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

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.

NMR characterization of an assembling RHIM (RIP homotypic interaction motif) amyloid reveals a cryptic region for self-recognition.

The RIP homotypic interaction motif (RHIM) is an essential protein motif in inflammatory signaling and certain cell death pathways. RHIM signaling occurs following the assembly of functional amyloids, and while the structural biology of such higher-order RHIM complexes has started to emerge, the conformations and dynamics of nonassembled RHIMs remain unknown. Here, using solution NMR spectroscopy, we report the characterization of the monomeric form of the RHIM in receptor-interacting protein kinase 3 (RIPK3), a fundamental protein in human immunity. Our results establish that the RHIM of RIPK3 is an intrinsically disordered protein motif, contrary to prediction, and that exchange dynamics between free monomers and amyloid-bound RIPK3 monomers involve a 20-residue stretch outside the RHIM that is not incorporated within the structured cores of the RIPK3 assemblies determined by cryo-EM or solid-state NMR. Thus, our findings expand on the structural characterization of RHIM-containing proteins, specifically highlighting conformational dynamics involved in assembly processes.

iPSC-derived PSEN2 (N141I) astrocytes and microglia exhibit a primed inflammatory phenotype.

BACKGROUND: Widescale evidence points to the involvement of glia and immune pathways in the progression of Alzheimer's disease (AD). AD-associated iPSC-derived glial cells show a diverse range of AD-related phenotypic states encompassing cytokine/chemokine release, phagocytosis and morphological profiles, but to date studies are limited to cells derived from PSEN1, APOE and APP mutations or sporadic patients. The aim of the current study was to successfully differentiate iPSC-derived microglia and astrocytes from patients harbouring an AD-causative PSEN2 (N141I) mutation and characterise the inflammatory and morphological profile of these cells. METHODS: iPSCs from three healthy control individuals and three familial AD patients harbouring a heterozygous PSEN2 (N141I) mutation were used to derive astrocytes and microglia-like cells and cell identity and morphology were characterised through immunofluorescent microscopy. Cellular characterisation involved the stimulation of these cells by LPS and Aß42 and analysis of cytokine/chemokine release was conducted through ELISAs and multi-cytokine arrays. The phagocytic capacity of these cells was then indexed by the uptake of fluorescently-labelled fibrillar Aß42. RESULTS: AD-derived astrocytes and microglia-like cells exhibited an atrophied and less complex morphological appearance than healthy controls. AD-derived astrocytes showed increased basal expression of GFAP, S100ß and increased secretion and phagocytosis of Aß42 while AD-derived microglia-like cells showed decreased IL-8 secretion compared to healthy controls. Upon immunological challenge AD-derived astrocytes and microglia-like cells showed exaggerated secretion of the pro-inflammatory IL-6, CXCL1, ICAM-1 and IL-8 from astrocytes and IL-18 and MIF from microglia. CONCLUSION: Our study showed, for the first time, the differentiation and characterisation of iPSC-derived astrocytes and microglia-like cells harbouring a PSEN2 (N141I) mutation. PSEN2 (N141I)-mutant astrocytes and microglia-like cells presented with a 'primed' phenotype characterised by reduced morphological complexity, exaggerated pro-inflammatory cytokine secretion and altered Aß42 production and phagocytosis.

From Fundamental Amyloid Protein Self-Assembly to Development of Bioplastics.

Proteins can self-assemble into a range of nanostructures as a result of molecular interactions. Amyloid nanofibrils, as one of them, were first discovered with regard to the relevance of neurodegenerative diseases but now have been exploited as building blocks to generate multiscale materials with designed functions for versatile applications. This review interconnects the mechanism of amyloid fibrillation, the current approaches to synthesizing amyloid protein-based materials, and the application in bioplastic development. We focus on the fundamental structures of self-assembled amyloid fibrils and how external factors can affect protein aggregation to optimize the process. Protein self-assembly is essentially the autonomous congregation of smaller protein units into larger, organized structures. Since the properties of the self-assembly can be manipulated by changing intrinsic factors and external conditions, protein self-assembly serves as an excellent building block for bioplastic development. Building on these principles, general processing methods and pathways from raw protein sources to mature state materials are proposed, providing a guide for the development of large-scale production. Additionally, this review discusses the diverse properties of protein-based amyloid nanofibrils and how they can be utilized as bioplastics. The economic feasibility of the protein bioplastics is also compared to conventional plastics in large-scale production scenarios, supporting their potential as sustainable bioplastics for future applications.

Surface-Induced Hydrophobin Assemblies with Versatile Properties and Distinct Underlying Structures.

