), tetrameric native TTR appears to dissociate to monomers that maintain most of the native-like β-sheet structures. The amyloidogenic protein undergoes a conformational transition to largely unfolded states at more acidic conditions (pH 2.4), leading to amyloid with distinct molecular structures. Aggregation kinetics is also highly dependent upon the acidic conditions. TTR quickly forms moderately ordered amyloids at pH 4.4, while the aggregation kinetics is dramatically reduced at a lower pH of 2.4. The effect of the pathogenic mutations on aggregation kinetics is also markedly different under the two different acidic conditions. Pathogenic TTR variants (V30M and L55P) aggregate more aggressively than WT TTR at pH 4.4. In contrast, the single-point mutations do not affect the aggregation kinetics at the more acidic condition of pH 2.4. Given that the pathogenic mutations lead to more aggressive forms of TTR amyloidoses, the mildly acidic condition might be more suitable for mechanistic studies of TTR misfolding and aggregation.     

Biological functions of amyloids: Facts and hypotheses

Amyloids are fibrous protein aggregates that arise via polymerization of proteins with their concurrent conformational rearrangement and the formation of a specific cross-β structure. Amyloids are of particular interest as a cause of a vast group of human and animal diseases called amyloidoses. Some of these diseases are caused by prions, a specific type of amyloids, and are transmissible. Apart from mammals, prion amyloids are described in lower eukaryotes, where they act as nonchromosomal genetic determinants. Although amyloids are usually associated with pathologies in humans and animals, the increasing number of findings suggests that the acquisition of an amyloid or prion form by a protein is of biological significance in some cases. The review summarizes the data on the biological significance of prion and nonprion amyloids in a wide range of species from bacteria to mammals.     

In vitro antiamyloidogenic properties of 1,4-naphthoquinones

The aim of this study is to find out whether several 1,4-naphthoquinones (1,4-NQ) can interact with the amyloidogenic pathway of the amyloid precursor protein processing, particularly targeting at β-secretase (BACE), as well as at β-amyloid peptide (Aβ) aggregation and disaggregating preformed Aβ fibrils. Compounds bearing hydroxyl groups at the quinoid (2) or benzenoid rings (5, 6) as well as some 2- and 3-aryl derivatives (11-15) showed BACE inhibitory activity, without effect on amyloid aggregation or disaggregation. The halogenated compounds 8 and 10 were selective for the inhibition of amyloid aggregation. On the other hand, 1,4-naphthoquinone (1), 6-hydroxy-1,4-naphthoquinone (4) and 2-(3,4-dichlorophenyl)-1,4-naphthoquinone (26) did not show any BACE inhibitory activity but were active on amyloid aggregation and disaggregation preformed Aβ fibrils. Juglone (5-hydroxy-1,4-naphthoquinone (3), and 3-(p-hydroxyphenyl)-5-methoxy-1,4-napththoquinone (19) were active on all the three targets. Therefore, we suggest that 1,4-NQ derivatives, specially 3 and 19, should be explored as possible drug candidates or lead compounds for the development of drugs to prevent amyloid aggregation and neurotoxicity in Alzheimer’s disease.     

Unifying features of systemic and cerebral amyloidosis

Amyloidosis is a generic term for a group of clinically and biochemically diverse diseases that are characterized by the deposition of an insoluble fibrillar protein in the extracellular space. Over 16 biochemically distinct amyloids are known. Despite this diversity, all amyloids have a particular ultrastructural and tinctorial appearance, a β-pleated sheet structure, and are codeposited with a group of amyloid-associated proteins. The most common amyloidosis is Alzheimer’s disease (AD), where Aβ is the main component of the amyloid. Recently it has been found that Aβ exists as a normal soluble protein (sAβ) in biological fluids. This links AD more closely to some of the systemic amyloidoses, where the amyloid precursor is found in the circulation normally. Numerous mutations have been found in the Aβ precursor (βPP) gene, associated with familial AD. Many mutations are also found in some of the hereditary systemic amyloidoses. For example, over 40 mutations in the transthyretin (TTR) gene are associated with amyloid. However, both Aβ and TTR related amyloid deposition can occur with no mutation. The pathogenesis of amyloid is complex, and appears to be associated with genetic and environmental risk factors that can be similar in the systemic and cerebral amyloidoses.     

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.     

Clusterin in Alzheimer's disease: An amyloidogenic inhibitor of amyloid formation?

Clusterin is a heterodimeric glycoprotein (α- and β-chain), which has been described as an extracellular molecular chaperone. In humans, clusterin is an amyloid-associated protein, co-localizing with fibrillar deposits in several amyloidoses, including Alzheimer's disease. To clarify its potential implication in amyloid formation, we located aggregation-prone regions within the sequence of clusterin α-chain, via computational methods. We had peptide-analogues, which correspond to each of these regions, chemically synthesized and experimentally demonstrated that all of them can form amyloid-like fibrils. We also provide evidence that the same peptide-analogues can inhibit amyloid-β fibril formation, potentially making them appropriate drug candidates for Alzheimer's disease. At the same time, our findings hint that the respective aggregation-prone clusterin regions may be implicated in the molecular mechanism in which clusterin inhibits amyloid formation. Furthermore, we suggest that molecular chaperones with amyloidogenic properties might have a role in the regulation of amyloid formation, essentially acting as functional amyloids.     

