Emerging Paradigms for Synthetic Design of Functional Amyloids

Many living organisms make use of diverse amyloid proteins as functional building blocks to fulfill a variety of physiological applications. This fact, along with the intrinsic self-assembly and outstanding material properties of amyloids, has prompted a significant amount of research in the synthetic design of functional amyloids to form diverse nanoarchitectures, molecular materials, and hybrid or composite materials. In particular, a new research paradigm has recently been advanced that uses synthetic biology to harness functional amyloids with cells as living materials or functional devices. Here we outline important progress in the synthetic design of functional amyloids, in the context of both non-living and living systems. We propose several important tools and underline emerging techniques and principles that might be useful in advancing the future synthetic design of functional amyloids.     

Functional Amyloids: Where Supramolecular Amyloid Assembly Controls Biological Activity or Generates New Functionality

Functional amyloids are a rapidly expanding class of fibrillar protein structures, with a core cross-β scaffold, where novel and advantageous biological function is generated by the assembly of the amyloid. The growing number of amyloid structures determined at high resolution reveal how this supramolecular template both accommodates a wide variety of amino acid sequences and also imposes selectivity on the assembly process. The amyloid fibril can no longer be considered a generic aggregate, even when associated with disease and loss of function. In functional amyloids the polymeric β-sheet rich structure provides multiple different examples of unique control mechanisms and structures that are finely tuned to deliver assembly or disassembly in response to physiological or environmental cues. Here we review the range of mechanisms at play in natural, functional amyloids, where tight control of amyloidogenicity is achieved by environmental triggers of conformational change, proteolytic generation of amyloidogenic fragments, or heteromeric seeding and amyloid fibril stability. In the amyloid fibril form, activity can be regulated by pH, ligand binding and higher order protofilament or fibril architectures that impact the arrangement of associated domains and amyloid stability. The growing understanding of the molecular basis for the control of structure and functionality delivered by natural amyloids in nearly all life forms should inform the development of therapies for amyloid-associated diseases and guide the design of innovative biomaterials.     

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.     

The formation of Escherichia coli curli amyloid fibrils is mediated by prion-like peptide repeats

Amyloid fibril formation is the hallmark of major human maladies including Alzheimer's disease, type II diabetes, and prion diseases. Prion-like phenomena were also observed in yeast. Although not evolutionarily related, one similarity between the animal PrP and the yeast Sup35 prion proteins is the occurrence of short peptide repeats that are assumed to play a key role in the assembly of the amyloid structures. It was recently demonstrated that typical amyloid fibril formation is associated with biofilm formation by Escherichia coli. Here, we note the functional and structural similarity between oligopeptide repeats of the major curli protein and those of animal and yeast prions. We demonstrate that synthetic peptides corresponding to the repeats form fibrillar structures. Furthermore, conjugation of beta-breaker elements to the prion-like repeat significantly inhibits amyloid formation and cell invasion of curli-expressing bacteria. This implies a functional role of the repeat in the self-assembly of the fibrils. Since mammal prion, yeast prion, and curli protein are evolutionarily distinct, the conserved peptide repeats most likely define an optimized self-association motif that was independently evolved by diverse systems.     

The Tubular Sheaths Encasing Methanosaeta thermophila Filaments Are Functional Amyloids

Archaea are renowned for their ability to thrive in extreme environments, although they can be found in virtually all habitats. Their adaptive success is linked to their unique cell envelopes that are extremely resistant to chemical and thermal denaturation and that resist proteolysis by common proteases. Here we employ amyloid-specific conformation antibodies and biophysical techniques to show that the extracellular cell wall sheaths encasing the methanogenic archaea Methanosaeta thermophila PT are functional amyloids. Depolymerization of sheaths and subsequent MS/MS analyses revealed that the sheaths are composed of a single major sheath protein (MspA). The amyloidogenic nature of MspA was confirmed by in vitro amyloid formation of recombinant MspA under a wide range of environmental conditions. This is the first report of a functional amyloid from the archaeal domain of life. The amyloid nature explains the extreme resistance of the sheath, the elastic properties that allow diffusible substrates to penetrate through expandable hoop boundaries, and how the sheaths are able to split and elongate outside the cell. The archaeal sheath amyloids do not share homology with any of the currently known functional amyloids and clearly represent a new function of the amyloid protein fold.     

