RESUMEN
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.
Asunto(s)
Amiloidosis/patología , Apolipoproteínas A , Señales de Clasificación de Proteína , Anciano , Femenino , Humanos , Masculino , Placa Amiloide/patologíaRESUMEN
Systemic amyloidosis caused by extracellular deposition of insoluble fibrils derived from the pathological aggregation of circulating proteins, such as transthyretin, is a severe and usually fatal condition. Elucidation of the molecular pathogenic mechanism of the disease and discovery of effective therapies still represents a challenging medical issue. The in vitro preparation of amyloid fibrils that exhibit structural and biochemical properties closely similar to those of natural fibrils is central to improving our understanding of the biophysical basis of amyloid formation in vivo and may offer an important tool for drug discovery. Here, we compared the morphology and thermodynamic stability of natural transthyretin fibrils with those of fibrils generated in vitro either using the common acidification procedure or primed by limited selective cleavage by plasmin. The free energies for fibril formation were -12.36, -8.10, and -10.61 kcal mol-1, respectively. The fibrils generated via plasmin cleavage were more stable than those prepared at low pH and were thermodynamically and morphologically similar to natural fibrils extracted from human amyloidotic tissue. Determination of thermodynamic stability is an important tool that is complementary to other methods of structural comparison between ex vivo fibrils and fibrils generated in vitro Our finding that fibrils created via an in vitro amyloidogenic pathway are structurally similar to ex vivo human amyloid fibrils does not necessarily establish that the fibrillogenic pathway is the same for both, but it narrows the current knowledge gap between in vitro models and in vivo pathophysiology.
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Neuropatías Amiloides Familiares/patología , Amiloide/química , Prealbúmina/química , Amiloide/genética , Amiloide/ultraestructura , Neuropatías Amiloides Familiares/genética , Humanos , Mutación , Prealbúmina/genética , Agregado de Proteínas , Agregación Patológica de Proteínas/genética , Agregación Patológica de Proteínas/patología , Estabilidad Proteica , TermodinámicaRESUMEN
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.
Asunto(s)
Amiloidosis/diagnóstico , Biología Computacional , Difusión de la Información , Proteómica/métodos , Programas Informáticos , Algoritmos , Proteínas Amiloidogénicas/metabolismo , Amiloidosis/metabolismo , Humanos , Italia , Reino UnidoRESUMEN
Systemic amyloidosis is a serious disease which is caused when normal circulating proteins misfold and aggregate extracellularly as insoluble fibrillary deposits throughout the body. This commonly results in cardiac, renal and neurological damage. The tissue target, progression and outcome of the disease depends on the type of protein forming the fibril deposit, and its correct identification is central to determining therapy. Proteomics is now used routinely in our centre to type amyloid; over the past 7 years we have examined over 2000 clinical samples. Proteomics results are linked directly to our patient database using a simple algorithm to automatically highlight the most likely amyloidogenic protein. Whilst the approach has proved very successful, we have encountered a number of challenges, including poor sample recovery, limited enzymatic digestion, the presence of multiple amyloidogenic proteins and the identification of pathogenic variants. Our proteomics procedures and approaches to resolving difficult issues are outlined.
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Proteínas Amiloidogénicas/análisis , Amiloidosis/diagnóstico , Proteómica/métodos , Algoritmos , Secuencia de Aminoácidos , Humanos , Reino UnidoRESUMEN
ß2-microglobulin (ß2-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 ß2-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 ß2-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 ß2-m at lower concentrations. Both strains exhibit behavioral defects, the severity of which correlates with ß2-m levels rather than with the presence of mutations, being more pronounced in WT ß2-m worms. ß2-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 ß2-m at high concentration compared to D76N ß2-m worms. Altogether, these data show that ß2-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 ß2-m amyloidosis (high levels of non-mutated ß2-m vs. normal levels of variant ß2-m) and provide important clues on the molecular bases of these human diseases.
RESUMEN
High-dimensional cytometry is an innovative tool for immune monitoring in health and disease, and it has provided novel insight into the underlying biology as well as biomarkers for a variety of diseases. However, the analysis of large multiparametric datasets usually requires specialist computational knowledge. Here, we describe ImmunoCluster (https://github.com/kordastilab/ImmunoCluster), an R package for immune profiling cellular heterogeneity in high-dimensional liquid and imaging mass cytometry, and flow cytometry data, designed to facilitate computational analysis by a nonspecialist. The analysis framework implemented within ImmunoCluster is readily scalable to millions of cells and provides a variety of visualization and analytical approaches, as well as a rich array of plotting tools that can be tailored to users' needs. The protocol consists of three core computational stages: (1) data import and quality control; (2) dimensionality reduction and unsupervised clustering; and (3) annotation and differential testing, all contained within an R-based open-source framework.
