The C-terminal 32-mer fragment of hemoglobin alpha is an amyloidogenic peptide with antimicrobial properties.

Antimicrobial peptides (AMPs) are major components of the innate immune defense. Accumulating evidence suggests that the antibacterial activity of many AMPs is dependent on the formation of amyloid-like fibrils. To identify novel fibril forming AMPs, we generated a spleen-derived peptide library and screened it for the presence of amyloidogenic peptides. This approach led to the identification of a C-terminal 32-mer fragment of alpha-hemoglobin, termed HBA(111-142). The non-fibrillar peptide has membranolytic activity against various bacterial species, while the HBA(111-142) fibrils aggregated bacteria to promote their phagocytotic clearance. Further, HBA(111-142) fibrils selectively inhibited measles and herpes viruses (HSV-1, HSV-2, HCMV), but not SARS-CoV-2, ZIKV and IAV. HBA(111-142) is released from its precursor by ubiquitous aspartic proteases under acidic conditions characteristic at sites of infection and inflammation. Thus, HBA(111-142) is an amyloidogenic AMP that may specifically be generated from a highly abundant precursor during bacterial or viral infection and may play an important role in innate antimicrobial immune responses.

Lipid clearance and amyloid formation by serum amyloid A: exploring the links between beneficial and pathologic actions of an enigmatic protein.

Serum amyloid A (SAA) is named after a life-threatening disease, yet this small evolutionarily conserved protein must have played a vital role in host defense. Most circulating SAA binds plasma lipoproteins and modulates their metabolism. However, this hardly justifies the rapid and dramatic SAA upregulation in inflammation, which is concomitant with upregulation of secretory phospholipase A2 (sPLA2). We proposed that these proteins synergistically clear cell membrane debris from the sites of injury. The present study uses biochemical and biophysical approaches to further explore the beneficial function of SAA and its potential links to amyloid formation. We show that murine and human SAA1 are powerful detergents that solubilize diverse lipids, including mammalian biomembranes, converting them into lipoprotein-size nanoparticles. These nanoparticles provide ligands for cell receptors, such as scavenger receptor CD36 or heparin/heparan sulfate, act as substrates of sPLA2, and sequester toxic products of sPLA2. Together, these functions enable SAA to rapidly clear unprotected lipids. SAA can also adsorb, without remodeling, to lipoprotein-size nanoparticles such as exosomal liposomes, which are proxies for lipoproteins. SAA in complexes with zwitterionic phospholipids stabilizes α-helices, while SAA in complexes containing anionic lipids or micelle-forming sPLA2 products forms metastable ß-sheet-rich species that readily aggregate to form amyloid. Consequently, the synergy between SAA and sPLA2 extends from the beneficial lipid clearance to the pathologic amyloid formation. Furthermore, we show that lipid composition alters SAA conformation and thereby can influence the metabolic fate of SAA-lipid complexes, including their proamyloidogenic and proatherogenic binding to heparan sulfate.

Unraveling the complexity of amyloid polymorphism using gold nanoparticles and cryo-EM.

Increasing evidence suggests that amyloid polymorphism gives rise to different strains of amyloids with distinct toxicities and pathology-spreading properties. Validating this hypothesis is challenging due to a lack of tools and methods that allow for the direct characterization of amyloid polymorphism in hydrated and complex biological samples. Here, we report on the development of 11-mercapto-1-undecanesulfonate-coated gold nanoparticles (NPs) that efficiently label the edges of synthetic, recombinant, and native amyloid fibrils derived from different amyloidogenic proteins. We demonstrate that these NPs represent powerful tools for assessing amyloid morphological polymorphism, using cryogenic transmission electron microscopy (cryo-EM). The NPs allowed for the visualization of morphological features that are not directly observed using standard imaging techniques, including transmission electron microscopy with use of the negative stain or cryo-EM imaging. The use of these NPs to label native paired helical filaments (PHFs) from the postmortem brain of a patient with Alzheimer's disease, as well as amyloid fibrils extracted from the heart tissue of a patient suffering from systemic amyloid light-chain amyloidosis, revealed a high degree of homogeneity across the fibrils derived from human tissue in comparison with fibrils aggregated in vitro. These findings are consistent with, and strongly support, the emerging view that the physiologic milieu is a key determinant of amyloid fibril strains. Together, these advances should not only facilitate the profiling and characterization of amyloids for structural studies by cryo-EM, but also pave the way to elucidate the structural basis of amyloid strains and toxicity, and possibly the correlation between the pathological and clinical heterogeneity of amyloid diseases.

