Intrinsically disordered proteins in the formation of functional amyloids from bacteria to humans

CHAPTER FIVE

Intrinsically disordered proteins in the formation of functional amyloids from bacteria to humans Anamika Avni, Hema M. Swasthi, Anupa Majumdar, Samrat Mukhopadhyay* Centre for Protein Science, Design and Engineering, Department of Biological Sciences, and Department of Chemical Sciences, Indian Institute of Science Education and Research (IISER) Mohali, Punjab, India *Corresponding author: e-mail address: [email protected]

Contents 1. An introduction to intrinsically disordered proteins 2. Mechanism of protein aggregation and amyloid formation 3. Supramolecular architecture of amyloids 4. Amyloids as functional workhorses 5. Bacterial functional amyloids 6. Functional amyloids in yeast 7. Functional amyloids in long-term memory in Aplysia and Drosophila 8. Amyloid fibers in spider silk 9. Functional amyloids in vertebrates and higher organisms 10. Functional amyloids in mammals and humans 11. Functional amyloids in the plant kingdom 12. Conclusions and future directions Acknowledgments References

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Abstract Amyloids are nanoscopic ordered self-assemblies of misfolded proteins that are formed via aggregation of partially unfolded or intrinsically disordered proteins (IDPs) and are commonly linked to devastating human diseases. An enlarging body of recent research has demonstrated that certain amyloids can be beneficial and participate in a wide range of physiological functions from bacteria to humans. These amyloids are termed as functional amyloids. Like disease-associated amyloids, a vast majority of functional amyloids are derived from a range of IDPs or hybrid proteins containing ordered domains and intrinsically disordered regions (IDRs). In this chapter, we describe an account of recent studies on the aggregation behavior of IDPs resulting in the formation of functional amyloids in a diverse range of organisms from bacteria to human. We also discuss the strategies that are used by these organisms to regulate the spatiotemporal amyloid assembly in their physiological functions. Progress in Molecular Biology and Translational Science, Volume 166 ISSN 1877-1173 https://doi.org/10.1016/bs.pmbts.2019.05.005

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1. An introduction to intrinsically disordered proteins Proteins are versatile macromolecular workhorses of life. A mammalian cell typically contains 10,000–20,000 different proteins.1 A critical balance between protein synthesis, abundance, and degradation is essential to maintain cellular proteostasis. Most of the proteins need to adopt a well-defined 3D structure in order to attain the functionally active states.2 The folding information is encoded within the amino acid sequence of the nascent polypeptide chain. A newly synthesized polypeptide chain can possess a large number of conformational states; therefore, the process of protein folding is error-prone.3 Moreover, the biologically active state of a protein is often marginally stable under the physiological conditions.1 The cellular machinery consists of a complex network of molecular chaperones to prevent misfolding and to promote efficient folding.4a Nevertheless, under some conditions a metastable, misfolded, aberrant state of a protein can get accumulated during protein folding and can trigger aggregation resulting in the formation of either amorphous aggregates or thermodynamically more stable ordered amyloid aggregates as described by an energy landscape diagram (Fig. 1A).4b The proteostasis efficiency declines as a function of age and the expression of molecular chaperones are highly down-regulated upon aging.5 An age-related decline in the proteostasis network triggers protein misfolding that can result in the loss of function and/or the gain of toxic function of a protein.6 Such toxic functions facilitate the manifestation of many neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), prion disease as well as of non-neuropathic systemic and localized amyloidosis.3,7 Interestingly, disease-associated amyloids are often formed by the proteins which can readily undergo partial unfolding or by the proteins that lack well-defined 3D structure, namely, intrinsically disordered proteins (IDPs).1 In fact, the development of various protein misfolding diseases originates from conformational changes in corresponding amyloidogenic proteins, where ordered proteins (partially) unravel to form partially unfolded amyloidogenic conformers, whereas, the formation of such amyloidogenic conformers by extended IDPs might require their collapse and/or partial folding.8 IDPs confront the classical sequence-structurefunction paradigm which states that a function of a protein critically depends on its well-defined 3D structure. IDPs are highly flexible and exist in an ensemble of conformational states; nevertheless, they are functional.9,10 The amino acid content of IDPs is significantly different from that of

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Fig. 1 (A) Schematic representation of the protein folding energy landscape. The righthand part shows the aggregation funnel. (B) A schematic of nucleation-dependent polymerization model of protein aggregation and amyloid formation. Panel A reproduced and adapted with permission from Springer Nature, Hartl FU, Hayer-Hartl M. Converging concepts of protein folding in vitro and in vivo. Nat Struct Mol Biol. 2009;16(6):574–581. https://doi.org/10.1038/nsmb.1591. Panel B reproduced and adapted with permission from Springer Nature, Iadanza MG, Jackson MP, Hewitt EW, Ranson NA, Radford SE. A new era for understanding amyloid structures and disease. Nat Rev Mol Cell Biol. 2018;19(12):755–773. doi:10.1038/s41580-018-0060-8.

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globular proteins. They are usually rich in disorder-promoting amino acids and lack order-promoting/hydrophobic amino acids.6 Many IDPs can undergo folding coupled to binding, in other words, they can undergo a disorder-to-order structural transition upon interacting with the specific binding partners.10,11 They are known to be involved in many functions which include cellular signaling, regulation of translation and transcription, and protein phosphorylation and so forth.12–15 IDPs often do not undergo a complete structural transition even in the bound state, in other words, they remain partially disordered even in the presence of binding partners. Such complexes are termed as “fuzzy” complexes.16,17 The fuzziness allows the protein to have interactions with alternative partners and to have simultaneous interactions with different targets. Recent studies have revealed that membrane-less organelles are formed by IDPs/IDRs with lowcomplexity sequences.18–24 These intracellular liquid condensates are non-stoichiometric supramolecular complexes of proteins and nucleic acids and participate in many critical cellular functions.21 However, maturation of such biomolecular condensates can lead to the formation of solid-like aggregates and amyloid fibrils.25,26 Recent studies have suggested that aggregation of a diverse range of IDPs into amyloid fibrils has important functional implications in a wide range of organisms from bacteria to humans. In the subsequent sections, we will first discuss the key mechanistic and structural issues of amyloids and then will describe these fascinating biologically functional amyloids.

