In situ Sub-Cellular Identification of Functional Amyloids in Bacteria and Archaea by Infrared Nanospectroscopy.

Formation of amyloid structures is originally linked to human disease. However, amyloid materials are found extensively in the animal and bacterial world where they stabilize intra- and extra-cellular environments like biofilms or cell envelopes. To date, functional amyloids have largely been studied using optical microscopy techniques in vivo, or after removal from their biological context for higher-resolution studies in vitro. Furthermore, conventional microscopies only indirectly identify amyloids based on morphology or unspecific amyloid dyes. Here, the high chemical and spatial (≈20 nm) resolution of Infrared Nanospectroscopy (AFM-IR) to investigate functional amyloid from Escherichia coli (curli), Pseudomonas (Fap), and the Archaea Methanosaeta (MspA) in situ is exploited. It is demonstrated that AFM-IR identifies amyloid protein within single intact cells through their cross ß-sheet secondary structure, which has a unique spectroscopic signature in the amide I band of protein. Using this approach, nanoscale-resolved chemical images and spectra of purified curli and Methanosaeta cell wall sheaths are provided. The results highlight significant differences in secondary structure between E. coli cells with and without curli. Taken together, these results suggest that AFM-IR is a new and powerful label-free tool for in situ investigations of the biophysical state of functional amyloid and biomolecules in general.

Cryo-EM structures of hIAPP fibrils seeded by patient-extracted fibrils reveal new polymorphs and conserved fibril cores.

Amyloidosis of human islet amyloid polypeptide (hIAPP) is a pathological hallmark of type II diabetes (T2D), an epidemic afflicting nearly 10% of the world's population. To visualize disease-relevant hIAPP fibrils, we extracted amyloid fibrils from islet cells of a T2D donor and amplified their quantity by seeding synthetic hIAPP. Cryo-EM studies revealed four fibril polymorphic atomic structures. Their resemblance to four unseeded hIAPP fibrils varies from nearly identical (TW3) to non-existent (TW2). The diverse repertoire of hIAPP polymorphs appears to arise from three distinct protofilament cores entwined in different combinations. The structural distinctiveness of TW1, TW2 and TW4 suggests they may be faithful replications of the pathogenic seeds. If so, the structures determined here provide the most direct view yet of hIAPP amyloid fibrils formed during T2D.

Immunohistochemical typing of amyloid in fixed paraffin-embedded samples by an automatic procedure: Comparison with immunofluorescence data on fresh-frozen tissue.

Amyloidosis comprises a spectrum of disorders characterized by the extracellular deposition of amorphous material, originating from an abnormal serum protein. The typing of amyloid into its many variants represents a pivotal step for a correct patient management. Several methods are currently used, including mass spectrometry, immunofluorescence, immunohistochemistry, and immunogold labeling. The aim of the present study was to investigate the accuracy and reliability of immunohistochemistry by means of a recently developed amyloid antibody panel applicable on fixed paraffin-embedded tissues in an automated platform. Patients with clinically and pathologically proven amyloidosis were divided into two cohorts: a pilot one, which included selected amyloidosis cases from 2009 to 2018, and a retrospective one (comprising all consecutive amyloidosis cases analyzed between November 2018 and May 2020). The above-referred panel of antibodies for amyloid classification was tested in all cases using an automated immunohistochemistry platform. When fresh-frozen material was available, immunofluorescence was also performed. Among 130 patients, a total of 143 samples from different organs was investigated. They corresponded to 51 patients from the pilot cohort and 79 ones from the retrospective cohort. In 82 cases (63%), fresh-frozen tissue was tested by immunofluorescence, serving to define amyloid subtype only in 30 of them (36.6%). On the contrary, the automated immunohistochemistry procedure using the above-referred new antibodies allowed to establish the amyloid type in all 130 cases (100%). These included: ALλ (n = 60, 46.2%), ATTR (n = 29, 22.3%), AA (n = 19, 14.6%), ALκ (n = 18, 13.8%), ALys (n = 2, 1.5%), and Aß2M amyloidosis (n = 2, 1.5%). The present immunohistochemistry antibody panel represents a sensitive, reliable, fast, and low-cost method for amyloid typing. Since immunohistochemistry is available in most pathology laboratories, it may become the new gold standard for amyloidosis classification, either used alone or combined with mass spectrometry in selected cases.

