Amyloid and the Cross-Beta Architecture

Amyloid fibrils are made up of a misfolded, self-assembled protein organised into a well-defined, repetitive structure. They are well known for their ...

University of Sussex

Many critical biological processes take place at hydrophobic:hydrophilic interfaces, and a wide range of organisms produce surface-active proteins and peptides that reduce surface and interfacial tension and mediate growth and development at these boundaries. Microorganisms produce both small lipid-associated peptides and amphipathic proteins that allow growth across water:air boundaries, attachment to surfaces, predation, and improved bioavailability of hydrophobic substrates. Higher-order organisms produce surface-active proteins with a wide variety of functions, including the provision of protective foam environments for vulnerable reproductive stages, evaporative cooling, and gas exchange across airway membranes. In general, the biological functions supported by these diverse polypeptides require them to have an amphipathic nature, and this is achieved by a diverse range of molecular structures, with some proteins undergoing significant conformational change or intermolecular association to generate the structures that are surface active.

Molecular Characteristics and Biological Functions of Surface-Active and Surfactant Proteins.

Hydrophobins are small proteins secreted by fungi and which spontaneously assemble into amphipathic layers at hydrophilic-hydrophobic interfaces. We have examined the self-assembly of the Class I hydrophobins EAS and DewA, the Class II hydrophobin NC2 and an engineered chimeric hydrophobin. These Class I hydrophobins form layers composed of laterally associated fibrils with an underlying amyloid structure. These two Class I hydrophobins, despite showing significant conformational differences in solution, self-assemble to form fibrillar layers with very similar structures and require a hydrophilic-hydrophobic interface to trigger self-assembly. Addition of additives that influence surface tension can be used to manipulate the fine structure of the protein films. The Class II hydrophobin NC2 forms a mesh-like protein network and the engineered chimeric hydrophobin displays two multimeric forms, depending on assembly conditions. When formed on a graphite surface, the fibrillar EAS layers are resistant to alcohol, acid and basic washes. In contrast, the NC2 Class II monolayers are dissociated by alcohol treatment but are relatively stable towards acid and base washes. The engineered chimeric Class I/II hydrophobin shows increased stability towards alcohol and acid and base washes. Self-assembled hydrophobin films may have extensive applications in biotechnology where biocompatible; amphipathic coatings facilitate the functionalization of nanomaterials.

Fungal Hydrophobin Proteins Produce Self-Assembling Protein Films with Diverse Structure and Chemical Stability.

The Receptor-interacting protein kinase Homotypic Interaction Motif (RHIM) is an amino acid sequence that mediates multiple protein:protein interactions in the mammalian programmed cell death pathway known as necroptosis. At least one key RHIM-based complex has been shown to have a functional amyloid fibril structure, which provides a stable hetero-oligomeric platform for downstream signaling. RHIMs and related motifs are present in immunity-related proteins across nature, from viruses to fungi to metazoans. Necroptosis is a hallmark feature of cellular clearance of infection. For this reason, numerous pathogens, including viruses and bacteria, have developed varied methods to modulate necroptosis, focusing on inhibiting RHIM:RHIM interactions, and thus their downstream cell death effects. This review will discuss current understanding of RHIM:RHIM interactions in normal cellular activation of necroptosis, from a structural and cell biology perspective. It will compare the mechanisms by which pathogens subvert these interactions in order to maintain their replicative and infective cycles and consider the similarities between RHIMs and other functional amyloid-forming proteins associated with cell death and innate immunity. It will discuss the implications of the heteromeric nature and structure of RHIM-based amyloid complexes in the context of other functional amyloids.

RHIM-based protein:protein interactions in microbial defence against programmed cell death by necroptosis.

Hydrophobins are amphiphilic proteins secreted by filamentous fungi in a soluble form, which can self-assemble at hydrophilic/hydrophobic or water/air interfaces to form amphiphilic layers that have multiple biological roles. We have investigated the conformational changes that occur upon self-assembly of six hydrophobins that form functional amyloid fibrils with a rodlet morphology. These hydrophobins are present in the cell wall of spores from different fungal species. From available structures and NMR chemical shifts, we established the secondary structures of the monomeric forms of these proteins and monitored their conformational changes upon amyloid rodlet formation or thermal transitions using synchrotron radiation circular dichroism and Fourier-transform infrared spectroscopy (FT-IR). Thermal transitions were followed by synchrotron radiation circular dichroism in quartz cells that allowed for microbubbles and hence water/air interfaces to form and showed irreversible conformations that differed from the rodlet state for most of the proteins. In contrast, thermal transitions on hermetic calcium fluoride cells showed reversible conformational changes. Heating hydrophobin solutions with a water/air interface on a silicon crystal surface in FT-IR experiments resulted in a gain in β-sheet content typical of amyloid fibrils for all except one protein. Rodlet formation was further confirmed by electron microscopy. FT-IR spectra of pre-formed hydrophobin rodlet preparations also showed a gain in β-sheet characteristic of the amyloid cross-β structure. Our results indicate that hydrophobins are capable of significant conformational plasticity and the nature of the assemblies formed by these surface-active proteins is highly dependent on the interface at which self-assembly takes place.