Seeded fibrils of the germline variant of human λ-III immunoglobulin light chain FOR005 have a similar core as patient fibrils with reduced stability.

Systemic antibody light chains (AL) amyloidosis is characterized by deposition of amyloid fibrils derived from a particular antibody light chain. Cardiac involvement is a major risk factor for mortality. Using MAS solid-state NMR, we studied the fibril structure of a recombinant light chain fragment corresponding to the fibril protein from patient FOR005, together with fibrils formed by protein sequence variants that are derived from the closest germline (GL) sequence. Both analyzed fibril structures were seeded with ex-vivo amyloid fibrils purified from the explanted heart of this patient. We find that residues 11-42 and 69-102 adopt ß-sheet conformation in patient protein fibrils. We identify arginine-49 as a key residue that forms a salt bridge to aspartate-25 in the patient protein fibril structure. In the germline sequence, this residue is replaced by a glycine. Fibrils from the GL protein and from the patient protein harboring the single point mutation R49G can be both heterologously seeded using patient ex-vivo fibrils. Seeded R49G fibrils show an increased heterogeneity in the C-terminal residues 80-102, which is reflected by the disappearance of all resonances of these residues. By contrast, residues 11-42 and 69-77, which are visible in the MAS solid-state NMR spectra, show 13Cα chemical shifts that are highly like patient fibrils. The mutation R49G thus induces a conformational heterogeneity at the C terminus in the fibril state, whereas the overall fibril topology is retained. These findings imply that patient mutations in FOR005 can stabilize the fibril structure.

Methods to study the structure of misfolded protein states in systemic amyloidosis.

Systemic amyloidosis is defined as a protein misfolding disease in which the amyloid is not necessarily deposited within the same organ that produces the fibril precursor protein. There are different types of systemic amyloidosis, depending on the protein constructing the fibrils. This review will focus on recent advances made in the understanding of the structural basis of three major forms of systemic amyloidosis: systemic AA, AL and ATTR amyloidosis. The three diseases arise from the misfolding of serum amyloid A protein, immunoglobulin light chains or transthyretin. The presented advances in understanding were enabled by recent progress in the methodology available to study amyloid structures and protein misfolding, in particular concerning cryo-electron microscopy (cryo-EM) and nuclear magnetic resonance (NMR) spectroscopy. An important observation made with these techniques is that the structures of previously described in vitro formed amyloid fibrils did not correlate with the structures of amyloid fibrils extracted from diseased tissue, and that in vitro fibrils were typically more protease sensitive. It is thus possible that ex vivo fibrils were selected in vivo by their proteolytic stability.

Half a century of amyloids: past, present and future.

Amyloid diseases are global epidemics with profound health, social and economic implications and yet remain without a cure. This dire situation calls for research into the origin and pathological manifestations of amyloidosis to stimulate continued development of new therapeutics. In basic science and engineering, the cross-ß architecture has been a constant thread underlying the structural characteristics of pathological and functional amyloids, and realizing that amyloid structures can be both pathological and functional in nature has fuelled innovations in artificial amyloids, whose use today ranges from water purification to 3D printing. At the conclusion of a half century since Eanes and Glenner's seminal study of amyloids in humans, this review commemorates the occasion by documenting the major milestones in amyloid research to date, from the perspectives of structural biology, biophysics, medicine, microbiology, engineering and nanotechnology. We also discuss new challenges and opportunities to drive this interdisciplinary field moving forward.

Automatic identification of crossovers in cryo-EM images of murine amyloid protein A fibrils with machine learning.