Identification of amyloidogenic proteins in the microbiomes of a rat Parkinson's disease model and wild-type rats.

Cross seeding between amyloidogenic proteins in the gut is receiving increasing attention as a possible mechanism for initiation or acceleration of amyloid formation by aggregation-prone proteins such as αSN, which is central in the development of Parkinson's disease (PD). This is particularly pertinent in view of the growing number of functional (i.e., benign and useful) amyloid proteins discovered in bacteria. Here we identify two amyloidogenic proteins, Pr12 and Pr17, in fecal matter from PD transgenic rats and their wild type counterparts, based on their stability against dissolution by formic acid (FA). Both proteins show robust aggregation into ThT-positive aggregates that contain higher-order ß-sheets and have a fibrillar morphology, indicative of amyloid proteins. In addition, Pr17 aggregates formed in vitro showed significant resistance against FA, suggesting an ability to form highly stable amyloid. Treatment with proteinase K revealed a protected core of approx. 9 kDa. Neither Pr12 nor Pr17, however, affected αSN aggregation in vitro. Thus, amyloidogenicity does not per se lead to an ability to cross-seed fibrillation of αSN. Our results support the use of proteomics and FA to identify amyloidogenic protein in complex mixtures and suggests that there may be numerous functional amyloid proteins in microbiomes.

Trehalose Inhibits the Heat-Induced Formation of the Amyloid-Like Structure of Soluble Proteins Isolated from Human Cataract Lens.

The age-dependent loss of solubility and aggregation of crystallins constitute the pathological hallmarks of cataract. Several biochemical and biophysical factors are responsible for the reduction of crystallins' solubility and formation of irreversible protein aggregates, which display amyloid-like characteristics. The present study reports the heat-induced aggregation of soluble proteins isolated from human cataract lenses and the formation of amyloid-like structures. Exposure of protein at 55 °C for 4 h resulted in extensive (≈ 60%) protein aggregation. The heat-induced protein aggregates displayed substantial (≈ 20 nm) redshift in the wavelength of maximum absorption (λmax) of Congo red (CR) and increase in Thioflavin T (ThT) fluorescence emission intensity, indicating the presence of amyloid-like structures in the heat-induced protein aggregates. Subsequently, the addition of trehalose resulted in substantial inhibition of heat-induced aggregation and the formation of amyloid-like structure. The ability of trehalose to inhibit the heat-induced aggregation was found to be linearly dependent upon its concentration used. The optimum effect was observed in the presence of 30-40% (w/v) trehalose where the aggregated was found to be reduced from 60 to 30%. The present study demonstrated the ability to trehalose to inhibit the protein aggregation and interfere with the formation of amyloid-like structures.

Structural Characterization of Cardiac Ex Vivo Transthyretin Amyloid: Insight into the Transthyretin Misfolding Pathway In Vivo.

Structural characterization of misfolded protein aggregates is essential to understanding the molecular mechanism of protein aggregation associated with various protein misfolding disorders. Here, we report structural analyses of ex vivo transthyretin aggregates extracted from human cardiac tissue. Comparative structural analyses of in vitro and ex vivo transthyretin aggregates using various biophysical techniques revealed that cardiac transthyretin amyloid has structural features similar to those of in vitro transthyretin amyloid. Our solid-state nuclear magnetic resonance studies showed that in vitro amyloid contains extensive nativelike ß-sheet structures, while other loop regions including helical structures are disrupted in the amyloid state. These results suggest that transthyretin undergoes a common misfolding and aggregation transition to nativelike aggregation-prone monomers that self-assemble into amyloid precipitates in vitro and in vivo.