2. Mechanism of protein aggregation and amyloid formation Protein aggregation is typically described by a nucleation-dependent polymerization (NDP) model (Fig. 1B).27–31 NDP has three distinct phases: (a) Nucleation or lag phase: the soluble species associate to form thermodynamically unstable nuclei and addition of pre-formed aggregates may eliminate/shorten the lag phase of protein aggregation; (b) Elongation or log phase: the transient intermediates assemble and trigger the polymerization process; and (c) Saturation phase: fibrils mature and there will be no further addition of soluble species to the fibril end. The monomers, either partially unfolded (for ordered proteins) or collapsed/partially folded (for intrinsically disordered proteins),8 undergo early oligomerization and then reorganize to form the amyloid-competent nuclei. This type of nucleation process, termed as nucleated conformational conversion,32 has been described

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for the aggregation of various proteins such as yeast prion protein (Sup35NM), α-synuclein, Aβ, huntingtin exon 1, hen lysozyme, and IAPP to name a few. A large body of evidence suggests that transient oligomeric species exert more toxicity than mature fibrils.33–36 Another type of aggregation mechanism is isodesmic polymerization or linear polymerization. Isodesmic polymerization does not exhibit three separate phases like NDP. The rate constant of each association step is identical and is independent of the size of the polymer. The NDP mechanism is generally distinguished from linear polymerization based on the existence of a lag phase that can be abolished upon addition of pre-formed seeds and the presence of a critical monomer concentration in order to drive the aggregation process.29 In globular proteins, the aggregation-prone regions are buried within the core of the protein. Therefore, they undergo partial unfolding to give rise to a state that is competent for aggregation and this state follows any of the aggregation mechanisms described above.8,37 A growing body of evidence suggests that the pathogenicity is due to the oligomeric intermediates formed during the process of protein aggregation. Interaction of oligomeric intermediates with the cellular membrane can lead to pore formation that results in cellular dysfunction and cell death. The exposure of hydrophobic residues on the surface of transient intermediates seems to be the determinant of oligomeric toxicity.38,39 Oligomeric species of same size and morphology but with the different solvent-exposed hydrophobic surface have been shown to exhibit varying extents of toxicity. Size of the oligomers is another determinant of toxicity of the misfolded species. Studies on oligomers of different sizes but having the same extent of β-sheet and solvent-exposed hydrophobic surface have demonstrated that the oligomers of smaller size exert more toxicity.28 As the size of the oligomer increases the toxicity decreases. The possible explanation for the toxicity exhibited by small oligomers is that they can diffuse rapidly and can interact with different substrates more readily.40 For instance, in Alzheimer’s disease, it has been shown that extracellular protein oligomers can interact with phospholipids, insulin receptors, membrane receptors, and many other cellular components.33 Oligomers of α-synuclein are known to interact with the cell membrane resulting in the efflux of Ca2+, with mitochondria leading to their dysfunction, and can also interact with the components of the proteostasis network.36 Toxicity of amyloids might be contributed by aberrant interactions of oligomeric intermediates with endogenous cellular components such as phospholipids, protein receptors, RNA. Such altered interactions can potentially induce cellular dysfunction that can ultimately lead to cell death.

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3. Supramolecular architecture of amyloids Amyloids are nanoscopic protein aggregates that form long and unbranched fibrils with certain biophysical and biochemical properties. The morphology of fibrils visualized using transmission electron microscopy (TEM) and atomic force microscopy (AFM) has demonstrated that these fibrils are typically on the order of hundreds of nanometers to micrometers in length and 8–12 nm in width.41,42 Amyloids share a common cross-β structural motif, in which the β-sheets run parallel to the growing fibril axis with their strands perpendicular to the axis (Fig. 2). X-ray diffraction of ˚ corresponds to amyloid fibrils displays two diffraction patterns: 10 A ˚ inter-sheet spacing and 4.8 A corresponds to the inter-strand distance.41,42 A highly ordered arrangement in amyloids allows monitoring their formation using amyloid markers such as thioflavin T (ThT) and congo red.43,44 While a HET-S (2lbu)

d Tau ‘PHF’ (5o3l)

b Ab1–42 ‘LS’ (5oqv)

Ab1–42 (5kk3)

Tau ‘SF’ (5o3t)

c Ab1–40 ‘2A’ (2lmn)

Ab1–40 ‘3Q’ (2lmp)

e a-Synuclein (2n0a)

Fig. 2 The architecture of amyloid fibrils from a wide range of proteins and peptides associated with deadly neurogenerative diseases. The top layer of the polypeptide chain is shown in red. 2A: fibrils with twofold symmetry; 3Q: fibrils with threefold symmetry; PHF: paired fibrils; SF: straight fibrils. The PDB accession numbers are shown in parenthesis. Reproduced with permission from Springer Nature, Iadanza MG, Jackson MP, Hewitt EW, Ranson NA, Radford SE. A new era for understanding amyloid structures and disease. Nat Rev Mol Cell Biol. 2018;19(12):755–773. doi:10.1038/s41580-018-0060-8.

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ThT intercalates with β-rich amyloids and exhibits enhanced fluorescence, Congo red-stained amyloid fibrils exhibit apple-green birefringence under polarized light. Despite their highly ordered structure, amyloids do not lend themselves to crystallography due to the existence of intrinsic structural heterogeneity, polymorphism, and the presence of unstructured domains. X-ray and electron diffraction studies using short fibril-forming peptide of amyloidforming proteins have provided a better atomistic insight into amyloid structures.41 X-ray diffraction studies on microcrystal formed from seven-segment residue GNNQQNY of Sup35 and many other amyloid-forming segment demonstrated that two β-sheets mate to form a steric zipper, which extends throughout the crystal.45 The β-sheets of amyloid associate in pairs and the side-chains of one sheet interdigitate with the side-chains of its mating partner resulting in the formation of a tight steric-zipper interface.41 Such a mating pair of β-sheet is known as the protofilament and two or more protofilaments associate to form amyloid fibrils.41 The mating pair of β-sheets can be bonded in different ways. Aggregates of a single protein can give rise to different amyloid structures, which are termed as polymorphs. Polymorphism in amyloid can arise due to differences in the number of protofilaments association or due to the differences in the mode of their packing. Polymorphism exhibited by the amyloids might be related to prion strains.41,42,45,46 Amyloids possess astonishing tensile strength which is comparable to steel.47 This unusual stability of amyloid is due to the paired β-sheet motif, which is stabilized by many factors such as hydrophobic and van der Waals forces in the steric zipper interface, polarized hydrogen bonds that run up and down the β-sheet, a ladder of bonding by the side chain on the surface of sheets, and the entropy gain upon release of water molecules from the β-sheet upon steric zipper formation.41 Solid-state nuclear magnetic resonance (ssNMR) is a widely used technique to study the molecular structure of amyloid.48,49 ssNMR studies on amyloid fibrils made from isotopically labeled proteins can provide information on backbone conformation and site-specific information of amino acid residue, parallel and anti-parallel organization of β-sheets, and amino acid side chain contacts.50 The information from electron microscopy together with ssNMR can be used to construct full molecular models.49 ssNMR performed on amyloid fibrils formed by Aβ and α-synuclein, that are implicated in AD and PD, respectively, are known to exhibit in-register parallel packing.48,49,51 In-registry is defined as parallel if the translational repeat of the sheet is perpendicular to the strand direction. Additionally, steady-state and time-resolved fluorescence methods offer sensitive site-specific structural and dynamical information on amyloid fibrils.52–54