Detecting crossovers in cryo-electron microscopy images of protein fibrils is an important step towards determining the morphological composition of a sample. Currently, the crossover locations are picked by hand, which introduces errors and is a time-consuming procedure. With the rise of deep learning in computer vision tasks, the automation of such problems has become more and more applicable. However, because of insufficient quality of raw data and missing labels, neural networks alone cannot be applied successfully to target the given problem. Thus, we propose an approach combining conventional computer vision techniques and deep learning to automatically detect fibril crossovers in two-dimensional cryo-electron microscopy image data and apply it to murine amyloid protein A fibrils, where we first use direct image processing methods to simplify the image data such that a convolutional neural network can be applied to the remaining segmentation problem. LAY DESCRIPTION: The ability of protein to form fibrillary structures underlies important cellular functions but can also give rise to disease, such as in a group of disorders, termed amyloid diseases. These diseases are characterised by the formation of abnormal protein filaments, so-called amyloid fibrils, that deposit inside the tissue. Many amyloid fibrils are helically twisted, which leads to periodic variations in the apparent width of the fibril, when observing amyloid fibrils using microscopy techniques like cryogenic electron microscopy (cryo-EM). Due to the two-dimensional projection, parts of the fibril orthogonal to the projection plane appear narrower than parts parallel to the plane. The parts of small width are called crossovers. The distance between two adjacent crossovers is an important characteristic for the analysis of amyloid fibrils, because it is informative about the fibril morphology and because it can be determined from raw data by eye. A given protein can typically form different fibril morphologies. The morphology can vary depending on the chemical and physical conditions of fibril formation, but even when fibrils are formed under identical solution conditions, different morphologies may be present in a sample. As the crossovers allow to define fibril morphologies in a heterogeneous sample, detecting crossovers is an important first step in the sample analysis. In the present paper, we introduce a method for the automated detection of fibril crossovers in cryo-EM image data. The data consists of greyscale images, each showing an unknown number of potentially overlapping fibrils. In a first step, techniques from image analysis and pattern detection are employed to detect single fibrils in the raw data. Then, a convolutional neural network is used to find the locations of crossovers on each single fibril. As these predictions may contain errors, further postprocessing steps assess the quality and may slightly alter or reject the predicted crossovers.

Cellular mechanism of fibril formation from serum amyloid A1 protein.

Serum amyloid A1 (SAA1) is an apolipoprotein that binds to the high-density lipoprotein (HDL) fraction of the serum and constitutes the fibril precursor protein in systemic AA amyloidosis. We here show that HDL binding blocks fibril formation from soluble SAA1 protein, whereas internalization into mononuclear phagocytes leads to the formation of amyloid. SAA1 aggregation in the cell model disturbs the integrity of vesicular membranes and leads to lysosomal leakage and apoptotic death. The formed amyloid becomes deposited outside the cell where it can seed the fibrillation of extracellular SAA1. Our data imply that cells are transiently required in the amyloidogenic cascade and promote the initial nucleation of the deposits. This mechanism reconciles previous evidence for the extracellular location of deposits and amyloid precursor protein with observations the cells are crucial for the formation of amyloid.

Cryo-EM reveals the steric zipper structure of a light chain-derived amyloid fibril.

Amyloid fibrils are proteinaceous aggregates associated with diseases in humans and animals. The fibrils are defined by intermolecular interactions between the fibril-forming polypeptide chains, but it has so far remained difficult to reveal the assembly of the peptide subunits in a full-scale fibril. Using electron cryomicroscopy (cryo-EM), we present a reconstruction of a fibril formed from the pathogenic core of an amyloidogenic immunoglobulin (Ig) light chain. The fibril density shows a lattice-like assembly of face-to-face packed peptide dimers that corresponds to the structure of steric zippers in peptide crystals. Interpretation of the density map with a molecular model enabled us to identify the intermolecular interactions between the peptides and rationalize the hierarchical structure of the fibril based on simple chemical principles.

Electron tomography reveals the fibril structure and lipid interactions in amyloid deposits.

Electron tomography is an increasingly powerful method to study the detailed architecture of macromolecular complexes or cellular structures. Applied to amyloid deposits formed in a cell culture model of systemic amyloid A amyloidosis, we could determine the structural morphology of the fibrils directly in the deposit. The deposited fibrils are arranged in different networks, and depending on the relative fibril orientation, we can distinguish between fibril meshworks, fibril bundles, and amyloid stars. These networks are frequently infiltrated by vesicular lipid inclusions that may originate from the death of the amyloid-forming cells. Our data support the role of nonfibril components for constructing fibril deposits and provide structural views of different types of lipid-fibril interactions.

Cell model for the identification and characterization of prion-like components from Alzheimer brain tissue.

Intracerebral injection of brain extracts from Alzheimer's disease (AD) patients into appropriate mouse models was previously found to drastically accelerate the deposition of Aß amyloid in the recipient animals indicating a prion-like activity. In this study we show that this prion-like activity can be also identified by using a cell culture model of Aß plaque formation. Analysis of biochemical fractions of AD brain extract indicate that the seeding-activity correlated with the presence of Aß peptide and Aß-derived aggregates. In vitro-formed fibrils were also active but their activity was low and depending on the fibril structure and conditions of fibril formation. Our data indicate a conformational basis of the observed seeding effect and suggest the utility of our cell model for further studies on the prion-like activity of AD extracts.

Peptide dimer structure in an Aß(1-42) fibril visualized with cryo-EM.