AßT Amyloidogenesis: Unique,or Variation on a Systemic Theme

Abstract

For more than a century amyloid was considered to be an interesting, unique, but inconsequential pathologic entity that rarely caused significant clinical problems. We now recognize that amyloid is not one entity. In vivo it is a uniform organization of a disease, or process, specific protein co-deposited with a set of common structural components. Amyloid has been implicated in the pathogenesis of diseases affecting millions of patients. These range from Alzheimer's disease, adult-onset diabetes, consequences of prolonged renal dialysis, to the historically recognized systemic forms associated with inflammation and plasma cell disturbances. Strong evidence is emerging that even when deposited in local organ sites significant physiologic effects may ensue.

With emphasis on Aβ amyloid, we review the present definition, classification, and general in vivo pathogenetic events believed to be involved in the deposition of amyloids. This encompasses the need for an adequate amyloid precursor protein pool, whether precursor proteolysis is required prior to deposition, amyloidogenic amino acid sequences, fibrillogenic nucleating particles, and an in vivo microenvironment conducive to fibrillogenesis. The latter includes several components that seem to be part of all amyloids. The role these common components may play in amyloid accumulation, why amyloids tend to be associated with basement membranes, and how one may use these findings for anti-amyloid therapeutic strategies is also examined.     

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.     

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.     

When amyloids become prions

The conformational diseases, linked to protein aggregation into amyloid conformations, range from non-infectious neurodegenerative disorders, such as Alzheimer''s disease (AD), to highly infectious ones, such as human transmissible spongiform encephalopathies (TSEs). They are commonly known as prion diseases. However, since all amyloids could be considered prions (from those involved in cell-to-cell transmission to those responsible for real neuronal invasion), it is necessary to find an underlying cause of the different capacity to infect that each of the proteins prone to form amyloids has. As proposed here, both the intrinsic cytotoxicity and the number of nuclei of aggregation per cell could be key factors in this transmission capacity of each amyloid.     

In vitro polymerization of a functional Escherichia coli amyloid protein

Amyloid formation is characterized by the conversion of soluble proteins into biochemically and structurally distinct fibers. Although amyloid formation is traditionally associated with diseases such as Alzheimer disease, a number of biologically functional amyloids have recently been described. Curli are amyloid fibers produced by Escherichia coli that contribute to biofilm formation and other important physiological processes. We characterized the polymerization properties of the major curli subunit protein CsgA. CsgA polymerizes into an amyloid fiber in a sigmoidal kinetic fashion with a distinct lag, growth, and stationary phase. Adding sonicated preformed CsgA fibers to the polymerization reaction can significantly shorten the duration of the lag phase. We also demonstrate that the conversion of soluble CsgA into an insoluble fiber involves the transient formation of an intermediate similar to that characterized for several disease-associated amyloids. The CsgA core amyloid domain can be divided into five repeating units that share sequence and structural hallmarks. We show that peptides representing three of these repeating units are amyloidogenic in vitro. Although the defining characteristics of CsgA polymerization appear conserved with disease-associated amyloids, these proteins evolved in diverse systems and for different purposes. Therefore, amyloidogenesis appears to be an innate protein folding pathway that can be capitalized on to fulfill normal physiological tasks.     

De Novo Designed α-Sheet Peptides Inhibit Functional Amyloid Formation of Streptococcus mutans Biofilms

Streptococcus mutans is a bacterial species that predominates in the oral microbiome. S. mutans binds to the tooth surface, metabolizes sugars and produces acid, leading to cavity formation. S. mutans can also cause infectious endocarditis. Recent evidence suggests that S. mutans biofilms contain amyloid fibrils. Amyloids are insoluble fibrillar protein aggregates, and bacteria use functional amyloids to improve robustness of their biofilms. While the functional amyloids in bacteria such as Escherichia coli and Staphylococcus aureus have been heavily investigated, little is known about the mechanism of S. mutans amyloid formation. Previous results from our laboratory with the amyloidogenic proteins and peptides from the aforementioned bacteria and other mammalian amyloid systems suggest that amyloid formation progresses via an intermediate that adopts a unique secondary structure—α-sheet. De novo designed peptides with alternating l- and d-amino acid also adopt an α-sheet secondary structure and inhibit amyloid formation by binding to soluble oligomeric species during amyloidogenesis. Inhibition of fibrillization by α-sheet peptides suggests the presence of α-sheet during amyloid formation. To investigate the mechanism of functional amyloid formation in S. mutans, α-sheet peptides were compared to epigallocatechin gallate for their ability to inhibit fibril formation in S. mutans. Inhibition was demonstrated in a biofilm plate assay and on hydroxyapatite surfaces both in S. mutans alone and in bacteria from human saliva. The observed inhibition suggests that an α-sheet mediated mechanism may be operative during functional amyloid formation.     