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Alergia e Inmunología , Biología Computacional/métodos , Citometría de Flujo/métodos , Algoritmos , Linfocitos B/citología , Linfocitos B/inmunología , Análisis de Datos , HumanosRESUMEN
Dissection of the role and function of human γδ T cells and their heterogeneous subsets in cancer, inflammation, and auto-immune diseases is a growing and dynamic research field of increasing interest to the scientific community. Therefore, harmonization and standardization of techniques for the characterization of peripheral and tissue-resident γδ T cells is crucial to facilitate comparability between published and emerging research. The application of commercially available reagents to classify γδ T cells, in particular the combination of multiple Abs, is not always trouble-free, posing major demands on researchers entering this field. Occasionally, even entire γδ T cell subsets may remain undetected when certain Abs are combined in flow cytometric analysis with multicolor Ab panels, or might be lost during cell isolation procedures. Here, based on the recent literature and our own experience, we provide an overview of methods commonly employed for the phenotypic and functional characterization of human γδ T cells including advanced polychromatic flow cytometry, mass cytometry, immunohistochemistry, and magnetic cell isolation. We highlight potential pitfalls and discuss how to circumvent these obstacles.
Asunto(s)
Citometría de Flujo/normas , Separación Inmunomagnética/normas , Receptores de Antígenos de Linfocitos T gamma-delta/análisis , Subgrupos de Linfocitos T/inmunología , Anticuerpos/química , Carcinoma/diagnóstico , Carcinoma/inmunología , Carcinoma/patología , Estudios de Casos y Controles , Neoplasias del Colon/diagnóstico , Neoplasias del Colon/inmunología , Neoplasias del Colon/patología , Citometría de Flujo/métodos , Colorantes Fluorescentes/química , Expresión Génica , Enfermedad de Hodgkin/diagnóstico , Enfermedad de Hodgkin/inmunología , Enfermedad de Hodgkin/patología , Humanos , Inmunohistoquímica , Separación Inmunomagnética/métodos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/patología , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Receptores de Antígenos de Linfocitos T gamma-delta/inmunología , Subgrupos de Linfocitos T/patologíaRESUMEN
When Salmonella enterica is not in a planktonic state, it persists in organised communities encased in extracellular polymeric substances (EPS), defined as biofilms. Environmental conditions ultimately dictate the key properties of the biofilms such as porosity, density, water content, charge, sorption and ion exchange properties, hydrophobicity and mechanical stability. S. enterica has been extensively studied due to its ability to infect the gastrointestinal environment. However, only during the last decades studies on its persistence and replication in soil, plant and abiotic surfaces have been proposed. S. enterica is an environmental bacterium able to effectively persist outside the human host. It does so by using EPS as tools to cope with environmental fluctuations. We therefore address this mini-review to classify those EPS that are produced by Salmonella with focus on the environment (plant, soil, and abiotic surfaces) by using a classification of EPS proposed by Flemming and collaborators in 2007. The EPS are therefore classified as structural, sorptive, surface-active, active, and informative.
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Proteínas Bacterianas/metabolismo , Biopolímeros/metabolismo , Matriz Extracelular de Sustancias Poliméricas/metabolismo , Plantas/microbiología , Salmonella enterica/metabolismo , Proteínas Bacterianas/genética , Biopelículas , Biopolímeros/química , Biopolímeros/genética , Matriz Extracelular de Sustancias Poliméricas/química , Matriz Extracelular de Sustancias Poliméricas/genética , Salmonella enterica/química , Salmonella enterica/genéticaRESUMEN
The wild type protein, transthyretin (TTR), and over 120 genetic TTR variants are amyloidogenic and cause, respectively, sporadic and hereditary systemic TTR amyloidosis. The homotetrameric TTR contains two identical thyroxine binding pockets, occupation of which by specific ligands can inhibit TTR amyloidogenesis in vitro. Ligand binding stabilizes the tetramer, inhibiting its proteolytic cleavage and its dissociation. Here, we show with solution-state NMR that ligand binding induces long-distance conformational changes in the TTR that have not previously been detected by X-ray crystallography, consistently with the inhibition of the cleavage of the DE loop. The NMR findings, coupled with surface plasmon resonance measurements, have identified dynamic exchange processes underlying the negative cooperativity of binding of "monovalent" ligand tafamidis. In contrast, mds84, our prototypic "bivalent" ligand, which is a more potent stabilizer of TTR in vitro that occupies both thyroxine pockets and the intramolecular channel between them, has greater structural effects.