Primary cervical spine AL-κ amyloidoma: A case report and review of the literature.

Of the myriad of variants of amyloidoses where abnormally folded proteins damage native tissue, primary cervical spine amyloidoma represents one of the rarest forms. Since clinical presentations and imaging findings appear similar to other pathologies, including abscesses, metastatic lesions, and inflammatory lesions, a definitive diagnosis requires a biopsy with specific immunohistochemical stains. We present the first known case of primary cervical amyloid light-chain (AL)-κ subtype amyloidoma and compare the clinical presentations, imaging findings, treatment options, and immunohistochemical subtypes of primary, hemodialysis, and multiple myeloma cervical amyloidomas. Our case is of a 58-year-old man who developed neck pain radiating to the left arm with bilateral upper extremity weakness over several months. Magnetic resonance imaging revealed a circumferential C1-C2 mass extending into the neural foramina inducing severe mass effect. The patient underwent C2 laminectomy and resection of the lesion which was discovered during surgery to be completely epidural. Postoperatively, his pain and weakness improved. A complete work-up was negative for systemic amyloidosis or inflammatory conditions. In the setting of a long clinical history of hemodialysis, this patient required specific staining and laboratory testing to correctly diagnose his primary cervical AL-κ subtype amyloidoma. Cervical amyloidomas comprise a very small minority of amyloid pathology with an exceptional prognosis following successful surgical resection and stabilization. It is recommended these patients undergo surgical resection with appropriate characterization and a complete work-up to rule out systemic disease.

Mass Spectrometry Amyloid Typing Is Reproducible across Multiple Organ Sites.

We have determined patient's amyloid subtype through immunohistochemical and proteomic analyses of formalin-fixed, paraffin-embedded (FFPE) tissue samples from two affected organs per patient. Amyloid typing, via immunohistochemistry (IHC) and laser microdissection followed by the combination of liquid chromatography with mass spectrometry (LMD-LC-MS), was performed using tissue samples of the human heart, liver, kidney, tongue, and small intestine from 11 patients, and the results were compared with clinical data. LMD-LC-MS correctly typed AL amyloidosis in all 22 FFPE tissue samples despite tissue origin. In contrast, IHC was successful only in the analysis of eight FFPE tissue samples with differences between the examined organs. In the majority of LMD-LC-MS typed samples, the level of IHC staining intensity for transthyretin and serum amyloid A was the same as that for Ig κ and Ig λ antibodies, suggesting low Ig κ or Ig λ antibodies reactivity and the additional antibody clones were essential for correct typing. Both methods used in the study were found to be suitable for amyloid typing, although LMD-LC-MS yielded more promising results than IHC.

Preparation of α-Synuclein Amyloid Assemblies for Toxicity Experiments.

Amyloid assemblies of certain proteins, including the Parkinson disease-related protein α-synuclein, are commonly associated with the development and spreading of neurodegenerative diseases, although the nature of the most toxic forms and the mechanisms by which they trigger neurodegeneration remain largely unknown. This is at least in part due to the inherent challenges involved in the preparation of stable and structurally homogeneous samples of amyloid assemblies that could be used in toxicity experiments. Here, we describe the preparation of two different types of stable α-synuclein amyloid assemblies, namely a kinetically trapped oligomeric species and a propagating-competent fibrillar polymorph. The degree of heterogeneity in the samples has been defined and carefully minimized, thus allowing for meaningful structure-toxicity relationships in different α-synuclein amyloid assemblies to be established.

Selective and Sensitive Pull Down of Amyloid Fibrils Produced in Vitro and in Vivo by the Use of Pentameric-Thiophene-Coupled Resins.

