Direct assessment in bacteria of prionoid propagation and phenotype selection by Hsp70 chaperone

Protein amyloid aggregates epigenetically determine either advantageous or proteinopathic phenotypes. Prions are infectious amyloidogenic proteins, whereas prionoids lack infectivity but spread from mother to daughter cells. While prion amyloidosis has been studied in yeast and mammalian cells models, the dynamics of transmission of an amyloid proteinopathy has not been addressed yet in bacteria. Using time‐lapse microscopy and a microfluidic set‐up, we have assessed in Escherichia coli the vertical transmission of the amyloidosis caused by the synthetic bacterial model prionoid RepA‐WH1 at single cell resolution within their lineage context. We identify in vivo the coexistence of two strain‐like types of amyloid aggregates within a genetically identical population and a controlled homogeneous environment. The amyloids are either toxic globular particles or single comet‐shaped aggregates that split during cytokinesis and exhibit milder toxicity. Both segregate and propagate in sublineages, yet show interconversion. ClpB (Hsp104) chaperone, key for spreading of yeast prions, has no effect on the dynamics of the two RepA‐WH1 aggregates. However, the propagation of the comet‐like species is DnaK (Hsp70)‐dependent. The bacterial RepA‐WH1 prionoid thus provides key qualitative and quantitative clues on the biology of intracellular amyloid proteinopathies.     

Prion-like propagation of cytosolic protein aggregates

Amyloid formation is a hallmark of several systemic and neurodegenerative diseases. Extracellular amyloid deposits or intracellular inclusions arise from the conformational transition of normally soluble proteins into highly ordered fibrillar aggregates. Amyloid fibrils are formed by nucleated polymerization, a process also shared by prions, proteinaceous infectious agents identified in mammals and fungi. Unlike so called non-infectious amyloids, the aggregation phenotype of prion proteins can be efficiently transmitted between cells and organisms. Recent discoveries in vivo now implicate that even disease-associated intracellular protein aggregates consisting of α-synuclein or Tau have the capacity to seed aggregation of homotypic native proteins and might propagate their amyloid states in a prion-like manner. Studies in tissue culture demonstrate that aggregation of diverse intracellular amyloidogenic proteins can be induced by exogenous fibrillar seeds. Still, a prerequisite for prion-like propagation is the fragmentation of proteinaceous aggregates into smaller seeds that can be transmitted to daughter cells. So far efficient propagation of the aggregation phenotype in the absence of exogenous seeds was only observed for a yeast prion domain expressed in tissue culture. Intrinsic properties of amyloidogenic protein aggregates and a suitable host environment likely determine if a protein polymer can propagate in a prion-like manner in the mammalian cytosol.     

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.     

Prion-like propagation of cytosolic protein aggregates: Insights from cell culture models

Amyloid formation is a hallmark of several systemic and neurodegenerative diseases. Extracellular amyloid deposits or intracellular inclusions arise from the conformational transition of normally soluble proteins into highly ordered fibrillar aggregates. Amyloid fibrils are formed by nucleated polymerization, a process also shared by prions, proteinaceous infectious agents identified in mammals and fungi. Unlike so called non-infectious amyloids, the aggregation phenotype of prion proteins can be efficiently transmitted between cells and organisms. Recent discoveries in vivo now implicate that even disease-associated intracellular protein aggregates consisting of α-synuclein or Tau have the capacity to seed aggregation of homotypic native proteins and might propagate their amyloid states in a prion-like manner. Studies in tissue culture demonstrate that aggregation of diverse intracellular amyloidogenic proteins can be induced by exogenous fibrillar seeds. Still, a prerequisite for prion-like propagation is the fragmentation of proteinaceous aggregates into smaller seeds that can be transmitted to daughter cells. So far efficient propagation of the aggregation phenotype in the absence of exogenous seeds was only observed for a yeast prion domain expressed in tissue culture. Intrinsic properties of amyloidogenic protein aggregates and a suitable host environment likely determine if a protein polymer can propagate in a prion-like manner in the mammalian cytosol.Key words: prion, Sup35, huntingtin, polyglutamine, Tau, co-aggregation, amyloid, α-synuclein     

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.     