Hydrophobins are remarkable proteins due to their ability to self-assemble into amphipathic coatings that reverse surface wettability. Here, the versatility of the Class I hydrophobins EASΔ15 and DewY in diverse nanosuspension and coating applications is demonstrated. The hydrophobins are shown to coat or emulsify a range of substrates including oil, hydrophobic drugs, and nanodiamonds and alter their solution and surface behavior. Surprisingly, while the coatings confer new properties, only a subset is found to be resistant to hot detergent treatment, a feature previously thought to be characteristic of the functional amyloid form of Class I hydrophobins. These results demonstrate that substrate surface properties can influence the molecular structures and physiochemical properties of hydrophobin and possibly other functional amyloids. Functional amyloid assembly with different substrates and conditions may be analogous to the propagation of different polymorphs of disease-associated amyloid fibrils with distinct structures, stability, and clinical phenotypes. Given that amyloid formation is not required for Class I hydrophobins to serve diverse applications, our findings open up new opportunities for their use in applications requiring a range of chemical and physical properties. In hydrophobin nanotechnological applications where high stability of assemblies is required, simultaneous structural and functional characterization should be carried out. Finally, while results in this study pertain to synthetic substrates, they raise the possibility that at least some members of the pseudo-Class I and Class III hydrophobins, reported to form assemblies with noncanonical properties, may be Class I hydrophobins adopting alternative structures in response to environmental cues.

Single-Molecule Two-Color Coincidence Detection of Unlabeled alpha-Synuclein Aggregates.

Protein misfolding and aggregation into oligomeric and fibrillar structures is a common feature of many neurogenerative disorders. Single-molecule techniques have enabled characterization of these lowly abundant, highly heterogeneous protein aggregates, previously inaccessible using ensemble averaging techniques. However, they usually rely on the use of recombinantly-expressed labeled protein, or on the addition of amyloid stains that are not protein-specific. To circumvent these challenges, we have made use of a high affinity antibody labeled with orthogonal fluorophores combined with fast-flow microfluidics and single-molecule confocal microscopy to specifically detect α-synuclein, the protein associated with Parkinson's disease. We used this approach to determine the number and size of α-synuclein aggregates down to picomolar concentrations in biologically relevant samples.

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

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.

Varicella zoster virus encodes a viral decoy RHIM to inhibit cell death.

Herpesviruses are known to encode a number of inhibitors of host cell death, including RIP Homotypic Interaction Motif (RHIM)-containing proteins. Varicella zoster virus (VZV) is a member of the alphaherpesvirus subfamily and is responsible for causing chickenpox and shingles. We have identified a novel viral RHIM in the VZV capsid triplex protein, open reading frame (ORF) 20, that acts as a host cell death inhibitor. Like the human cellular RHIMs in RIPK1 and RIPK3 that stabilise the necrosome in TNF-induced necroptosis, and the viral RHIM in M45 from murine cytomegalovirus that inhibits cell death, the ORF20 RHIM is capable of forming fibrillar functional amyloid complexes. Notably, the ORF20 RHIM forms hybrid amyloid complexes with human ZBP1, a cytoplasmic sensor of viral nucleic acid. Although VZV can inhibit TNF-induced necroptosis, the ORF20 RHIM does not appear to be responsible for this inhibition. In contrast, the ZBP1 pathway is identified as important for VZV infection. Mutation of the ORF20 RHIM renders the virus incapable of efficient spread in ZBP1-expressing HT-29 cells, an effect which can be reversed by the inhibition of caspases. Therefore we conclude that the VZV ORF20 RHIM is important for preventing ZBP1-driven apoptosis during VZV infection, and propose that it mediates this effect by sequestering ZBP1 into decoy amyloid assemblies.

Don't Turn Off the Tap! The Importance of Discovery Science to the Australian Cardiovascular Sector and Improving Clinical Outcomes Into the Future.

Despite significant advances in interventional and therapeutic approaches, cardiovascular disease (CVD) remains the leading cause of death and mortality. To lower this health burden, cardiovascular discovery scientists need to play an integral part in the solution. Successful clinical translation is achieved when built upon a strong foundational understanding of the disease mechanisms involved. Changes in the Australian funding landscape, to place greater emphasis on translation, however, have increased job insecurity for discovery science researchers and especially early-mid career researchers. To highlight the importance of discovery science in cardiovascular research, this review compiles six science stories in which fundamental discoveries, often involving Australian researchers, has led to or is advancing to clinical translation. These stories demonstrate the importance of the role of discovery scientists and the need for their work to be prioritised now and in the future. Australia needs to keep discovery scientists supported and fully engaged within the broader cardiovascular research ecosystem so they can help realise the next game-changing therapy or diagnostic approach that diminishes the burden of CVD on society.