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In recent years, Cryo-EM has evolved as a prominent structural tool that yields high-resolution (2–3 A˚) structures of amyloid fibrils and can also discern the structural nuances associated with the different cross-β sheet rich polymorphs of amyloid fibrils or filaments derived from brains of patients diagnosed with several neurodegenerative diseases.50,55–59 For instance, the ordered assembly of full-length tau along with five of its isoforms in the human brain contribute toward the formation of the neurofibrillary tangles and inclusion bodies in Alzheimer’s disease (AD) and Pick’s disease (PiD).55,57 Cryo-EM studies of the two distinct types of amyloid filaments, the paired helical filaments and the straight filaments, isolated from an AD patient’s brain, each comprising two C-shaped protofilaments, reveal ˚ characteristic longitudinal crossover distances ranging from 650 to 900 A 57 ˚ and filament widths ranging from 100 to 150 A. In the case of PiD, the narrow Pick filaments (NPF) and the wide Pick filaments (WPF), comprising single elongated protofilaments, exhibit longitudinal crossover distances ˚ , widths ranging from 50 to 300 A ˚ , and show a characteristic packof 1000 A ing of the amyloid core which is completely different from that observed in case of AD55 (Fig. 2). Recently, another novel amyloid fold of tau was identified in the filaments isolated from a patient diagnosed with chronic traumatic encephalopathy, exhibiting a relatively more open conformation encompassing the β-helix region, in comparison to filaments observed in AD.56 Several recent Cryo-EM studies also report on the structure of two distinct amyloid polymorphs, the rod and the twister polymorphs, formed from alpha-synuclein.58,59 The ability of this technique to delineate the distinct amyloid architectures and amino acid side-chain overlaps within the fibrils opens up avenues for its use in the structural investigation of other neurodegenerative diseases. Recent advances in these techniques have also raised the possibility of elucidating the different polymorphic structures of the plaques and intracellular inclusions associated with different disease phenotypes in situ which will help in designing therapeutics against these debilitating diseases.50

4. Amyloids as functional workhorses Protein misfolding leading to amyloid deposition is primarily associated with neurodegenerative diseases. A growing body of evidence suggests that amyloid structure is widespread in nature for beneficial purposes and these amyloids are termed as functional amyloids.60–63 Understanding the mechanism of functional amyloid formation can provide insights into

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Table 1 Select examples of functional amyloids in a host of organisms ranging from bacteria to humans including plants. IDPs Organisms Amyloid functions

Curli

Escherichia coli

Biofilm formation and host cell adhesion and invasion

FapC

Pseudomonas fluorescens

Biofilm formation

Sup35p

Saccharomyces cerevisiae

Regulation of stop-codon readthrough

CPEB

Aplysia and Drosophila

Long-term memory

Spidroin

Araneus diadematus

Structural (spider silk)

Rubber elongation factor (REF)

Hevea brasiliensis

Biosynthesis of natural rubber

Xvelo

Xenopus laevis

Regulation in oogenesis

Pmel17

Homo sapiens

Melanin biogenesis

Semenogelin1 and 2; SEVI 1 and 2

Homo sapiens

Sperm selection, clearance, and antimicrobial activity

how a cell regulates and propagates their formation without contributing toxicity. Functional amyloids are evolved to regulate the spatiotemporal amyloid assembly and sequester the transient toxic oligomeric intermediates. This can be achieved by speeding up the amyloid formation kinetics, often by altering the mechanism from NDP to isodesmic aggregation. The change in the mechanism abolishes the prolonged lag phase and can minimize the accumulation of toxic oligomers. Functional amyloids are found in species ranging from bacteria to humans (Table 1). In the following sections, we discuss a wide range of functional amyloids derived primarily from IDPs.

5. Bacterial functional amyloids Curli is amyloid fimbriae produced by Gram-negative bacteria, Escherichia coli (E. coli). Curli is known to be involved in functions such as biofilm formation, cell-cell adhesion, and is also known to induce inflammatory responses in the host cell.64–67 Curli is the first reported functional amyloid and it is a proteinaceous component expressed on the extracellular