Probing Structural Changes during Self-assembly of Surface-Active Hydrophobin Proteins that Form Functional Amyloids in Fungi.

The murine cytomegalovirus protein M45 protects infected mouse cells from necroptotic death and, when heterologously expressed, can protect human cells from necroptosis induced by tumour necrosis factor receptor (TNFR) activation. Here, we show that the N-terminal 90 residues of the M45 protein, which contain a RIP homotypic interaction motif (RHIM), are sufficient to confer protection against TNFR-induced necroptosis. This N-terminal region of M45 drives rapid self-assembly into homo-oligomeric amyloid fibrils and interacts with the RHIMs of the human kinases RIPK1 and RIPK3, and the Z-DNA binding protein 1 (ZBP1), to form heteromeric amyloid fibrils  Mutation of the tetrad residues in the M45 RHIM attenuates homo- and hetero-amyloid assembly by M45, suggesting that the amyloidogenic nature of the M45 RHIM underlies its biological activity. The M45 RHIM preferentially interacts with RIPK3 and ZBP1 over RIPK1 and alters the properties of the host RHIM protein assemblies. Our results indicate that M45 mimics the interactions made by RIPK1 or ZBP1 with RIPK3, thereby forming heteromeric amyloid structures, which may explain its ability to inhibit necroptosis.

Viral M45 and necroptosis-associated proteins form heteromeric amyloid assemblies.

The functional amyloid state of proteins has in recent years garnered much attention for its role in serving crucial and diverse biological roles. Amyloid is a protein fold characterised by fibrillar morphology, binding of the amyloid-specific dyes Thioflavin T and Congo Red, insolubility and underlying cross-β structure. Amyloids were initially characterised as an aberrant protein fold associated with mammalian disease. However, in the last two decades, functional amyloids have been described in almost all biological systems, from viruses, to bacteria and archaea, to humans. Understanding the structure and role of these amyloids elucidates novel and potentially ancient mechanisms of protein function throughout nature. Many of these microbial functional amyloids are utilised by pathogens for invasion and maintenance of infection. As such, they offer novel avenues for therapies. This review examines the structure and mechanism of known microbial functional amyloids, with a particular focus on the pathogenicity conferred by the production of these structures and the strategies utilised by microbes to interfere with host amyloid structures. The biological importance of microbial amyloid assemblies is highlighted by their ubiquity and diverse functionality.

Microbial functional amyloids serve diverse purposes for structure, adhesion and defence.

The fungal hydrophobins are small proteins that are able to self-assemble spontaneously into amphipathic monolayers at hydrophobic:hydrophilic interfaces. These protein monolayers can reverse the wettability of a surface, making them suitable for increasing the biocompatibility of many hydrophobic nanomaterials. One subgroup of this family, the class I hydrophobins, forms monolayers that are composed of extremely robust amyloid-like fibrils, called rodlets. Here, we describe the protocols for the production and purification of recombinant hydrophobins and oxidative refolding to a biologically active, soluble, monomeric form. We describe methods to trigger the self-assembly into the fibrillar rodlet state and techniques to characterize the physicochemical properties of the polymeric forms.

Formation of Amphipathic Amyloid Monolayers from Fungal Hydrophobin Proteins.

In recent years, much attention has focused on incorporating biological and bio-inspired nanomaterials into various applications that range from functionalising surfaces and enhancing biomolecule binding properties, to coating drugs for improved bioavailability and delivery. Hydrophobin proteins, which can spontaneously assemble into amphipathic layers at hydrophobic:hydrophilic interfaces, are exciting candidates for use as nanomaterials. These unique proteins, which are only expressed by filamentous fungi, have been the focus of increasing interest from the biotechnology industry, as evidenced by the sharply growing number of hydrophobin-associated publications and patents. Here, we explore the contribution of different hydrophobins to supporting fungal growth and development. We describe the key structural elements of hydrophobins and the molecular characteristics that underlie self-assembly of these proteins at interfaces. We outline the multiple roles that hydrophobins can play in supporting aerial growth of filamentous structures, facilitating spore dispersal and preventing an immune response in the infected host. The growing understanding of the hydrophobin protein structure and self-assembly process highlights the potential for hydrophobin proteins to be engineered for use in a variety of novel applications that require biocompatible coatings.

