Diana Canetti

BackgroundProteomics is routinely used to type clinical amyloid deposits, and offers additional benefit of identifying genetic variants, which can be diagnostically useful. Reviewing the proteomics data for ATTR patients attending our Centre revealed an unusually large number of samples containing a rare pathogenic H90D TTR variant alongside the more common H90N variant.MethodsThese findings raised questions to their source. Proteomics was used to monitor the generation of H90D/H90N variants in fresh, frozen, stored samples during extraction and digestion, and also following Cu2+-mediated oxidation.ResultsThere was no evidence that the variants were present in the circulation, except in one patient with genetically confirmed H90D TTR, in fresh fat aspirates or tissues from an ATTR amyloid mouse model. The variant could be generated in vitro from both wild-type TTR and ex vivo ATTR fibrils by non-enzymic oxidation of histidine at position 90. These data suggest that the H90D variant can be generated artefactually from wild-type 90H TTR through a radical-mediated oxidation of histidine, followed by its conversion to asparagine and aspartic acid. This probably occurs during storage.ConclusionsIn the absence of genetic data, the identification of H90D TTR in stored tissue by proteomics should be treated with caution.

**Author Listing - Lead/ Submitting Author (we will be communicating with this individual): Name, Institution, City and State where the work was | POSTER

**Author Listing - Lead/ Submitting Author (we will be communicating with this individual): Name, Institution, City and State where the work was | POSTER

β₂‐microglobulin (β₂‐m) is a plasma protein derived from physiological shedding of the class I major histocompatibility complex (MHCI), causing human systemic amyloidosis either due to persistently high concentrations of the wild‐type (WT) protein in hemodialyzed patients, or in presence of mutations, such as D76N β₂‐m, which favor protein deposition in the adulthood, despite normal plasma levels. Here we describe a new transgenic Caenorhabditis elegans (C. elegans) strain expressing human WT β₂‐m at high concentrations, mimicking the condition that underlies dialysis‐related amyloidosis (DRA) and we compare it to a previously established strain expressing the highly amyloidogenic D76N β₂‐m at lower concentrations. Both strains exhibit behavioral defects, the severity of which correlates with β₂‐m levels rather than with the presence of mutations, being more pronounced in WT β₂‐m worms. β₂‐m expression also has a deep impact on the nematodes' proteomic and metabolic profiles. Most significantly affected processes include protein degradation and stress response, amino acids metabolism, and bioenergetics. Molecular alterations are more pronounced in worms expressing WT β₂‐m at high concentration compared to D76N β₂‐m worms. Altogether, these data show that β₂‐m is a proteotoxic protein in vivo also in its wild‐type form, and that concentration plays a key role in modulating pathogenicity. Our transgenic nematodes recapitulate the distinctive features subtending DRA compared to hereditary β₂‐m amyloidosis (high levels of non‐mutated β₂‐m vs. normal levels of variant β₂‐m) and provide important clues on the molecular bases of these human diseases.

AL amyloidosis is caused by the misfolding of immunoglobulin light chains leading to an impaired function of tissues and organs in which they accumulate. Due to the paucity of -omics profiles from undissected samples, few studies have addressed amyloid-related damage system wide. To fill this gap, we evaluated proteome changes in the abdominal subcutaneous adipose tissue of patients affected by the AL isotypes κ and λ. Through our retrospective analysis based on graph theory, we have herein deduced new insights representing a step forward from the pioneering proteomic investigations previously published by our group. ECM/cytoskeleton, oxidative stress and proteostasis were confirmed as leading processes. In this scenario, some proteins, including glutathione peroxidase 1 (GPX1), tubulins and the TRiC complex, were classified as biologically and topologically relevant. These and other results overlap with those already reported for other amyloidoses, supporting the hypothesis that amyloidogenic proteins could induce similar mechanisms independently of the main fibril precursor and of the target tissues/organs. Of course, further studies based on larger patient cohorts and different tissues/organs will be essential, which would be a key point that would allow for a more robust selection of the main molecular players and a more accurate correlation with clinical aspects.