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matrix of enteric bacteria.65,68 Amyloid fibrils formed by curli subunits are composed of β-helical structure69 as opposed to disease-associated amyloids of α-synuclein (Parkinson’s disease), Aβ, and tau (Alzheimer’s disease) which form in-register parallel β-sheet structure. Curli shows tinctorial properties upon binding to congo red and exhibits enhanced fluorescence upon interacting with ThT.68 Unlike many disease-associated amyloids, curli is not SDS soluble and requires formic acid for its monomerization.68 Many disease-associated amyloids are formed as a consequence of aberrant misfolding and aggregation. However, a highly orchestrated machinery is associated with curli biogenesis.60,65,70 The secretion pathway associated with the curli formation is known as nucleation-precipitation mechanism or type VIII secretion system.65,68,70,71 Seven proteins are involved in the curli formation and are encoded by divergently transcribed operons csgBAC and csgDEFG (CSG: curli specific gene) (Fig. 3).65,68,70 Curli comprises aggregates of CsgA and CsgB. CsgA forms the major constituent of curli and the nucleator protein CsgB forms the minor subunit of curli.72,73 CsgD is the master regulator of biofilm formation. CsgD controls the curli expression as well as the extracellular polysaccharide production in E. coli. Five accessory proteins control the spatiotemporal assembly of curli subunits.60,70 CsgC assists like a molecular chaperone, which prevents aggregation and maintains the solubility of curli subunits in the periplasm.74 At a substoichiometric ratio 1:500, CsgC completely inhibits CsgA assembly in vitro. However, CsgC prevents CsgB (truncated) assembly at a high molar ratio (1:10). The molecular mechanism of CsgC mediated inhibition of CsgA aggregation is poorly understood. CsgC might be transiently interacting with soluble and prefibrillar intermediates of CsgA and prevents the ordered amyloid assembly of the latter.74 CsgC is a β-rich soluble protein and it shares an immunoglobulin-like fold. A recent study has demonstrated that human transthyretin (TTR) inhibits CsgA aggregation. Interestingly, TTR possesses structural similarity with CsgC.75 CsgG is a pore-forming lipoprotein and acts as a secretion channel for CsgA, CsgB, and CsgF. CsgG, a 262 amino acid residue protein forms nanomeric transport channel which comprises 36-stranded β-barrel that traverses the outer membrane.76,77 CsgG is selective for CsgA and CsgB under physiological concentration but at an elevated concentration, it can trigger the leakage of periplasmic proteins in vivo.78 CsgE is a periplasmic soluble protein and it is known to inhibit CsgA aggregation in vitro.78 Cells deficient in CsgE impairs translocation of CsgA to the outer membrane which results in less curli production. Studies suggest that CsgG and CsgE

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Fig. 3 Model of curli biogenesis. Seven proteins, encoded by two divergent operons csgDEFG and csgACB are involved in the curli assembly. CsgD is the regulator of csgBAC operon. All the proteins, except CsgD has secretory signal peptide for targeting to the periplasm. The solubility of curli subunits is maintained by CsgC. CsgG is a nanomeric secretion channel which is capped by nine CsgE molecules. CsgA and CsgB are secreted to the cell surface in CsgG-dependent manner. On the cell surface the nucleator protein anchors on the membrane and nucleates CsgA aggregation. CsgF associates with CsgG on the outer surface and is essential for efficient curli assembly.

exist as a dynamic complex of 9:9 stoichiometric ratio. CsgE nanomer acts as a capping adaptor of CsgG secretion channel.77 CsgF is a 12.9 kDa protein and is secreted to the outer surface in CsgG-dependent manner. CsgA fails to polymerize on the cell surface in the absence of CsgF. CsgF might be essential for proper localization of the nucleator protein CsgB or might have a chaperone-like activity that generates fully functional CsgB.79 CsgA and CsgB are composed of three distinct domains, secreted signal peptide, N22 domain, and five imperfect repeats. These proteins are directed to the periplasm via SecYEG and the signal peptide gets proteolytically cleaved

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in the periplasm. Thus, mature CsgA and CsgB are devoid of the secretory signal peptide. N-terminal 22 amino acid is essential for the translocation of CsgA to the outer membrane but this domain is not strictly required for CsgB secretion.80–82 N-22 domain is followed by five imperfect repeats that form the amyloid core. CsgA shares 30% sequence identity and 50% sequence similarity with CsgB. The R1 to R5 repeats of CsgA share a common motif Ser-X5-Gln-X-Gly-X-Gly-Asn-X-Ala-X3-Gln.80 Studies using synthetic peptides have demonstrated that R1 and R5 form long and straight fibrils, whereas R3 forms short aggregates. The repeats R2 and R4 comprise gatekeeper residues and these peptides do not aggregate in vitro.83 R1–R4 repeats of CsgB have Ala-X3-Gln-X-Gly-X2-Asn-X-Ala-X3-Gln. R1, R2, and R4 synthetic peptides aggregate, whereas R3 and R5 do not assemble into amyloids in vitro. Without R4/R5 repeats of CsgB, the protein fails to perform its function and curli subunits are secreted away from the cell surface as an SDS-soluble form.81 The outer membrane of Gram-negative bacteria comprises lipopolysaccharide which is a negatively charged glycophospholipid. Curli subunits, CsgA and CsgB, display a lag phase during in vitro aggregation. In vitro aggregation studies have shown that the interaction of C-terminal region of CsgB (R4 and R5) with lipopolysaccharide (LPS) surface results in the charge neutralization on the protein that triggers a spontaneous formulation of oligomers. These membrane-induced CsgB oligomers heteronucleate the aggregation of CsgA and allow the latter to aggregate without a nucleation phase. Elimination of lag phase might be the plausible mechanism to bypass the toxic events during curli biogenesis on the bacterial membrane84 (Fig. 4A). The major protein component of bacterial biofilm is attributed to curli and the other components include cellulose and DNA. In addition to strengthening and stabilizing the matrix, these components of enteric biofilms individually and in combination contribute to bacterial virulence and transmission.85 Systemic recognition of these pathogen associated biofilm components activates several immune receptors against enteric bacteria.85 The enteric biofilm as well as the DNA enhance the systemic lupus erythematosus and causes the aggregation of α-synuclein involved in Parkinson’s disease.85 Curli is known to interact with a wide range of human proteins which includes plasma proteins and MHC 1. The presence of curli expressing bacteria elongates the blood clotting process. Curliated bacteria exhibit a high binding toward fibrinogen, an important constituent of a blood clot. A recent study has shown the presence of human fibrinogen inhibits the amyloid assembly of the major subunit of curli (Fig. 4B).