Protein aggregation is a hallmark of several degenerative diseases, including Alzheimer's disease, Parkinson's disease and familial amyloidosis (Finnish type) (FAF). A method to isolate and detect amyloids is desired for the diagnosis of amyloid diseases. Here, we report the synthesis of pentameric thiophene amyloid ligand (p-FTAA) linked to agarose resin for selective purification of amyloid aggregates produced in vitro and in vivo. Using amyloid fibrils produced in vitro from α-synuclein, gelsolin, and Aß1-40 and gelsolin amyloid aggregates extracted from tissue homogenates of a mouse model of FAF, we observed that p-FTAA resin was able to pull down amyloid aggregates. The functionalized resin was also able to pull down oligomers produced in vitro from the A30P variant of α-synuclein. The methodology described here can be useful for the diagnosis of amyloidogenic disease and also can be used to purify amyloid fibrils from biological samples, rendering the fibrils available for more accurate structural and biochemical characterization.

Isolation and Analysis of Prion and Amyloid Aggregates from Yeast Cells.

Amyloid fibers are large and extremely stable structures that can resist denaturation by strong anionic detergents, such as sodium dodecyl sulfate or sarkosyl. Here, we present two complementary analytical methods that exploit these properties, enabling the isolation and characterization of amyloid/prion aggregates. The first technique, known as semidenaturating detergent agarose gel electrophoresis, is an immunoblotting technique, conceptually similar to conventional western blotting. It enables the targeted identification of large detergent-resistant protein aggregates using antibodies specific to the protein of interest. The second method, called the technique for amyloid purification and identification, is a nontargeted approach that can isolate amyloid aggregates for analysis by tandem mass spectrometry. The latter approach requires no special genetic tools or antibodies, and can identify amyloid-forming proteins, such as prions, as well as proteins tightly associated with amyloid, from a variety of cell sources.

Protein Aggregate-Ligand Binding Assays Based on Microfluidic Diffusional Separation.

The measurement of molecular interactions with pathological protein aggregates, including amyloid fibrils, is of central importance in the context of the development of diagnostic and therapeutic strategies against protein misfolding disorders. Probing such interactions by conventional methods can, however, be challenging because of the supramolecular nature of protein aggregates, their heterogeneity, and their often dynamic nature. Here we demonstrate that direct measurement of diffusion on a microfluidic platform enables the determination of affinity and kinetics data for ligand binding to amyloid fibrils in solution. This method yields rapid binding information from only microlitres of sample, and is therefore a powerful technique for identifying and characterising molecular species with potential therapeutic or diagnostic application.

Preparation of Amyloid Fibrils for Magic-Angle Spinning Solid-State NMR Spectroscopy.

Solid-state NMR spectroscopy (SSNMR) is an established and invaluable tool for the study of amyloid fibril structure with atomic-level detail. Optimization of the homogeneity and concentration of fibrils enhances the resolution and sensitivity of SSNMR spectra. Here, we present a fibrillization and fibril processing protocol, starting from purified monomeric α-synuclein, that enables the collection of high-resolution SSNMR spectra suitable for site-specific structural analysis. This protocol does not rely on any special features of α-synuclein and should be generalizable to any other amyloid protein.

Purification and Refolding to Amyloid Fibrils of (His)6-tagged Recombinant Shadoo Protein Expressed as Inclusion Bodies in E. coli.

The Escherichia coli expression system is a powerful tool for the production of recombinant eukaryotic proteins. We use it to produce Shadoo, a protein belonging to the prion family. A chromatographic method for the purification of (His)6-tagged recombinant Shadoo expressed as inclusion bodies is described. The inclusion bodies are solubilized in 8 M urea and bound to a Ni(2+)-charged column to perform ion affinity chromatography. Bound proteins are eluted by a gradient of imidazole. Fractions containing Shadoo protein are subjected to size exclusion chromatography to obtain a highly purified protein. In the final step purified Shadoo is desalted to remove salts, urea and imidazole. Recombinant Shadoo protein is an important reagent for biophysical and biochemical studies of protein conformation disorders occurring in prion diseases. Many reports demonstrated that prion neurodegenerative diseases originate from the deposition of stable, ordered amyloid fibrils. Sample protocols describing how to fibrillate Shadoo into amyloid fibrils at acidic and neutral/basic pHs are presented. The methods on how to produce and fibrillate Shadoo can facilitate research in laboratories working on prion diseases, since it allows for production of large amounts of protein in a rapid and low cost manner.