The RHIM of the Immune Adaptor Protein TRIF Forms Hybrid Amyloids with Other Necroptosis-Associated Proteins.

TIR-domain-containing adapter-inducing interferon-ß (TRIF) is an innate immune protein that serves as an adaptor for multiple cellular signalling outcomes in the context of infection. TRIF is activated via ligation of Toll-like receptors 3 and 4. One outcome of TRIF-directed signalling is the activation of the programmed cell death pathway necroptosis, which is governed by interactions between proteins that contain a RIP Homotypic Interaction Motif (RHIM). TRIF contains a RHIM sequence and can interact with receptor interacting protein kinases 1 (RIPK1) and 3 (RIPK3) to initiate necroptosis. Here, we demonstrate that the RHIM of TRIF is amyloidogenic and supports the formation of homomeric TRIF-containing fibrils. We show that the core tetrad sequence within the RHIM governs the supramolecular organisation of TRIF amyloid assemblies, although the stable amyloid core of TRIF amyloid fibrils comprises a much larger region than the conserved RHIM only. We provide evidence that RHIMs of TRIF, RIPK1 and RIPK3 interact directly to form heteromeric structures and that these TRIF-containing hetero-assemblies display altered and emergent properties that likely underlie necroptosis signalling in response to Toll-like receptor activation.

A Fluorescent Sensor for Quantitative Super-Resolution Imaging of Amyloid Fibril Assembly.

Many soluble proteins can self-assemble into macromolecular structures called amyloids, a subset of which are implicated in a range of neurodegenerative disorders. The nanoscale size and structural heterogeneity of prefibrillar and early aggregates, as well as mature amyloid fibrils, pose significant challenges for the quantification of amyloid morphologies. We report a fluorescent amyloid sensor AmyBlink-1 and its application in super-resolution imaging of amyloid structures. AmyBlink-1 exhibits a 5-fold increase in ratio of the green (thioflavin T) to red (Alexa Fluor 647) emission intensities upon interaction with amyloid fibrils. Using AmyBlink-1, we performed nanoscale imaging of four different types of amyloid fibrils, achieving a resolution of ≈30 nm. AmyBlink-1 enables nanoscale visualization and subsequent quantification of morphological features, such as the length and skew of individual amyloid aggregates formed at different times along the amyloid assembly pathway.

Hydrophobin Rodlets on the Fungal Cell Wall.

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 ß-sheet core.

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

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 in vitro 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.

Cell-free expression of natively folded hydrophobins.

Hydrophobins are a family of cysteine-rich proteins unique to filamentous fungi. The proteins are produced in a soluble form but self-assemble into organised amphipathic layers at hydrophilic:hydrophobic interfaces. These layers contribute to transitions between wet and dry environments, spore dispersal and attachment to surfaces for growth and infection. Hydrophobins are characterised by four disulphide bonds that are critical to their structure and function. Thus, obtaining correctly folded, soluble and functional hydrophobins directly from bacterial recombinant expression is challenging and in most cases, initial denaturation from inclusion bodies followed by oxidative refolding are required to obtain folded proteins. Here, we report the use of cell-free expression with E. coli cell lysate to directly obtain natively folded hydrophobins. All six of the hydrophobins tested could be expressed after optimisation of redox conditions. For some hydrophobins, the inclusion of the disulfide isomerase DsbC further enhanced expression levels. We are able to achieve a yield of up to 1 mg of natively folded hydrophobin per mL of reaction. This has allowed the confirmation of the correct folding of hydrophobins with the use of 15N-cysteine and 15N-1H nuclear magnetic resonance experiments within 24 h of starting from plasmid stocks.

Fungal Hydrophobins and Their Self-Assembly into Functional Nanomaterials.

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.

Self-assembly of functional, amphipathic amyloid monolayers by the fungal hydrophobin EAS.

The hydrophobin EAS from the fungus Neurospora crassa forms functional amyloid fibrils called rodlets that facilitate spore formation and dispersal. Self-assembly of EAS into fibrillar rodlets occurs spontaneously at hydrophobic:hydrophilic interfaces and the rodlets further associate laterally to form amphipathic monolayers. We have used site-directed mutagenesis and peptide experiments to identify the region of EAS that drives intermolecular association and formation of the cross-ß rodlet structure. Transplanting this region into a nonamyloidogenic hydrophobin enables it to form rodlets. We have also determined the structure and dynamics of an EAS variant with reduced rodlet-forming ability. Taken together, these data allow us to pinpoint the conformational changes that take place when hydrophobins self-assemble at an interface and to propose a model for the amphipathic EAS rodlet structure.