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Fig. 4 (A) Model of curli amyloid formation on the lipopolysaccharide surface. (B) Inhibition of CsgA aggregation by human fibrinogen (bottom). Inset shows AFM images of CsgA in the absence (6 h) and in the presence of fibrinogen (24 h). Panel A reproduced and adapted with permission from the American Society for Biochemistry (Continued)

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An interplay between CsgA and fibrinogen might affect the curli biogenesis and biofilm formation. Thus, CsgA and fibrinogen interaction could be a host-defense against bacterial colonization and infection.86 In addition to E. coli fimbriae, amyloid-like fimbriae (ALF) are also produced by Pseudomonas strain UK4. TEM image showed that the cells are encompassed in a thick extracellular matrix that contains embedded fimbriae “similar to, but less distinct than, curli produced by E. coli.”87 Biophysical characterization of purified ALF using far-UV synchrotron radiation circular dichroism (SRCD) and FT-IR showed extensive β-sheet rich structures having morphology identical to E. coli curli.87 The amyloid structure was further supported by ThT fluorescence portraying the tendency of fimbriae to bind ThT. Full genomic sequencing of ALF precursor protein fapC revealed that it contains N-terminal signal sequence followed by three imperfect repeats (R1–3) separated by two linker regions (L1 2) and a C-terminal tail.87 The repeats found are notably different from other fibrins, curli and Tafi. They lack aromatic residues and are rich in glutamine and asparagine residues. Like curli in E. coli, Pseudomonas ALF is also involved in biofilm formation. The heterologous expression of Pseudomonas fapA-F operon in E. coli BL(21)DE3 results in “highly aggregative phenotype.”87

6. Functional amyloids in yeast Human prion protein (PrP) encoded by the PRNP gene is 253 amino acid long and is made mostly of α-helices and is commonly found in the cell membrane of neurons. Misfolded prion undergoes a conformation transition from an α-helical cellular form (PrPc) to a β-rich state. This aberrantly folded infectious prion is termed as scrapie prion (PrPSc).88 When misfolded protein (PrPSc) interacts with normal prion protein, it acts as a template in inducing misfolding in the normal prion protein (PrPc). These prions are resistant to proteases and have an affinity for the brain. Accumulation of misfolded prion can trigger apoptosis and cyst formation that results in the spongy appearance of brain spongiform which ultimately leads to spongiform encephalopathy.89 Fig. 4—Cont’d and Molecular Biology, Swasthi HM, Mukhopadhyay S. Electrostatic lipid-protein interactions sequester the curli amyloid fold on the lipopolysaccharide membrane surface. J Biol Chem. 2017;292(48):19861–19872. doi:10.1074/jbc.M117.815522. Panel B reproduced and adapted with permission from the American Chemical Society, Swasthi HM, Bhasne K, Mahapatra S, Mukhopadhyay S. Human fibrinogen inhibits amyloid assembly of biofilm-forming CsgA. Biochemistry. 2018;57(44):6270–6273. doi:10.1021/acs. biochem.8b00841.

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The presence of the prion protein in yeast and fungus, in addition to mammals, provides a broader definition to prions than just being a proteinaceous infectious particle. Ure2p and Sup35p are prion proteins in Saccharomyces cerevisiae and they give rise to distinct phenotypes in their soluble and misfolded state.90–92 These proteins have a globular domain and an unstructured region which is rich in glutamine and asparagine residues.93 Conversion of the normal prion protein to the misfolded state affects the protein function and creates a host of new phenotypes. These phenotypes are transferred from mother to daughter cells and thus yeast prions, without a change in the genetic material, is capable of changing the biological phenotypes and are regarded as epigenetic modifiers.94 Sup35 is a translation termination factor. The soluble form of the protein recognizes the stop codon, whereas the aggregated state [PSI+] causes readthrough and leads to new biological phenotypes.95 The prion protein normally contains regions required for the formation of the prion state called the prion domains (PrDs).96 These domains are intrinsically disordered and rich in glutamine and asparagine except Mod5p prion domain.97,98 [PSI+] prion state can be detected by introducing a nonsense mutation in the ADE1 gene of adenine biosynthesis pathway.99 In [PSI+] cells, Sup35 is rendered in the amyloid state, that causes a read-through of the stop-codon resulting in the complete synthesis of Ade1 protein and forming white color colonies on YPD medium.99 On the contrary, in [psi ] cells, nonsense mutation blocks the adenine biosynthesis pathway and leads to the accumulation of an intermediate product which upon oxidation gives rise to red color colonies on YPD medium. Structural variations exhibited by different amyloid fibrils formed by a polypeptide chain are referred to as polymorphism. Amyloid fibrils formed by yeast prion protein at 4 °C (Sc4) and 37 °C (Sc37) in vitro can induce two distinct phenotypes in Saccharomyces cerevisiae.100 Sc4 and Sc37 give rise to strong [PSI+] and weak [PSI+], respectively. The NM-domain of Sup35 governs the prion status and this region is sufficient for prion-based inheritance. Picosecond fluorescence depolarization measurements on the single tryptophan (Trp) variants of Sup35 NM have allowed us to construct a sitespecific conformational mobility map of these two distinct amyloid forms. Sc4 fibrils exhibit an overall higher local flexibility compared to Sc37. Trp residues 7 and 21 at the N-terminal end display a higher amplitude of flexibility in the Sc4 fibrils (Fig. 5). The residue positions 184, 221, and 250 in the M-domain are flexible in both Sc4 and Sc37 states. However, these locations in Sc37 are slightly less mobile indicating a relatively tight

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Fig. 5 Time-resolved fluorescence anisotropy decays of different Trp residue positions in two different amyloid forms, Sc4 (blue) and Sc37 (red) and a plot of local flexibility vs. residue positions in two forms of amyloids. Reproduced and adapted with permission from the American Chemical Society, Narang D, Swasthi HM, Mahapatra S, Mukhopadhyay S. Site-specific fluorescence depolarization kinetics distinguishes the amyloid folds responsible for distinct yeast prion strains. J Phys Chem B. 2017;121(36):8447–8453. doi:10.1021/ acs.jpcb.7b05550.

packing within its amyloid fibrils. Higher local mobility of Sc4 suggests a lower degree of packing within the fibrils. This accounts for the fragility and for the exhibition of strong [PSI+] by Sc4 fibrils54.101 The filamentous fungus Podospora anserine contains a prion protein HET-s that is 289 residues long and contains a C-terminal prion-forming domain (218–289).94,102 This protein can exist in the prion state [Het-s] that is responsible for a phenomenon called heterokaryon incompatibility.103,104 Both the protein and the prion domain are capable of forming amyloid fibrils

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in vitro that is contagious to the cells in the non-prion state [Het-s*].105–107 It was proposed that the core of the fibrils of HET-s (218–289) consists of two β-strand-turn-β-strand motifs.102 More recent studies have shown that HET-s fibrils adopt a characteristic left-handed β solenoid structure in which each molecule forms two helical windings with a triangular hydrophobic core.108