Rapid chemical decontamination of infectious CJD and scrapie particles parallels treatments known to disrupt microbes and biofilms.

Neurodegenerative human CJD and sheep scrapie are diseases caused by several different transmissible encephalopathy (TSE) agents. These infectious agents provoke innate immune responses in the brain, including late-onset abnormal prion protein (PrP-res) amyloid. Agent particles that lack detectable PrP sequences by deep proteomic analysis are highly infectious. Yet these agents, and their unusual resistance to denaturation, are often evaluated by PrP amyloid disruption. To reexamine the intrinsic resistance of TSE agents to denaturation, a paradigm for less resistant viruses and microbes, we developed a rapid and reproducible high yield agent isolation procedure from cultured cells that minimized PrP amyloid and other cellular proteins. Monotypic neuronal GT1 cells infected with the FU-CJD or 22L scrapie agents do not have complex brain changes that can camouflage infectious particles and prevent their disruption, and there are only 2 reports on infectious titers of any human CJD strain treated with chemical denaturants. Infectious titers of both CJD and scrapie were reduced by >4 logs with Thiourea-urea, a treatment not previously tested. A mere 5 min exposure to 4M GdnHCl at 22°C reduced infectivity by >5 logs. Infectious 22L particles were significantly more sensitive to denaturation than FU-CJD particles. A protocol using sonication with these chemical treatments may effectively decontaminate complicated instruments, such as duodenoscopes that harbor additional virulent microbes and biofilms associated with recent iatrogenic infections.

Amyloids: from Pathogenesis to Function.

The term "amyloids" refers to fibrillar protein aggregates with cross-ß structure. They have been a subject of intense scrutiny since the middle of the previous century. First, this interest is due to association of amyloids with dozens of incurable human diseases called amyloidoses, which affect hundreds of millions of people. However, during the last decade the paradigm of amyloids as pathogens has changed due to an increase in understanding of their role as a specific variant of quaternary protein structure essential for the living cell. Thus, functional amyloids are found in all domains of the living world, and they fulfill a variety of roles ranging from biofilm formation in bacteria to long-term memory regulation in higher eukaryotes. Prions, which are proteins capable of existing under the same conditions in two or more conformations at least one of which having infective properties, also typically have amyloid features. There are weighty reasons to believe that the currently known amyloids are only a minority of their real number. This review provides a retrospective analysis of stages in the development of amyloid biology that during the last decade resulted, on one hand, in reinterpretation of the biological role of amyloids, and on the other hand, in the development of systems biology of amyloids, or amyloidomics.

Sedimentation Velocity Analysis of the Size Distribution of Amyloid Oligomers and Fibrils.

Amyloid fibrils result from the self-assembly of proteins into large aggregates with fibrillar morphology and common structural features. These fibrils form the major component of amyloid plaques that are associated with a number of common and debilitating diseases, including Alzheimer's disease. While a range of unrelated proteins and peptides are known to form amyloid fibrils, a common feature is the formation of aggregates of various sizes, including mature fibrils of differing length and/or structural morphology, small oligomeric precursors, and other less well-understood forms such as amorphous aggregates. These various species can possess distinct biochemical, biophysical, and pathological properties. Sedimentation velocity analysis can characterize amyloid fibril formation in exceptional detail, providing a particularly useful method for resolving the complex heterogeneity present in amyloid systems. In this chapter, we describe analytical methods for accurate quantification of both total amyloid fibril formation and the formation of distinct amyloid structures based on differential sedimentation properties. We also detail modern analytical ultracentrifugation methods to determine the size distribution of amyloid aggregates. We illustrate examples of the use of these techniques to provide biophysical and structural information on amyloid systems that would otherwise be difficult to obtain.