Fluorescent N-arylaminonaphthalene sulfonate probes for amyloid aggregation of alpha-synuclein

The deposition of fibrillar structures (amyloids) is characteristic of pathological conditions including Alzheimer's and Parkinson's diseases. The detection of protein deposits and the evaluation of their kinetics of aggregation are generally based on fluorescent probes such as thioflavin T and Congo red. In a search for improved fluorescence tools for studying amyloid formation, we explored the ability of N-arylaminonaphthalene sulfonate (NAS) derivatives to act as noncovalent probes of α-synuclein (AS) fibrillation, a process linked to Parkinson's disease and other neurodegenerative disorders. The compounds bound to fibrillar AS with micromolar K_ds, and exhibited fluorescence enhancement, hyperchromism, and high anisotropy. We conclude that the probes experience a hydrophobic environment and/or restricted motion in a polar region. Time- and spectrally resolved emission intensity and anisotropy provided further information regarding structural features of the protein and the dynamics of solvent relaxation. The steady-state and time-resolved parameters changed during the course of aggregation. Compared with thioflavin T, NAS derivatives constitute more sensitive and versatile probes for AS aggregation, and in the case of bis-NAS detect oligomeric as well as fibrillar species. They can function in convenient, continuous assays, thereby providing useful tools for studying the mechanisms of amyloid formation and for high-throughput screening of factors inhibiting and/or reversing protein aggregation in neurodegenerative diseases.     

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.     

Could yeast prion domains originate from polyQ/N tracts?

A significant body of evidence shows that polyglutamine (polyQ) tracts are important for various biological functions. The characteristic polymorphism of polyQ length is thought to play an important role in the adaptation of organisms to their environment. However, proteins with expanded polyQ are prone to form amyloids, which cause diseases in humans and animals and toxicity in yeast. Saccharomyces cerevisiae contain at least 8 proteins which can form heritable amyloids, called prions, and most of them are proteins with glutamine- and asparagine-enriched domains. Yeast prion amyloids are susceptible to fragmentation by the protein disaggregase Hsp104, which allows them to propagate and be transmitted to daughter cells during cell divisions. We have previously shown that interspersion of polyQ domains with some non-glutamine residues stimulates fragmentation of polyQ amyloids in yeast and that yeast prion domains are often enriched in one of these residues. These findings indicate that yeast prion domains may have derived from polyQ tracts via accumulation and amplification of mutations. The same hypothesis may be applied to polyasparagine (polyN) tracts, since they display similar properties to polyQ, such as length polymorphism, amyloid formation and toxicity. We propose that mutations in polyQ/N may be favored by natural selection thus making prion domains likely by-products of the evolution of polyQ/N.     

Substoichiometric Hsp104 regulates the genesis and persistence of self-replicable amyloid seeds of Sup35 prion domain

Prion-like self-perpetuating conformational conversion of proteins is involved in both transmissible neurodegenerative diseases in mammals and non-Mendelian inheritance in yeast. The transmissibility of amyloid-like aggregates is dependent on the stoichiometry of chaperones such as heat shock proteins (Hsps), including disaggregases. To provide the mechanistic underpinnings of the formation and persistence of prefibrillar amyloid seeds, we investigated the role of substoichiometric Hsp104 on the in vitro amyloid aggregation of the prion domain (NM-domain) of Saccharomyces cerevisiae Sup35. At low substoichiometric concentrations, we show Hsp104 exhibits a dual role: it considerably accelerates the formation of prefibrillar species by shortening the lag phase but also prolongs their persistence by introducing unusual kinetic halts and delaying their conversion into mature amyloid fibers. Additionally, Hsp104-modulated amyloid species displayed a better seeding capability compared to NM-only amyloids. Using biochemical and biophysical tools coupled with site-specific dynamic readouts, we characterized the distinct structural and dynamical signatures of these amyloids. We reveal that Hsp104-remodeled amyloidogenic species are compositionally diverse in prefibrillar aggregates and are packed in a more ordered fashion compared to NM-only amyloids. Finally, we show these Hsp104-remodeled, conformationally distinct NM aggregates display an enhanced autocatalytic self-templating ability that might be crucial for phenotypic outcomes. Taken together, our results demonstrate that substoichiometric Hsp104 promotes compositional diversity and conformational modulations during amyloid formation, yielding effective prefibrillar seeds that are capable of driving prion-like Sup35 propagation. Our findings underscore the key functional and pathological roles of substoichiometric chaperones in prion-like propagation.     

Protein aggregation and amyloid fibril formation prediction software from primary sequence: towards controlling the formation of bacterial inclusion bodies

Proteins might aggregate into ordered or amorphous structures, utilizing relatively short sequence stretches, usually organized in β-sheet-like assemblies. Here, we attempt to list all available software, developed during the last decade or so, for the prediction of such aggregation-prone stretches from protein primary structure, without distinguishing whether these algorithms predict amino acid sequences destined to be involved in ordered fibrillar amyloids or amorphous aggregates. The results of application of four of these programs on 23 proteins related to amyloidoses are compared. Because protein aggregation during protein production in bacterial cell factories has been shown to resemble amyloid formation, the algorithms might become useful tools to improve the solubility of recombinant proteins and for screening therapeutic approaches against amyloidoses under conditions that mimic physiologically relevant environments. One such example is given.     

Specific Chaperones and Regulatory Domains in Control of Amyloid Formation

Many proteins can form amyloid-like fibrils in vitro, but only about 30 amyloids are linked to disease, whereas some proteins form physiological amyloid-like assemblies. This raises questions of how the formation of toxic protein species during amyloidogenesis is prevented or contained in vivo. Intrinsic chaperoning or regulatory factors can control the aggregation in different protein systems, thereby preventing unwanted aggregation and enabling the biological use of amyloidogenic proteins. The molecular actions of these chaperones and regulators provide clues to the prevention of amyloid disease, as well as to the harnessing of amyloidogenic proteins in medicine and biotechnology.     

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.