The globular to fibrillar transition of proteins represents a key pathogenic event in the development of amyloid diseases. Although systemic amyloidoses share the common characteristic of amyloid deposition in the extracellular matrix, they are clinically heterogeneous as the affected organs may vary. The observation that precursors of amyloid fibrils derived from circulating globular plasma proteins led to huge efforts in trying to elucidate the structural events determining the protein metamorphosis from their globular to fibrillar state. Whereas the process of metamorphosis has inspired poets and writers from Ovid to Kafka, protein metamorphism is a more recent concept. It is an ideal metaphor in biochemistry for studying the protein folding paradigm and investigating determinants of folding dynamics. Although we have learned how to transform both normal and pathogenic globular proteins into fibrillar polymers in vitro, the events occurring in vivo, are far more complex and yet to be explained. A major gap still exists between in vivo and in vitro models of fibrillogenesis as the biological complexity of the disease in living organisms cannot be reproduced at the same extent in the test tube. Reviewing the major scientific attempts to monitor the amyloidogenic metamorphosis of globular proteins in systems of increasing complexity, from cell culture to human tissues, may help to bridge the gap between the experimental models and the actual pathological events in patients.

Apolipoprotein A‐IV amyloidosis is an uncommon form of the disease normally resulting in renal and cardiac dysfunction. ApoA‐IV amyloidosis was identified in 16 patients attending the National Amyloidosis Centre and in eight clinical samples received for histology review. Unexpectedly, proteomics identified the presence of ApoA‐IV signal sequence residues (p.18‐43 to p.20‐43) in 16/24 trypsin‐digested amyloid deposits but in only 1/266 non‐ApoA‐IV amyloid samples examined. These additional signal residues were also detected in the cardiac sample from the Swedish patient in which ApoA‐IV amyloid was first described, and in plasma from a single cardiac ApoA‐IV amyloidosis patient. The most common signal‐containing peptide observed in ApoA‐IV amyloid, p.20‐43, and to a far lesser extent the N‐terminal peptide, p.21‐43, were fibrillogenic in vitro at physiological pH, generating Congo red‐positive fibrils. The addition of a single signal‐derived alanine residue to the N‐terminus has resulted in markedly increased fibrillogenesis. If this effect translates to the mature circulating protein in vivo, then the presence of signal may result in preferential deposition as amyloid, perhaps acting as seed for the main circulating native form of the protein; it may also influence other ApoA‐IV‐associated pathologies. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Amyloidosis is a relatively rare human disease caused by the deposition of abnormal protein fibres in the extracellular space of various tissues, impairing their normal function. Proteomic analysis of patients’ biopsies, developed by Dogan and colleagues at the Mayo Clinic, has become crucial for clinical diagnosis and for identifying the amyloid type. Currently, the proteomic approach is routinely used at National Amyloidosis Centre (NAC, London, UK) and Istituto di Tecnologie Biomediche-Consiglio Nazionale delle Ricerche (ITB-CNR, Milan, Italy). Both centres are members of the European Proteomics Amyloid Network (EPAN), which was established with the aim of sharing and discussing best practice in the application of amyloid proteomics. One of the EPAN’s activities was to evaluate the quality and the confidence of the results achieved using different software and algorithms for protein identification. In this paper, we report the comparison of proteomics results obtained by sharing NAC proteomics data with the ITB-CNR centre. Mass spectrometric raw data were analysed using different software platforms including Mascot, Scaffold, Proteome Discoverer, Sequest and bespoke algorithms developed for an accurate and immediate amyloid protein identification. Our study showed a high concordance of the obtained results, suggesting a good accuracy of the different bioinformatics tools used in the respective centres. In conclusion, inter-centre data exchange is a worthwhile approach for testing and validating the performance of software platforms and the accuracy of results, and is particularly important where the proteomics data contribute to a clinical diagnosis.