7. Functional amyloids in long-term memory in Aplysia and Drosophila Functional amyloid-forming proteins are predominantly IDPs that may be functional both in their soluble as well as in aggregated forms. The aggregated states of a few proteins have shown to exhibit (functional) prion-like behavior, in other words, the aggregates self-perpetuate and inherit in a non-Mendelian fashion. A growing body of evidence suggests that the prion-like domains are associated with long-term memory in a wide range of organisms.109 Cytoplasmic polyadenylation element-binding protein (CPEB) is a neuronal RNA-binding protein that is known to be a functional prion-like protein and undergoes a conformational switch in the brain.110,111 In the aggregated, self-templating form, it can control the growth of protein chains at the synapses and is involved in the stabilization of long-term memory.109,111,112 Human CPEB3 comprises a low-complexity glutamine-rich N-terminal RNA-binding domain that resembles a prionlike state of RNA-binding form of CPEB of Aplysia.113 Studies in a yeast model have revealed the prion-like behavior of CPEB3.112,114 In the basal state, CPEB3 is SUMOylated (SUMO: small ubiquitin-like modifiers) and exists as a synaptic soluble protein.115,116 CPEB3 is known to repress translation of target mRNA in the synaptic cytosol.115,117 Upon neuronal stimulation, CPEB3 is ubiquitylated and deSUMOylated that facilitates fibril formation which further promotes polyadenylation and increases translation of proteins involved in long-term potentiation.112 This functional protein has been identified in a host of organisms like Aplysia CPEB, Drosophila Orb2, CPEB3, and mammals including humans.118–120 They are involved in a range of physiological functions by undergoing a conformational switch regulated by physiological signals, unlike the conventional pathogenic prions where transitions from the soluble to the aggregated state are uncontrolled. This type of mechanism can potentially be present in higher organisms including in humans.

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8. Amyloid fibers in spider silk The silk producing gland of spider spins a host of silk types that are composed of different types of intrinsically unstructured proteins. Dragline silk is the major ampullate silk formed by spidroin protein in the major ampullate gland of the garden spider Araneus diadematus.121,122 Highly repetitive alanineand glycine-rich motifs dominate the polypeptide sequence of the dragline silk proteins.123 These regions contribute to the elasticity, extensibility, and flexibility to the silk threads. Poly(alanine) forms β-sheets and crystalline particles that are responsible for the thread’s strength.124,125 The major ampullate gland consists of three major sections: A-zone, B-zone and a duct.126 A-zone is composed of secretory cells packed with secretory granules that secrete an aqueous solution of about 50% spidroin proteins. The so-called spinning dope flows into the B-zone and further flows into the narrow duct. After a host of protein assembly processes, silk is extended in spinning channels to form extremely tough silk threads. The spider’s silk gland consists of silk nanofibrils having an amyloidogenic character.126 Recombinant spider silk proteins often self-assemble under nondenaturing conditions to form fibrils having morphological similarities with amyloid-like fibrils of yeast prion protein Sup35p-NM.123 Sup35p-NM follows a nucleation conformation conversion aggregation mechanism127 whereas the mechanism of silk nanofibril formation has still not been unraveled. Silk protein aggregation can potentially be monitored using amyloidophilic fluorophores like ThT and congo red.123 A plethora of structural techniques were employed to ascertain the amyloid structure of silk fibrils. The CD and FT-IR spectra of these silk protein nanofibrils manifest the presence β-sheet structure in addition to β turns and a large amount of 31 helical and/or random coil structures. In addition to this, silk threads also exhibit X-Ray diffraction pattern typical of amyloids underlining the fact that spider silk nanofibrils have a core cross-β sheet structure.123

9. Functional amyloids in vertebrates and higher organisms As we discussed before, apart from their association with debilitating human neuropathies, amyloids are also known to perform several physiological functions in prokaryotes and eukaryotes.128–130 Recently, it has been shown that by the formation of a subcellular compartment, called Balbiani

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bodies, functional amyloids can be used to maintain dormant state in vertebrate oocytes.131 The oocytes of Xenopus laevis frog are known to contain Balbiani bodies.132 They are membrane-less compartments possessing mitochondria, E.R, RNA, and other organelles and are found in the cytoplasm of all vertebrate oocytes.131 These transient species are devoid of containing a lipid bilayer and exist only in dormant oocytes and disperses once the oocytes mature. Xvelo, an IDP, is an abundant component of Balbiani bodies. It has an N-terminal prion-like domain that is rich in aromatic amino acids and devoid in charged amino acids.131,133 Exposure to stimuli causes Xvelo to self-assemble to a self-promoting amyloid state.133 This stimuli-mediated amyloidogenesis of Xvelo leads to Balbiani body formation and forms a matrix that helps to keep organelles in place.131 The reversible nature of the formation of the Balbiani body makes the study of physiological amyloids more fascinating. Once the oocytes mature to form eggs, the assembled Xvelo diffuses back to its soluble state diminishing its amyloid-like properties.

10. Functional amyloids in mammals and humans Transmembrane protein Pmel17 forms functional amyloid structures that template melanogenesis from reactive melanin precursors.134 Synthesis of tyrosine-based polymer melanin occurs in highly abundant cellular lysosome-related organelles called melanosomes.135,136 These melanosomes reside in highly specialized cells like melanocytes and retinal pigment epithelium found in skin and eyes.137,138 Proprotein convertase cleavage of full-length transmembrane Pmel17 protein in post-Golgi compartment yields two fragments, namely, a luminal fragment Mα and a transmembrane fragment Mβ.139,140 Ex vivo melanosomes containing Mα fibers stain with amyloidophilic dyes, like congo red and thioflavin S as done by recombinant Mα. Mα fragment of Pmel17 self assembles to forms amyloid fibers within melanosome organelle exhibiting X-Ray diffraction pattern, morphology, and CD, FT-IR spectra typical of amyloids.134 These Mα fibers act as templates upon which the tyrosine-based indole quinone monomers organize and ultimately get polymerized enabling melanin synthesis (Figs. 6 and 7). The ability of activated melanin precursors to concentrate on these Pmel17 fibers come from the fact that amyloid-binding dye ThT and the reactive melanin quinone precursors like 5,6-indolequinone (DHQ) share a similar core structure.