Alzheimer's disease (AD) is a fatal neurodegenerative disorder in humans and the main cause of dementia in aging societies. The disease is characterized by the aberrant formation of ß-amyloid (Aß) peptide oligomers and fibrils. These structures may damage the brain and give rise to cerebral amyloid angiopathy, neuronal dysfunction, and cellular toxicity. Although the connection between AD and Aß fibrillation is extensively documented, much is still unknown about the formation of these Aß aggregates and their structures at the molecular level. Here, we combined electron cryomicroscopy, 3D reconstruction, and integrative structural modeling methods to determine the molecular architecture of a fibril formed by Aß(1-42), a particularly pathogenic variant of Aß peptide. Our model reveals that the individual layers of the Aß fibril are formed by peptide dimers with face-to-face packing. The two peptides forming the dimer possess identical tilde-shaped conformations and interact with each other by packing of their hydrophobic C-terminal ß-strands. The peptide C termini are located close to the main fibril axis, where they produce a hydrophobic core and are surrounded by the structurally more flexible and charged segments of the peptide N termini. The observed molecular architecture is compatible with the general chemical properties of Aß peptide and provides a structural basis for various biological observations that illuminate the molecular underpinnings of AD. Moreover, the structure provides direct evidence for a steric zipper within a fibril formed by full-length Aß peptide.

Generation and Characterization of Virus-Enhancing Peptide Nanofibrils Functionalized with Fluorescent Labels.

Retroviral gene transfer is the method of choice for the stable introduction of genetic material into the cellular genome. However, efficient gene transfer is often limited by low transduction rates of the viral vectors. We have recently described a 12-mer peptide, termed EF-C, that forms amyloid-like peptide nanofibrils (PNF), strongly increasing viral transduction efficiencies. These nanofibrils are polycationic and bind negatively charged membranes of virions and cells, thereby overcoming charge repulsions and resulting in increased rates of virion attachment and gene transfer. EF-C PNF enhance vector transduction more efficiently than other soluble additives and offer prospects for clinical applications. However, while the transduction-enhancing activity of PNF has been well-characterized, the exact mechanism and the kinetics underlying infection enhancement as well as the cellular fate of the fibrils are hardly explored. This is partially due to the fact that current labeling techniques for PNF rely on amyloid probes that cause high background staining or lose signal intensities after cellular uptake. Here, we sought to generate EF-C PNF covalently coupled with fluorescent labels. To achieve such covalent bioconjugates, the free amino groups of the EF-C peptide were coupled to the ATTO 495 or 647N NHS ester dyes. When small amounts of the labeled peptides were mixed with a 100- to 10 000-fold excess of the native peptide, PNF formed that were morphologically indistinguishable from those derived from the unlabeled peptide. The fluorescence of the fibrils could be readily detected using fluorescence spectroscopy, microscopy, and flow cytometry. In addition, labeled and nonlabeled fibrils captured viral particles and increased retroviral transduction with similar efficacy. These covalently fluorescence-labeled PNF are valuable tools with which to elucidate the mechanism(s) underlying transduction attachment and the fate of the fibrils in cells, tissues, and animal models.

Inhibition of Aß(1-40) fibril formation by cyclophilins.

Cyclophilins interact directly with the Alzheimer's disease peptide Aß (amyloid ß-peptide) and are therefore involved in the early stages of Alzheimer's disease. Aß binding to CypD (cyclophilin D) induces dysfunction of human mitochondria. We found that both CypD and CypA suppress in vitro fibril formation of Aß(1-40) at substoichiometric concentrations when present early in the aggregation process. The prototypic inhibitor CsA (cyclosporin A) of both cyclophilins as well as the new water-soluble MM258 derivative prevented this suppression. A SPOT peptide array approach and NMR titration experiments confirmed binding of Aß(1-40) to the catalytic site of CypD mainly via residues Lys(16)-Glu(22) The peptide Aß(16-20) representing this section showed submicromolar IC50 values for the peptidyl prolyl cis-trans isomerase activity of CypD and CypA and low-micromolar KD values in ITC experiments. Chemical cross-linking and NMR-detected hydrogen-deuterium exchange experiments revealed a shift in the populations of small Aß(1-40) oligomers towards the monomeric species, which we investigated in the present study as being the main process of prevention of Aß fibril formation by cyclophilins.

Common Fibril Structures Imply Systemically Conserved Protein Misfolding Pathways In†Vivo.