Curli biogenesis: Order out of disorder

Many bacteria assemble extracellular amyloid fibers on their cell surface. Secretion of proteins across membranes and the assembly of complex macromolecular structures must be highly coordinated to avoid the accumulation of potentially toxic intracellular protein aggregates. Extracellular amyloid fiber assembly poses an even greater threat to cellular health due to the highly aggregative nature of amyloids and the inherent toxicity of amyloid assembly intermediates. Therefore, temporal and spatial control of amyloid protein secretion is paramount. The biogenesis and assembly of the extracellular bacterial amyloid curli is an ideal system for studying how bacteria cope with the many challenges of controlled and ordered amyloid assembly. Here, we review the recent progress in the curli field that has made curli biogenesis one of the best-understood functional amyloid assembly pathways. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

Natural osmolytes remodel the aggregation pathway of mutant huntingtin exon 1

In response to stress small organic compounds termed osmolytes are ubiquitously accumulated in all cell types to regulate the intracellular solvent quality and to counteract the deleterious effect on the stability and function of cellular proteins. Given the evidence that destabilization of the native state of a protein either by mutation or by environmental changes triggers the aggregation in the neurodegenerative pathologies, the modulation of the intracellular solute composition with osmolytes is an attractive strategy to stabilize an aggregating protein. Here we report the effect of three natural osmolytes on the in vivo and in vitro aggregation landscape of huntingtin exon 1 implicated in the Huntington's disease. Trimethylamine N-oxide (TMAO) and proline redirect amyloid fibrillogenesis of the pathological huntingtin exon 1 to nonamyloidogenic amorphous assemblies via two dissimilar molecular mechanisms. TMAO causes a rapid formation of bulky amorphous aggregates with minimally exposed surface area, whereas proline solubilizes the monomer and suppresses the accumulation of early transient aggregates. Conversely, glycine-betaine enhances fibrillization in a fashion reminiscent of the genesis of functional amyloids. Strikingly, none of the natural osmolytes can completely abrogate the aggregate formation; however, they redirect the amyloidogenesis into alternative, nontoxic aggregate species. Our study reveals new insights into the complex interactions of osmoprotectants with polyQ aggregates.     

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.     

SynAggreg: A Multifunctional High-Throughput Technology for Precision Study of Amyloid Aggregation and Systematic Discovery of Synergistic Inhibitor Compounds

Numerous proteins can coalesce into amyloid self-assemblies, which are responsible for a class of diseases called amyloidoses, but which can also fulfill important biological functions and are of great interest for biotechnology. Amyloid aggregation is a complex multi-step process, poorly prone to detailed structural studies. Therefore, small molecules interacting with amyloids are often used as tools to probe the amyloid aggregation pathway and in some cases to treat amyloidoses as they prevent pathogenic protein aggregation. Here, we report on SynAggreg, an in vitro high-throughput (HT) platform dedicated to the precision study of amyloid aggregation and the effect of modulator compounds. SynAggreg relies on an accurate bi-fluorescent amyloid-tracer readout that overcomes some limitations of existing HT methods. It allows addressing diverse aspects of aggregation modulation that are critical for pathomechanistic studies, such as the specificity of compounds toward various amyloids and their effects on aggregation kinetics, as well as the co-assembly propensity of distinct amyloids and the influence of prion-like seeding on self-assembly. Furthermore, SynAggreg is the first HT technology that integrates tailored methodology to systematically identify synergistic compound combinations—an emerging strategy to improve fatal amyloidoses by targeting multiple steps of the aggregation pathway. To this end, we apply analytical combinatorial scores to rank the inhibition efficiency of couples of compounds and to readily detect synergism. Finally, the SynAggreg platform should be suited for the characterization of a broad class of amyloids, whether of interest for drug development purposes, for fundamental research on amyloid functions, or for biotechnological applications.     

A DNA‐promoted amyloid proteinopathy in Escherichia coli

Protein amyloids arise from the conformational conversion and assembly of a soluble protein into fibrilar aggregates with a crossed β‐sheet backbone. Amyloid aggregates are able to replicate by acting as a template for the structural transformation and accretion of further protein molecules. In physicochemical terms, amyloids arguably constitute the simplest self‐replicative macromolecular assemblies. Similarly to the mammalian proteins PrP and α‐synuclein, the winged‐helix dimerization (WH1) domain of the bacterial, plasmid‐encoded protein RepA can assemble into amyloid fibres upon binding to DNA in vitro. Here we report that a hyper‐amyloidogenic functional variant (A31V) of RepA, fused to a red fluorescent protein, causes an amyloid proteinopathy in Escherichia coli with the following features: (i) in the presence of multiple copies of the specific DNA sequence opsp, WH1(A31V) accumulates as cytoplasmatic inclusions segregated from the nucleoid; (ii) such aggregates are amyloid in nature; (iii) bacteria carrying the amyloid inclusions age, exhibiting a fivefold expanded generation time; (iv) before cytokinesis, small inclusions are assembled de novo and transferred to the daughter cells, in which transmission failures cure amyloidosis; and (v) in the absence of inducer DNA, purified cellular WH1(A31V) inclusions seed amyloid fibre growth in vitro from the soluble protein. RepA‐WH1 is a suitable bacterial model system for amyloid proteinopathies.     