Structure-activity relationship of amyloid fibrils

Protein aggregation is a process in which proteins self-associate into imperfectly ordered macroscopic entities. Such aggregates are generally classified as either amorphous or highly ordered, the most common form of the latter being amyloid fibrils. Amyloid fibrils composed of cross-β-sheet structure are the pathological hallmarks of several diseases including Alzheimer’s disease, but are also associated with functional states such as the fungal HET-s prion. This review aims to summarize the recent high-resolution structural studies of amyloid fibrils in light of their (potential) activities. We propose that the repetitive nature of the cross-β-sheet structure of amyloids is key for their multiple properties: the repeating motifs can translate a rather non-specific interaction into a specific one through cooperativity.     

The Hofmeister effect on amyloid formation using yeast prion protein

A variety of proteins are capable of converting from their soluble forms into highly ordered fibrous cross‐β aggregates (amyloids). This conversion is associated with certain pathological conditions in mammals, such as Alzheimer disease, and provides a basis for the infectious or hereditary protein isoforms (prions), causing neurodegenerative disorders in mammals and controlling heritable phenotypes in yeast. The N‐proximal region of the yeast prion protein Sup35 (Sup35NM) is frequently used as a model system for amyloid conversion studies in vitro. Traditionally, amyloids are recognized by their ability to bind Congo Red dye specific to β‐sheet rich structures. However, methods for quantifying amyloid fibril formation thus far were based on measurements linking Congo Red absorbance to concentration of insulin fibrils and may not be directly applicable to other amyloid‐forming proteins. Here, we present a corrected formula for measuring amyloid formation of Sup35NM by Congo Red assay. By utilizing this corrected procedure, we explore the effect of different sodium salts on the lag time and maximum rate of amyloid formation by Sup35NM. We find that increased kosmotropicity promotes amyloid polymerization in accordance with the Hofmeister series. In contrast, chaotropes inhibit polymerization, with the strength of inhibition correlating with the B‐viscosity coefficient of the Jones‐Dole equation, an increasingly accepted measure for the quantification of the Hofmeister series.     

On the evolutionary trajectories of signal-transducing amyloids in fungi and beyond

In the last decade, multiple reports have established that amyloids can bear important functional roles in a variety of biological processes and in distant taxonomic clades. In filamentous fungi, amyloids are involved in a signal transducing mechanism in which a group of NOD-like receptors (NLRs) controls downstream effector proteins to induce a programmed cell death reaction. A structurally characterized example of fungal signal-transducing amyloid is the prion-forming domain (PFD) of the HET-S toxin from Podospora anserina. Amyloid-mediated programmed cell death is equally reported in metazoans in the context of innate immunity and antiviral response. The cell death reaction, described as programmed necrosis, is dependent on an amyloid-forming RHIM motif (RIP homotypic interaction motif). An evolutionary link between the RHIM and the PFD signaling amyloids has been previously reported. Our recent study ties further the signaling amyloids in fungi and metazoans, reporting a fungal signal-transducing domain with amyloid and prion-like properties, which shows significant sequence similarity to the metazoan RHIM motif. Here, I discuss the expanding class of the signal-transducing amyloids and reflect on the possible evolutionary scenarios of their diversification.     

From arthritis to Alzheimer's disease: current concepts on the pathogenesis of amyloidosis

Amyloid is a generic term referring to a group of diverse but specific extracellular protein deposits which all have common morphologic properties, staining characteristics, and x-ray diffraction and infrared spectra. This review considers the new classification of amyloids, based on the underlying peptides forming the protein fibril, and their diversity. The pathogenesis of inflammation-associated amyloid and its relationship to high density lipoprotein metabolism is examined in detail. The lessons learned from models of inflammation-associated amyloid are extended to the other amyloids (e.g., Alzheimer's disease), and potential reasons for the common structural properties of all amyloids are explored.     