Systemic amyloidosis is caused by the misfolding of a circulating amyloid precursor protein and the deposition of amyloid fibrils in multiple organs. Chemical and biophysical analysis of amyloid fibrils from human AL and murine AA amyloidosis reveal the same fibril morphologies in different tissues or organs of one patient or diseased animal. The observed structural similarities concerned the fibril morphology, the fibril protein primary and secondary structures, the presence of post-translational modifications and, in case of the AL fibrils, the partially folded characteristics of the polypeptide chain within the fibril. Our data imply for both analyzed forms of amyloidosis that the pathways of protein misfolding are systemically conserved; that is, they follow the same rules irrespective of where inside one body fibrils are formed or accumulated.

Structural characterization of amyloid fibrils from the human parathyroid hormone.

Amyloid deposits are common in various tissues as a consequence of misfolded proteins. However, secretory protein and peptides are often stored in membrane coated granules as functional amyloids. In this article, we present a detailed characterization of in vitro generated amyloid fibrils from human parathyroid hormone (hPTH(1-84)). Fully mature fibrils could be obtained after a short lag phase within less than one hour at 65°C. These fibrils showed all characteristic of a cross-ß structure. Protease cleavage combined with mass spectrometry identified the central region of the peptide hormone involved in the fibril core formation. EGCG, an inhibitor of amyloid fibril formation, showed binding to residues in the peptide monomers corresponding to the later fibril core and thus explaining the inhibition of the fibril growth. Conformational and dynamic studies by solid-state NMR further corroborated the cross-ß core of the fibrils, but also identified highly mobile segments with a random coil structure not belonging to the rigid fibril core.

Age-dependent defects of alpha-synuclein oligomer uptake in microglia and monocytes.

Extracellular alpha-synuclein (αsyn) oligomers, associated to exosomes or free, play an important role in the pathogenesis of Parkinson's disease (PD). Increasing evidence suggests that these extracellular moieties activate microglia leading to enhanced neuronal damage. Despite extensive efforts on studying neuroinflammation in PD, little is known about the impact of age on microglial activation and phagocytosis, especially of extracellular αsyn oligomers. Here, we show that microglia isolated from adult mice, in contrast to microglia from young mice, display phagocytosis deficits of free and exosome-associated αsyn oligomers combined with enhanced TNFα secretion. In addition, we describe a dysregulation of monocyte subpopulations with age in mice and humans. Accordingly, human monocytes from elderly donors also show reduced phagocytic activity of extracellular αsyn. These findings suggest that these age-related alterations may contribute to an increased susceptibility to pathogens or abnormally folded proteins with age in neurodegenerative diseases.

Hydrostatic Pressure Increases the Catalytic Activity of Amyloid Fibril Enzymes.

We studied the combined effects of pressure (0.1-200 MPa) and temperature (22, 30, and 38 °C) on the catalytic activity of designed amyloid fibrils using a high-pressure stopped-flow system with rapid UV/Vis absorption detection. Complementary FT-IR spectroscopic data revealed a remarkably high pressure and temperature stability of the fibrillar systems. High pressure enhances the esterase activity as a consequence of a negative activation volume at all temperatures (about -14 cm(3) mol(-1) ). The enhancement is sustained in the whole temperature range covered, which allows a further acceleration of the enzymatic activity at high temperatures (activation energy 45-60 kJ mol(-1) ). Our data reveal the great potential of using both pressure and temperature modulation to optimize the enzyme efficiency of catalytic amyloid fibrils.

Polymorphism of Amyloid Fibrils In Vivo.

Polymorphism is a wide-spread feature of amyloid-like fibrils formed in vitro, but it has so far remained unclear whether the fibrils formed within a patient are also affected by this phenomenon. In this study we show that the amyloid fibrils within a diseased individual can vary considerably in their three-dimensional architecture. We demonstrate this heterogeneity with amyloid fibrils deposited within different organs, formed from sequentially non-homologous polypeptide chains and affecting human or animals. Irrespective of amyloid type or source, we found in vivo fibrils to be polymorphic. These data imply that the chemical principles of fibril assembly that lead to such polymorphism are fundamentally conserved in vivo and in vitro.

Enhanced Fibril Fragmentation of N-Terminally Truncated and Pyroglutamyl-Modified Aß Peptides.

N-terminal truncation and pyroglutamyl (pE) formation are naturally occurring chemical modifications of the Aß peptide in Alzheimer's disease. We show herein that these two modifications significantly reduce the fibril length and the transition midpoint of thermal unfolding of the fibrils, but they do not substantially perturb the fibrillary peptide conformation. This observation implies that the N terminus of the unmodified peptide protects Aß fibrils against mechanical stress and fragmentation and explains the high propensity of pE-modified peptides to form small and particularly toxic aggregates.