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Fig. 6 Different stages of melanosome maturation. Stage I: Nascent melanosome in Golgi area. Stage II: Generation of amyloid-like Pmel17 fibers within the immature melanosome. Stage III: Pmel17 fibers templating melanin polymerization. Stage IV: Melanin pigment in mature melanosome. PM: plasma membrane, GA: Golgi apparatus, m: mitochondria, ER: endoplasmic reticulum. Reproduced with permission. Copyright (2008) National Academy of Sciences, U.S.A. Hurbain I, Geerts WJ, Boudier T, Marco S, Verkleij AJ, Marks MS, Raposo G. Electron tomography of early melanosomes: implications for melanogenesis and the generation of fibrillar amyloid sheets. Proc Natl Acad Sci U S A. 2008;105 (50):19726–19731. doi:10.1073/pnas.0803488105.

There are essentially four stages of melanosome formation/maturation.140–142 Stage I consists of immature melanosome harboring the luminal Mα fragment and thin fibrils originating from intraluminal vesicles (ILVs). Stage II consists of long, mature fibrils across the length of the melanosome. This is followed by stage III and stage IV where Pmel17 fibers template melanin polymerization from tyrosine-based monomers with the help of the enzyme tyrosinase (Fig. 6). In addition to enhancing the polymerization of small molecule quinone precursors like DHQ into melanin, Mα fragment plays an important role in maintaining the cellular functions of melanocytes

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Fig. 7 Examples of MVBs (multivesicular bodies) harboring ILVs of varying sizes (A–D). Fibrils emanating from intralumenal vesicles (ILVs) within nascent MVBs and fibrils originating from them (arrows) (C and D). E: Stage II premelanosome showing generation of amyloid-like Pmel17 fibers. F: Mature Pmel17 fibers lying parallel to the plane of the section. Reproduced with permission. Copyright (2008) National Academy of Sciences, U.S.A. Hurbain I, Geerts WJ, Boudier T, Marco S, Verkleij AJ, Marks MS, Raposo G. Electron tomography of early melanosomes: implications for melanogenesis and the generation of fibrillar amyloid sheets. Proc Natl Acad Sci U S A. 2008;105(50):19726–19731. doi:10.1073/ pnas.0803488105.

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during melanin generation.143 The quinone precursors and several melanogenic intermediates being potent oxidizers have lethal effects on the cell. These compounds can diffuse out of melanosomes into the cytosol and can have several harmful effects in the cell such as disrupting the oxidative balance of the cell and ultimately cell death. Mα fibers potentially bind these tyrosine-based indolequinone monomers thereby mitigating melaninassociated toxicity by minimizing their diffusion out of the melanosomes. The luminal fragment of Pmel17 consists of mainly four domains—SP, NTR, PKD, and RPT.142 PKD and RPT domains form the major component of the fibrillar matrix within the melanosome. The RPT domain consists of 10 imperfect repeats and sites for O-glycosylation.141 Previous studies have highlighted the importance of the RPT domain in forming the functional amyloid fibrils which template melanin biosynthesis, where the absence of this domain completely obliterates this fibrillization process in vivo.141,144 The assembly of the highly soluble RPT domain into fibrils under mildly acidic pH conditions revealed a pH-dependent shift in the mechanism resulting in the formation of fibrils having distinct nanoscale morphologies. At lower values of pH (<4.5), this protein undergoes rapid assembly into initial oligomers which then mature without a lag phase into dendritic nanostructures, displaying an isodesmic polymerization behavior.145 On the contrary, at pH values >4.5, the RPT domain follows a nucleation-dependent polymerization mechanism to form longer threadlike fibrils which share structural attributes with fibrils observed within stage II melanosomes.146 In addition to their distinct morphologies, the fibrils formed above pH 4.5 exhibited stronger packing in the cross-β sheet structure in comparison to the dendritic nanostructures formed at pH 3 or 4.145 Interestingly, while the dendritic structures formed at pH 4 slowly matured into the amyloid fibrils on being transferred to a pH 5 solution, this structural conversion was not observed on incubation of the thread-like fibrils formed at pH 5 in pH 3 (or 4) solution conditions (Fig. 8). This intricate pH-dependent modulation of both mechanism and morphology associated with the aggregation of the RPT domain clearly recapitulated the pH-dependent melanosomal maturation during melanin biosynthesis. Another example of human functional amyloid is the semen amyloid. The semen amyloids have been found to be present in other life forms indicating that these fibrils are evolutionarily conserved.147 This is sufficient to shed light on the fact that these fibrils indeed play a key role in stimulating conception and embryonic development. The membrane of sperm cells is known to interact with semen amyloids as demonstrated by single-cell

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Fig. 8 Schematic representation of the pH-mediated switch from an isodesmic polymerization mechanism at pH 4 to a nucleation-dependent polymerization mechanism at pH 5 observed during aggregation of RPT domain of Pmel17, giving rise to the dendritic nanostructures at pH 4 and relatively more ordered thread-like fibrils at pH 5. Reproduced and adapted with permission from the American Chemical Society, Dogra P, Bhattacharya M, Mukhopadhyay S. pH-Responsive mechanistic switch regulates the formation of dendritic and fibrillar nanostructures of a functional amyloid. J Phys Chem B. 2017;121(2):412–419. doi:10.1021/acs.jpcb.6b11281.

analyses by immunofluorescence and electron microscopy. Experimentally these fibrils are known to encourage phagocytosis of sperm cells by macrophages.148,149 This leads to the rapid removal of damaged or dead sperm over motile ones by infiltrating phagocytosis from the lower female reproductive tract (FRT).148–150 This allows only the most efficient spermatozoa make it to the oviduct. In this manner semen amyloid fibrils promote sperm selection and clearance and thereby reproductive success. Furthermore, semen amyloid fibrils are known to display antimicrobial activity. They bind electrostatically to both gram-positive and gram-negative bacteria analogs to their interaction with viruses. This entrapping of bacteria can promote phagocytosis by macrophages.