Fungal Hydrophobins and Their Self-Assembly into Functional Nanomaterials.

The conidia of airborne fungi are protected by a hydrophobic protein layer that coats the cell wall polysaccharides and renders the spores resistant to wetting and desiccation. A similar layer is presented on the outer surface of the aerial hyphae of some fungi. This layer serves multiple purposes, including facilitating spore dispersal, mediating the growth of hyphae into the air from moist environments, aiding host interactions in symbiotic relationships and increasing infectivity in pathogenic fungi. The layer consists of tightly packed, fibrillar structures termed "rodlets", which are approximately 10 nm in diameter, hundreds of nanometres long and grouped in fascicles. Rodlets are an extremely stable protein structure, being resistant to detergents, denaturants and alcohols and requiring strong acids for depolymerisation. They are produced through the self-assembly of small, surface-active proteins that belong to the hydrophobin protein family. These small proteins are expressed by all filamentous fungi and are characterised by a high proportion of hydrophobic residues and the presence of eight cysteine residues. Rodlets are a form of the functional amyloid fibril, where the hydrophobin monomers are held together in the rodlets by intermolecular hydrogen bonds that contribute to a stable β-sheet core.

Hydrophobin Rodlets on the Fungal Cell Wall.

The viral protein ICP6, encoded by herpes simplex virus 1 (HSV-1), harbours a RIP-homotypic interaction motif (RHIM), that plays a role in viral inhibition of host cell death pathways. Other members of the Herpesviridae family also encode RHIM-containing proteins that interfere with host-cell death pathways, including the M45 protein from murine cytomegalovirus, and ORF20 protein from varicella zoster virus. We have used amyloid assembly assays, electron microscopy and single molecule fluorescence spectroscopy to show that the ICP6 RHIM is amyloidogenic and can interact with host RHIM-containing proteins to form heteromeric amyloid complexes, in a manner similar to that of M45 and ORF20 RHIMs. The core tetrad sequence of the ICP6 RHIM is important for both amyloid formation and interaction with host RHIM-containing proteins. Notably, we show that the amyloid forming capacity of the ICP6 RHIM is affected by the redox environment. We propose that the formation of an intramolecular disulfide bond within ICP6 triggers the formation of amyloid assemblies that are distinct from previously characterised viral amyloids M45 and ORF20. Formation of viral-host heteromeric amyloid assemblies may underlie a general mechanism of viral adaptation against host immune machineries.

Herpes simplex virus encoded ICP6 protein forms functional amyloid assemblies with necroptosis-associated host proteins.

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.

Article	Year
Molecular Characteristics and Biological Functions of Surface-Active and Surfactant Proteins. Annual review of biochemistry\| PubMed ID: 28125290	2017
Fungal Hydrophobin Proteins Produce Self-Assembling Protein Films with Diverse Structure and Chemical Stability. Nanomaterials (Basel, Switzerland)\| PubMed ID: 28344251	2014
RHIM-based protein:protein interactions in microbial defence against programmed cell death by necroptosis. Seminars in cell & developmental biology\| PubMed ID: 29738881	2020
Probing Structural Changes during Self-assembly of Surface-Active Hydrophobin Proteins that Form Functional Amyloids in Fungi. Journal of molecular biology\| PubMed ID: 30096347	2018
Viral M45 and necroptosis-associated proteins form heteromeric amyloid assemblies. EMBO reports\| PubMed ID: 30498077	2019
Microbial functional amyloids serve diverse purposes for structure, adhesion and defence. Biophysical reviews\| PubMed ID: 31049855	2019
Formation of Amphipathic Amyloid Monolayers from Fungal Hydrophobin Proteins. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 31612436	2020
Fungal Hydrophobins and Their Self-Assembly into Functional Nanomaterials. Advances in experimental medicine and biology\| PubMed ID: 31713199	2019
Hydrophobin Rodlets on the Fungal Cell Wall. Current topics in microbiology and immunology\| PubMed ID: 31875265	2020
Herpes simplex virus encoded ICP6 protein forms functional amyloid assemblies with necroptosis-associated host proteins. Biophysical chemistry\| PubMed ID: 33348174	2021
Functional Amyloids: Where Supramolecular Amyloid Assembly Controls Biological Activity or Generates New Functionality. Journal of molecular biology\| PubMed ID: 37330295	2023

Margaret Sunde

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