Intrinsic disorder modulates protein self-assembly and aggregation.

Protein molecules have evolved to adopt distinctive and well-defined functional and soluble states under physiological conditions. In some circumstances, however, proteins can self-assemble into fibrillar aggregates designated as amyloid fibrils. In vivo these processes are normally associated with severe pathological conditions but can sometimes have functional relevance. One such example is the hydrophobins, whose aggregation at air-water interfaces serves to create robust protein coats that help fungal spores to resist wetting and thus facilitate their dispersal in the air. We have performed multiscale simulations to address the molecular determinants governing the formation of functional amyloids by the class I fungal hydrophobin EAS. Extensive samplings of full-atom replica-exchange molecular dynamics and coarse-grained simulations have allowed us to identify factors that distinguish aggregation-prone from highly soluble states of EAS. As a result of unfavourable entropic terms, highly dynamical regions are shown to exert a crucial influence on the propensity of the protein to aggregate under different conditions. More generally, our findings suggest a key role that specific flexible structural elements can play to ensure the existence of soluble and functional states of proteins under physiological conditions.

Solution structure and interface-driven self-assembly of NC2, a new member of the Class II hydrophobin proteins.

Hydrophobins are fungal proteins that self-assemble spontaneously to form amphipathic monolayers at hydrophobic:hydrophilic interfaces. Hydrophobin assemblies facilitate fungal transitions between wet and dry environments and interactions with plant and animal hosts. NC2 is a previously uncharacterized hydrophobin from Neurospora crassa. It is a highly surface active protein and is able to form protein layers on a water:air interface that stabilize air bubbles. On a hydrophobic substrate, NC2 forms layers consisting of an ordered network of protein molecules, which dramatically decrease the water contact angle. The solution structure and dynamics of NC2 have been determined using nuclear magnetic resonance spectroscopy. The structure of this protein displays the same core fold as observed in other hydrophobin structures determined to date, including the Class II hydrophobins HFBI and HFBII from Trichoderma reesei, but certain features illuminate the structural differences between Classes I and II hydrophobins and also highlight the variations between structures of Class II hydrophobin family members. The unique properties of hydrophobins have attracted much attention for biotechnology applications. The insights obtained through determining the structure, biophysical properties and assembly characteristics of NC2 will facilitate the development of hydrophobin-based applications.

Solid-state NMR structure determination from diagonal-compensated, sparsely nonuniform-sampled 4D proton-proton restraints.

We report acquisition of diagonal-compensated protein structural restraints from four-dimensional solid-state NMR spectra on extensively deuterated and (1)H back-exchanged proteins. To achieve this, we use homonuclear (1)H-(1)H correlations with diagonal suppression and nonuniform sampling (NUS). Suppression of the diagonal allows the accurate identification of cross-peaks which are otherwise obscured by the strong autocorrelation or whose intensity is biased due to partial overlap with the diagonal. The approach results in unambiguous spectral interpretation and relatively few but reliable restraints for structure calculation. In addition, the diagonal suppression produces a spectrum with low dynamic range for which ultrasparse NUS data sets can be readily reconstructed, allowing straightforward application of NUS with only 2% sampling density with the advantage of more heavily sampling time-domain regions of high signal intensity. The method is demonstrated here for two proteins, α-spectrin SH3 microcrystals and hydrophobin functional amyloids. For the case of SH3, suppression of the diagonal results in facilitated identification of unambiguous restraints and improvement of the quality of the calculated structural ensemble compared to nondiagonal-suppressed 4D spectra. For the only partly assigned hydrophobin rodlets, the structure is yet unknown. Applied to this protein of biological significance with large inhomogeneous broadening, the method allows identification of unambiguous crosspeaks that are otherwise obscured by the diagonal.

A Coumarin-Based Array for the Discrimination of Amyloids.

Self-assembly of misfolded proteins can lead to the formation of amyloids, which are implicated in the onset of many pathologies including Alzheimer's disease and Parkinson's disease. The facile detection and discrimination of different amyloids are crucial for early diagnosis of amyloid-related pathologies. Here, we report the development of a fluorescent coumarin-based two-sensor array that is able to correctly discriminate between four different amyloids implicated in amyloid-related pathologies with 100% classification. The array was also applied to mouse models of Alzheimer's disease and was able to discriminate between samples from mice corresponding to early (6 months) and advanced (12 months) stages of Alzheimer's disease. Finally, the flexibility of the array was assessed by expanding the analytes to include functional amyloids. The same two-sensor array was able to correctly discriminate between eight different disease-associated and functional amyloids with 100% classification.