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In addition to their role played in reproduction, semen from healthy men contain amyloids that are known to amplify HIV infection.149,151 Peptides derived from C-terminal region of human seminal plasma protein, prostatic acid phosphatase (PAP) is termed as a semen-derived enhancer of viral infection (SEVI)149,151 and the peptide derived from the N-proximal region of PAP is named SEVI-2.152 Both these peptides are known to form amyloid fibrils, i.e., they exhibit apple-green birefringence under cross polarized light when stained with congo red, in X-ray diffraction gives two classical reflections of amyloids and exhibit fibrillar morphology visible by AFM.151–153 Additionally, amyloids derived from human semenogelin 1 and 2 (main protein present in human semen coagulum) are also present in human semen. These fibrils are collectively termed as SEM fibrils.154 Both the SEM and SEVI fibrils share structural and biophysical properties but differ in sequence homology. These fibrils are normally found to possess a high net positive charge at neutral pH.154,155 This allows them to strongly cohere electrostatically to negatively charged membranes of HIV virions and cells and thereby affecting their fusion to target cells. Stabilities of SEVI and SEM fibrils depend on the pH conditions. These fibrils tend to disassemble at acidic pH 2–3.156,157 Therefore, women suffering from bacterial vaginosis (vaginal fluid becomes less acidic) are at greater risk of infection.158 Moreover, attempts to nullify the positive charge of these fibrils with polyanions can reduce the infection rates.154,155,159 The amyloidogenic property of proteins can also be exploited to regulate cell adaptation to adverse environmental insults by entering a dormant state on subjecting to a stressor. Upon the application of apoptotic conditions, the amyloid converting motif (ACM) of heterogeneous proteins interacts with ribosomal intergenic noncoding RNA (rIGSRNA) and assembles to insoluble amyloid-like state thereby forming subnuclear amyloid bodies (A-bodies) that shows positive staining with an amyloidophilic dye.160 On cessation of environmental stress, the heat shock proteins disaggregate the A-bodies into the soluble, mobile state making the formation of A-bodies reversible. The unique feature of ACM of proteins is that they possess two arginine/histidine-rich domains bordering the amyloidogenic sequence of amino acids. These flanking arginine/histidine-rich domains are essential for RNA-seeded amyloidogenesis.160 Additionally, a line of evidence shows in secretory granules, many peptides and protein hormones are stored at a high concentration in the amyloid state.161 Amyloid formations of these hormones are highly regulated by factors such as pH, salt, and glycosaminoglycans. The high concentration of amyloid fibrils delays the disassembly of hormones from the fibrils after secretion.113

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11. Functional amyloids in the plant kingdom In addition to animals, the plant kingdom also utilizes functional amyloids. For instances, the proteins, namely, Small Rubber Particle Protein (SRPP) and Rubber Elongation Factor (REF) are the major allergen proteins found in the latex of Hevea brasiliensis (rubber tree) belonging to the Euphorbiaceae family.162–165 REF and SRPP are acidic proteins of 14.6 and 24 kDa, respectively, and are well known to take part in the biosynthesis of natural rubber. Hevea brasiliensis is a tropical plant grown predominantly in Southeast Asia to produce natural rubber. Natural rubber is obtained by polymerization of cis-1,4-polyisoprene from latex. Latex refers to the white sap produced by plant laticifer layers and consists of rubber and non-rubber particles, proteins, enzymes, organelles and so forth. REF and SRPP are the major hydrophobic paralogous latex proteins but have different aggregation properties and structural features. REF is found to rapidly aggregate under physiological conditions.166 The foremost report illustrating the presence of amyloidogenic proteins in the plant kingdom demonstrates that REF forms micrometer-long, unbranched fibrils at pH 7.4.166 Aggregative properties of REF and SRPP can be monitored using amyloidbinding dyes like ThT, congo red, and Bis-ANS. SRPP binds to these dyes to a very small extent pointing toward the presence of extremely small assemblies. X-ray diffraction (WAXS) studies of REF shows the presence of reflections at around 4.7 and 10 A˚, characteristic of amyloids. Further secondary structural analysis of these proteins using CD spectroscopy and ATR-FTIR spectroscopy evince the large β-sheet content in REF aggregates in contrast to soluble SRPP that exists largely in α-helices and random coils.166 Additionally, these two plant proteins also differ in their interaction with lipid monolayers used as a model to imitate the membrane of rubber particles. It is found that SRPP weakly interacts to the surface of small rubber particles (SRP)167 while REF is deeply inserted into the large rubber particles (LRP) and coat the rubber particles in more than one layer.168,169 This difference of interaction with lipid membranes could possibly lead to different functions of REF and SRPP in the biosynthesis of natural rubber.

12. Conclusions and future directions Amyloids have been extensively scrutinized due to their causative role in deadly human diseases. However, amyloids are now being recognized to

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play a much broader role in biology. They constitute functional entities that are thought to perform several fascinating biological functions in lower organisms like bacteria to higher organisms such as vertebrates, plants, and humans. This shows that amyloids possess an “evolutionary conserved quaternary structure” to carry out a diverse range of functions.62 They are much more than a “misfolded” toxic protein structure and can be formed in relation to either disease or function. The field of functional amyloid is undergoing a rapid transformation. A vast majority of functional amyloids are formed by a wide variety of IDPs in a diverse range of organisms. These functional amyloids also underscore the importance of functional roles of IDPs. We speculate that many more functional amyloids and their novel regulatory and functional strategies will be discovered in the near future. The state-of-the-art tools involving super-resolution microscopy, cryo-EM, advanced NMR, single-molecule methodologies, and other emerging molecular and cellular tools will allow us to study this fascinating class of amyloids. Our deeper understanding of the molecular mechanisms will also help us develop therapeutic strategies for the amyloid diseases as well as decipher a complex array of physiological functions performed by functional amyloids.

Acknowledgments We thank IISER Mohali, the Department of Science and Technology and the Ministry of Human Resource Development, Govt. of India for the financial support (SERB National Postdoctoral Fellowship to A.M.; MHRD Centre of Excellence grant and DST NanoMission grant to S.M.). The work described here has been contributed by several former and present members of the Mukhopadhyay laboratory including Drs. M. Bhattacharya, N. Jain, D. Narang, K. Bhasne, Ms. P. Dogra, and Mr. S. Mahapatra, and has been supported by several research grants from the Council of Scientific and Industrial Research, the Department of Science and Technology and the Department of Biotechnology, Govt. of India (to S.M.).

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