Functional diversity of protein fibrillar aggregates from physiology to RNA granules to neurodegenerative diseases

Many proteins exhibit propensities to form fibrillar aggregates called amyloids that are rich in β-sheet structures. Abnormal accumulation of amyloids in the brain and spinal cords is well known as a major pathological change in neurodegenerative diseases; therefore, amyloids have long been considered as disease culprits formed via protein misfolding and should be avoided in healthy cells. Recently, however, increasing numbers of proteins have been identified that require formation of fibrillar states for exertion of their physiological functions, and the critical roles of such functional amyloids include a molecular switch for environmental adaptation, a structural template for catalysis, and a regulator of intracellular signaling. Protein amyloids will, therefore, be more prevailed in our physiologies than we have expected so far. Here, we have reviewed recent studies on such regulatory roles of protein fibrillar aggregates in various physiologies and further discussed possible relations of functional to pathological amyloids.     

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.     

Amyloidogenesis of Tau protein

The role of microtubule‐associated protein Tau in neurodegeneration has been extensively investigated since the discovery of Tau amyloid aggregates in the brains of patients with Alzheimer's disease (AD). The process of formation of amyloid fibrils is known as amyloidogenesis and attracts much attention as a potential target in the prevention and treatment of neurodegenerative conditions linked to protein aggregation. Cerebral deposition of amyloid aggregates of Tau is observed not only in AD but also in numerous other tauopathies and prion diseases. Amyloidogenesis of intrinsically unstructured monomers of Tau can be triggered by mutations in the Tau gene, post‐translational modifications, or interactions with polyanionic molecules and aggregation‐prone proteins/peptides. The self‐assembly of amyloid fibrils of Tau shares a number of characteristic features with amyloidogenesis of other proteins involved in neurodegenerative diseases. For example, in vitro experiments have demonstrated that the nucleation phase, which is the rate‐limiting stage of Tau amyloidogenesis, is shortened in the presence of fragmented preformed Tau fibrils acting as aggregation templates (“seeds”). Accordingly, Tau aggregates released by tauopathy‐affected neurons can spread the neurodegenerative process in the brain through a prion‐like mechanism, originally described for the pathogenic form of prion protein. Moreover, Tau has been shown to form amyloid strains—structurally diverse self‐propagating aggregates of potentially various pathological effects, resembling in this respect prion strains. Here, we review the current literature on Tau aggregation and discuss mechanisms of propagation of Tau amyloid in the light of the prion‐like paradigm.     

Inclusion bodies: specificity in their aggregation process and amyloid-like structure

The accumulation of aggregated protein in the cell is associated with the pathology of many diseases and constitutes a major concern in protein production. Intracellular aggregates have been traditionally regarded as nonspecific associations of misfolded polypeptides. This view is challenged by studies demonstrating that, in vitro, aggregation often involves specific interactions. However, little is known about the specificity of in vivo protein deposition. Here, we investigate the degree of in vivo co-aggregation between two self-aggregating proteins, Abeta42 amyloid peptide and foot-and-mouth disease virus VP1 capsid protein, in prokaryotic cells. In addition, the ultrastructure of intracellular aggregates is explored to decipher whether amyloid fibrils and intracellular protein inclusions share structural properties. The data indicate that in vivo protein aggregation exhibits a remarkable specificity that depends on the establishment of selective interactions and results in the formation of oligomeric and fibrillar structures displaying amyloid-like properties. These features allow prokaryotic Abeta42 intracellular aggregates to act as effective seeds in the formation of Abeta42 amyloid fibrils. Overall, our results suggest that conserved mechanisms underlie protein aggregation in different organisms. They also have important implications for biotechnological and biomedical applications of recombinant polypeptides.     

