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.

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.

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.

Isolation, characterization, and aggregation of a structured bacterial matrix precursor.

Biofilms are surface-associated groups of microbial cells that are embedded in an extracellular matrix (ECM). The ECM is a network of biopolymers, mainly polysaccharides, proteins, and nucleic acids. ECM proteins serve a variety of structural roles and often form amyloid-like fibers. Despite the extensive study of the formation of amyloid fibers from their constituent subunits in humans, much less is known about the assembly of bacterial functional amyloid-like precursors into fibers. Using dynamic light scattering, atomic force microscopy, circular dichroism, and infrared spectroscopy, we show that our unique purification method of a Bacillus subtilis major matrix protein component results in stable oligomers that retain their native α-helical structure. The stability of these oligomers enabled us to control the external conditions that triggered their aggregation. In particular, we show that stretched fibers are formed on a hydrophobic surface, whereas plaque-like aggregates are formed in solution under acidic pH conditions. TasA is also shown to change conformation upon aggregation and gain some ß-sheet structure. Our studies of the aggregation of a bacterial matrix protein from its subunits shed new light on assembly processes of the ECM within bacterial biofilms.

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.

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.

Microcin amyloid fibrils A are reservoir of toxic oligomeric species.

Microcin E492 (Mcc), a low molecular weight bacteriocin produced by Klebsiella pneumoniae RYC492, has been shown to exist in two forms: soluble forms that are believed to be toxic to the bacterial cell by forming pores and non-toxic fibrillar forms that share similar biochemical and biophysical properties with amyloids associated with several human diseases. Here we report that fibrils polymerized in vitro from soluble forms sequester toxic species that can be released upon changing environmental conditions such as pH, ionic strength, and upon dilution. Our results indicate that basic pH (≥8.5), low NaCl concentrations (≤50 mm), and dilution (>10-fold) destabilize Mcc fibrils into more soluble species that are found to be toxic to the target cells. Additionally, we also found a similar conversion of non-toxic fibrils into highly toxic oligomers using Mcc aggregates produced in vivo. Moreover, the soluble protein released from fibrils is able to rapidly polymerize into amyloid fibrils under fibril-forming conditions and to efficiently seed aggregation of monomeric Mcc. Our findings indicate that fibrillar forms of Mcc constitute a reservoir of toxic oligomeric species that is released into the medium upon changing the environmental conditions. These findings may have substantial implications to understand the dynamic process of interconversion between toxic and non-toxic aggregated species implicated in protein misfolding diseases.

Bacterial expression and purification of the amyloidogenic peptide PAPf39 for multidimensional NMR spectroscopy.

PAPf39 is a 39 residue peptide fragment from human prostatic acidic phosphatase that forms amyloid fibrils in semen. These fibrils have been implicated in facilitating HIV transmission. To enable structural studies of PAPf39 by NMR spectroscopy, efficient methods allowing the production of milligram quantities of isotopically labeled peptide are essential. Here, we report the high-yield expression and purification of uniformly (13)C- and (15)N-labeled PAPf39 peptide, through expression as a fusion to ubiquitin at the N-terminus and an intein at the C-terminus. This allows the study of the PAPf39 monomer conformational ensemble by NMR spectroscopy. To this end, we performed the NMR chemical shift assignment of the PAPf39 peptide in the monomeric state at low pH.

Gelation in protein extracts from cold acclimated and non-acclimated winter rye (Secale cereale L. cv Musketeer).

A protein gel is a three-dimensional network consisting of molecular interactions between biopolymers that entrap a significant volume of a continuous liquid phase (water). Molecular interactions in gels occur at junction zones within and between protein molecules through electrostatic forces, hydrogen bonding, hydrophobic associations (van der Waals attractions) and covalent bonding. Gels have the physicochemical properties of both solids and liquids, and are extremely important in the production and stability of a variety of foods, bioproducts and pharmaceuticals. In this study, gelation was induced in phenol extracted protein fractions from non-acclimated (NA) and cold-acclimated (CA) winter rye (Secale cereale L. cv Musketeer) leaf tissue after repeated freeze-thaw treatments. Gel formation only occurred at high pH (pH 12.0) and a minimum of 3-4 freeze-thaw cycles were required. The gel was thermally stable and only a specific combination of chemical treatments could disrupt the gel network. SDS-PAGE analysis identified ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) as the major protein component in the gel, although Rubisco itself did not appear to be a factor in gelation. Raman spectroscopy suggested changes in protein secondary structure during freeze-thaw cycles. Overall, the NA and CA gels were similar in composition and structure, with the exception that the CA gel appeared to be amyloidic in nature based on thioflavin T (ThT) fluorescence. Protein gelation, particularly in the apoplast, may confer protection against freeze-induced dehydration and potentially have a commercial application to improve frozen food quality.

On the use of size exclusion chromatography for the resolution of mixed amyloid aggregate distributions: I. equilibrium partition models.