Dewetting transition assisted clearance of (NFGAILS) amyloid fibrils from cell membranes by graphene.

Clearance of partially ordered oligomers and monomers deposited on cell membrane surfaces is believed to be an effective route to alleviate many potential protein conformational diseases (PCDs). With large-scale all-atom molecular dynamics simulations, here we show that graphene nanosheets can easily and quickly win a competitive adsorption of human islet amyloid polypeptides (hIAPP22-28) NFGAILS and associated fibrils against cell membrane, due to graphene's unique two-dimensional, highly hydrophobic surface with its all-sp(2) hybrid structure. A nanoscale dewetting transition was observed at the interfacial region between the fibril (originally deposited on the membrane) and the graphene nanosheet, which significantly assisted the adsorption of fibrils onto graphene from the membrane. The π-π stacking interaction between Phe23 and graphene played a crucial role, providing the driving force for the adsorption at the graphene surface. This study renders new insight towards the importance of water during the interactions between amyloid peptides, the phospholipidic membrane, and graphene, which might shed some light on future developments of graphene-based nanomedicine for preventing/curing PCDs like type II diabetes mellitus.

Expression and purification of the aortic amyloid polypeptide medin.

The 50-amino acid protein medin is the main fibrillar component of human aortic medial amyloid (AMA), the most common form of localised amyloid which affects 97% of Caucasians over the age of 50. Structural models for several amyloid assemblies, including the Alzheimer's amyloid-ß peptides, have been defined from solid-state nuclear magnetic resonance (SSNMR) measurements on (13)C- and (15)N-labelled protein fibrils. SSNMR-derived structural information on fibrillar medin is scant, however, because studies to date have been restricted to limited measurements on site-specifically labelled protein prepared by solid-phase synthesis. Here we report a procedure for the expression of a SUMO-medin fusion protein in Escherichia coli and IMAC purification yielding pure, uniformly (13)C,(15)N-labelled medin in quantities required for SSNMR analysis. Thioflavin T fluorescence and dynamic light scattering measurements and transmission electron microscopy analysis confirm that recombinant medin assembles into amyloid-like fibrils over a 48-h period. The first (13)C and (15)N SSNMR spectra obtained for uniformly-labelled fibrils indicate that medin adopts a predominantly ß-sheet conformation with some unstructured elements, and provide the basis for further, more detailed structural investigations.

Label free sensing platform for amyloid fibrils effect on living cells.

This study presents a multiparametric label-free analysis gathering surface plasmon resonance (SPR) and electrical impedance spectroscopy (EIS) for monitoring the progress of a model epithelial cell culture (Madin Darbey Canine Kidney - MDCK) exposed to a peptide with high bio-medical relevance, amyloid ß (Aß42). The approach surpasses the limitations in using the SPR angle for analyzing confluent cell monolayers and proposes a novel quantitative analysis of the SPR dip combined with advanced EIS as a tool for dynamic cell assessment. Long, up to 48h time series of EIS and SPR data reveal a biphasic cellular response upon Aß42 exposure corresponding to changes in cell-substrate adherence, cell-cell tightening or cytoskeletal remodeling. The equivalent circuit used for fitting the EIS spectra provided substantiation of SPR analysis on the progress of cell adhesion as well as insight on dynamics of cell-cell junction. Complementary endpoint assays: western blot analysis and atomic force microscopy experiments have been performed for validation. The proposed label free sensing of nonlethal effect of model amyloid protein at cellular level provides enhanced resolution on cell-surface and cell-cell interactions modulated by membrane related protein apparatus, applicable as well to other adherent cell types and amyloid compounds.