MALDI-Mass Spectrometry Imaging Identifies Vitronectin as a Common Constituent of Amyloid Deposits

Amyloids are pathological intra- and extracellular fibrillar aggregates of polypeptides with a cross-β-sheet structure and characteristic tinctorial properties. The amyloid deposits commonly enclose several non-fibrillar components of the extracellular matrix. Their potential to regulate the formation and aggregation process of amyloid fibrils is still poorly understood. For a better understanding of the role of the extracellular matrix in amyloidosis, it is essential to gain deeper insights into the composition of amyloid deposits. Here, we utilized matrix-assisted laser desorption and ionization mass spectrometry imaging to identify extracellular matrix compounds in amyloid deposits. Using this technique, we identified and determined the spatial distribution of vitronectin within AApoAI-, ALλ-, ATTR- and AIns amyloid deposits and, using immunohistochemistry, validated the spatial overlap of vitronectin with amyloids in 175 cases with diverse types of amyloid in several different tissues.     

Challenges in sample preparation and structure determination of amyloids by cryo-EM

Amyloids share a common architecture but play disparate biological roles in processes ranging from bacterial defense mechanisms to protein misfolding diseases. Their structures are highly polymorphic, which makes them difficult to study by X-ray diffraction or NMR spectroscopy. Our understanding of amyloid structures is due in large part to recent advances in the field of cryo-EM, which allows for determining the polymorphs separately. In this review, we highlight the main stepping stones leading to the substantial number of high-resolution amyloid fibril structures known today as well as recent developments regarding automation and software in cryo-EM. We discuss that sample preparation should move closer to physiological conditions to understand how amyloid aggregation and disease are linked. We further highlight new approaches to address heterogeneity and polymorphism of amyloid fibrils in EM image processing and give an outlook to the upcoming challenges in researching the structural biology of amyloids.     

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.     

Facilitated aggregation of FG nucleoporins under molecular crowding conditions

Intrinsically disordered and phenylalanine–glycine‐rich nucleoporins (FG Nups) form a crowded and selective transport conduit inside the NPC that can only be transited with the help of nuclear transport receptors (NTRs). It has been shown in vitro that FG Nups can assemble into two distinct appearances, amyloids and hydrogels. If and how these phenomena are linked and if they have a physiological role still remains unclear. Using a variety of high‐resolution fluorescence and electron microscopic (EM) tools, we reveal that crowding conditions mimicking the NPC environment can accelerate the aggregation and amyloid formation speed of yeast and human FG Nups by orders of magnitude. Aggregation can be inhibited by NTRs, providing a rationale on how the cell might control amyloid formation of FG Nups. The superb spatial resolving power of EM also reveals that hydrogels are enlaced amyloid fibres, and these findings have implications for existing transport models and for NPC assembly.     

Polymerizing the fibre between bacteria and host cells: the biogenesis of functional amyloid fibres

Amyloid fibres are proteinaceous aggregates associated with several human diseases, including Alzheimer's, Huntington's and Creutzfeldt Jakob's. Disease-associated amyloid formation is the result of proteins that misfold and aggregate into β sheet-rich fibre polymers. Cellular toxicity is readily associated with amyloidogenesis, although the molecular mechanism of toxicity remains unknown. Recently, a new class of 'functional' amyloid fibres was discovered that demonstrates that amyloids can be utilized as a productive part of cellular biology. These functional amyloids will provide unique insights into how amyloid formation can be controlled and made less cytotoxic. Bacteria produce some of the best-characterized functional amyloids, including a surface amyloid fibre called curli. Assembled by enteric bacteria, curli fibres mediate attachment to surfaces and host tissues. Some bacterial amyloids, like harpins and microcinE492, have exploited amyloid toxicity in a directed and functional manner. Here, we review and discuss the functional amyloids assembled by bacteria. Special emphasis will be paid to the biology of functional amyloid synthesis and the connections between bacterial physiology and pathology.