Large proteins have a great tendency to aggregate but a low propensity to form amyloid fibrils

The assembly of soluble proteins into ordered fibrillar aggregates with cross-β structure is an essential event of many human diseases. The polypeptides undergoing aggregation are generally small in size. To explore if the small size is a primary determinant for the formation of amyloids under pathological conditions we have created two databases of proteins, forming amyloid-related and non-amyloid deposits in human diseases, respectively. The size distributions of the two protein populations are well separated, with the systems forming non-amyloid deposits appearing significantly larger. We have then investigated the propensity of the 486-residue hexokinase-B from Saccharomyces cerevisiae (YHKB) to form amyloid-like fibrils in vitro. This size is intermediate between the size distributions of amyloid and non-amyloid forming proteins. Aggregation was induced under conditions known to be most effective for amyloid formation by normally globular proteins: (i) low pH with salts, (ii) pH 5.5 with trifluoroethanol. In both situations YHKB aggregated very rapidly into species with significant β-sheet structure, as detected using circular dichroism and X-ray diffraction, but a weak Thioflavin T and Congo red binding. Moreover, atomic force microscopy indicated a morphology distinct from typical amyloid fibrils. Both types of aggregates were cytotoxic to human neuroblastoma cells, as indicated by the MTT assay. This analysis indicates that large proteins have a high tendency to form toxic aggregates, but low propensity to form regular amyloid in vivo and that such a behavior is intrinsically determined by the size of the protein, as suggested by the in vitro analysis of our sample protein.     

Localized amyloids important in diseases outside the brain--lessons from the islets of Langerhans and the thoracic aorta

It has long been understood that amyloids can be lethal in systemic diseases. More recently, it has been accepted that local cerebral aggregation of the small peptide Aβ is involved in the pathogenesis of Alzheimer's disease. Protein aggregation, with the generation of small amyloid deposits in specific organs, also occurs outside the central nervous system and often is associated with increased cell death. In this review, we discuss two lesser known but common localized amyloid fibril-forming proteins: the polypeptide hormone islet amyloid polypeptide (IAPP) and the lactadherin-derived peptide medin. IAPP aggregates and induces the depletion of islet β-cells in type 2 diabetes and in islets transplanted into type 1 diabetic subjects. Initial amyloid deposition occurs intracellularly and parts of this amyloid consist of proIAPP. Medin derived from lactadherin expressed by smooth muscle cells aggregates into amyloid in certain arteries, particularly the thoracic aortic media layer, and may have a role in the generation of the potentially lethal conditions of thoracic aortic aneurysm and dissection.     

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.     

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.     

Usage of a dataset of NMR resolved protein structures to test aggregation versus solubility prediction algorithms

There has been an increased interest in computational methods for amyloid and (or) aggregate prediction, due to the prevalence of these aggregates in numerous diseases and their recently discovered functional importance. To evaluate these methods, several datasets have been compiled. Typically, aggregation‐prone regions of proteins, which form aggregates or amyloids in vivo, are more than 15 residues long and intrinsically disordered. However, the number of such experimentally established amyloid forming and non‐forming sequences are limited, not exceeding one hundred entries in existing databases. In this work, we parsed all available NMR‐resolved protein structures from the PDB and assembled a new, sevenfold larger, dataset of unfolded sequences, soluble at high concentrations. We proposed to use these sequences as a negative set for evaluating methods for predicting aggregation in vivo. We also present the results of benchmarking cutting edge tools for the prediction of aggregation versus solubility propensity.     

The effect of amyloidogenic peptides on bacterial aging correlates with their intrinsic aggregation propensity

The formation of aggregates by misfolded proteins is thought to be inherently toxic, affecting cell fitness. This observation has led to the suggestion that selection against protein aggregation might be a major constraint on protein evolution. The precise fitness cost associated with protein aggregation has been traditionally difficult to evaluate. Moreover, it is not known if the detrimental effect of aggregates on cell physiology is generic or depends on the specific structural features of the protein deposit. In bacteria, the accumulation of intracellular protein aggregates reduces cell reproductive ability, promoting cellular aging. Here, we exploit the cell division defects promoted by the intracellular aggregation of Alzheimer's-disease-related amyloid β peptide in bacteria to demonstrate that the fitness cost associated with protein misfolding and aggregation is connected to the protein sequence, which controls both the in vivo aggregation rates and the conformational properties of the aggregates. We also show that the deleterious impact of protein aggregation on bacterial division can be buffered by molecular chaperones, likely broadening the sequential space on which natural selection can act. Overall, the results in the present work have potential implications for the evolution of proteins and provide a robust system to experimentally model and quantify the impact of protein aggregation on cell fitness.