In this study, we investigated the theoretical potential of size exclusion chromatography (SEC) for resolving mixtures of protein aggregates (of various sizes and shapes) produced in the generation of amyloid fibrils. We present our findings in the form of an equilibrium partition model. We first review the general characteristics of SEC and discuss the physicochemical features affecting solute transport and partition. We then develop new methods for estimating the transport and partition coefficients of protein aggregates on the basis of their molecular dimensions and the SEC column properties. We detail how these calculated properties can be used to estimate the likely resolving power of an SEC column. Model predictions were found to be in general agreement with experimental data gained from the measurement of the elution profile of sheared amyloid fibrils prepared from bovine insulin and passed through a Superose 6 precision SEC column. Our formalism should provide a basic appreciation of the competing factors at work and allow an informed choice to be made for optimal selection of SEC column medium to separate a desired size range of aggregate.

Removal versus fragmentation of amyloid-forming precursors via membrane filtration.

The presence of even minute amounts of protein aggregates in solution can significantly alter the kinetics of amyloid formation. Removal of such pre-existing aggregates is critical for reproducible analysis of amyloid formation. Here we examine the effects of membrane filtration on insulin fibrillization. We find that filtration of insulin with large pore membranes (≥ 100 nm) generally slows fibril formation relative to unfiltered solutions by removing pre-aggregated protein. Unexpectedly, filtration with small pore membranes (< 100 nm) showed no beneficial effect and, in some cases, accelerated insulin fibril formation. This effect may be due to fragmentation of pre-existing aggregates during filtration through small pore membranes, which can increase the number of amyloid-forming precursors. These findings reveal the complexity of removing protein aggregates via filtration and suggest optimal filtration protocols for conducting fibril formation analysis of insulin and similar amyloidogenic proteins.

Cutaneous amyloidosis: a concept review.

Concepts and semantics are crucial for good communication between clinicians and pathologists. Amyloidosis was described more than 150 years ago. Therefore, the terminology related to it is abundant, varied, and sometimes complex. In this report, we intend to discuss several terms related to the disease, with special emphasis on cutaneous amyloidosis. We present a review, from Virchow to present, of the concepts related to amyloidosis: its nature, the classification of cutaneous forms of the disease, and the techniques used in its diagnosis.

Isolating toxic insulin amyloid reactive species that lack ß-sheets and have wide pH stability.

Amyloid diseases, including Alzheimer's disease, are characterized by aggregation of normally functioning proteins or peptides into ordered, ß-sheet rich fibrils. Most of the theories on amyloid toxicity focus on the nuclei or oligomers in the fibril formation process. The nuclei and oligomers are transient species, making their full characterization difficult. We have isolated toxic protein species that act like an oligomer and may provide the first evidence of a stable reactive species created by disaggregation of amyloid fibrils. This reactive species was isolated by dissolving amyloid fibrils at high pH and it has a mass >100 kDa and a diameter of 48 ± 15 nm. It seeds the formation of fibrils in a dose dependent manner, but using circular dichroism and deep ultraviolet resonance Raman spectroscopy, the reactive species was found to not have a ß-sheet rich structure. We hypothesize that the reactive species does not decompose at high pH and maintains its structure in solution. The remaining disaggregated insulin, excluding the toxic reactive species that elongated the fibrils, returned to native structured insulin. This is the first time, to our knowledge, that a stable reactive species of an amyloid reaction has been separated and characterized by disaggregation of amyloid fibrils.

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.

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.

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.

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.

Isolation and identification by sequence analysis of experimentally induced guinea pig amyloid fibrils.

Amyloidosis was produced experimentally in guinea pigs by multiple casein injections. Amyloid fibrils were isolated and fractionated and a protein obtained that had an amino acid composition comparable with A protein, a unique nonimmunoglobulin constituent of secondary amyloid deposits. N-terminal sequence analysis demonstrated a sequence homologous with that of A proteins from human and monkey preparations but preceded by a 5-residue peptide which had an N-terminal histidine. A definite species specificity in A protein from human and guinea pig was identified on immunologic analysis.

Characterization of an amyloid fibril protein from senile cardiac amyloid.

A protein, ASCA, is isolated from amyloid fibrils extracted from heart tissue of five different patients with senile cardiac amyloidosis (SCA). The proteins of all five patients showed immunological identity when reacted with an antiserum raised against one of the proteins. In contrast, no reaction was obtained with antisera against a variety of other amyloid proteins. The antiserum against the subunit protein of senile cardiac amyloid did not react with any other amyloid preparations tested, nor with extracts of normal heart tissue. Thus, the subunit protein appeared to be unique to senile heart amyloid. The protein could form fibrils in vitro, had a mol wt of about 6,000 daltons and the amino acid compositions investigated in two cases showed extensive similarities but were clearly different from that of protein AA of secondary amyloid fibrils.