Palma Mangione
@unipv.it
Unit of Biochemistry, Department of Molecular Medicine, University of Pavia
University of Pavia, Italy
Scopus Publications
Scopus Publications
Francesca Lavatelli, Antonino Natalello, Loredana Marchese, Diletta Ami, Alessandra Corazza, Sara Raimondi, Maria Chiara Mimmi, Silvia Malinverni, P. Patrizia Mangione, Manel Terrones Palmer,et al.
Elsevier BV
Guglielmo Verona, Sara Raimondi, Diana Canetti, P. Patrizia Mangione, Loredana Marchese, Alessandra Corazza, Francesca Lavatelli, Julian D. Gillmore, Graham W. Taylor, Vittorio Bellotti,et al.
Wiley
AbstractThe mechanism that converts native human transthyretin into amyloid fibrils in vivo is still a debated and controversial issue. Commonly, non‐physiological conditions of pH, temperature, or organic solvents are used in in vitro models of fibrillogenesis of globular proteins. Transthyretin amyloid formation can be achieved under physiological conditions through a mechano‐enzymatic mechanism involving specific serine proteases such as trypsin or plasmin. Here, we investigate S52P and L111M transthyretin variants, both causing a severe form of systemic amyloidosis mostly targeting the heart at a relatively young age with heterogeneous phenotype among patients. Our studies on thermodynamics show that both proteins are significantly less stable than other amyloidogenic variants. However, despite a similar thermodynamic stability, L111M variant seems to have enhanced susceptibility to cleavage and a lower tendency to form fibrils than S52P in the presence of specific proteases and biomechanical forces. Heparin strongly enhances the fibrillogenic capacity of L111M transthyretin, but has no effect on the S52P variant. Fibrillar seeds similarly affect the fibrillogenesis of both proteins, with a stronger effect on the L111M variant. According to our model of mechano‐enzymatic fibrillogenesis, both full‐length and truncated monomers, released after the first cleavage, can enter into fibrillogenesis or degradation pathways. Our findings show that the kinetics of the two processes can be affected by several factors, such as intrinsic amyloidogenicity due to the specific mutations, environmental factors including heparin and fibrillar seeds that significantly accelerate the fibrillogenic pathway.
Sara Raimondi, Giulia Faravelli, Paola Nocerino, Valentina Mondani, Alma Baruffaldi, Loredana Marchese, Maria Chiara Mimmi, Diana Canetti, Guglielmo Verona, Marianna Caterino,et al.
Wiley
Abstractβ2‐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.
Cristina Cantarutti, Maria Chiara Mimmi, Guglielmo Verona, Walter Mandaliti, Graham W. Taylor, P. Patrizia Mangione, Sofia Giorgetti, Vittorio Bellotti, and Alessandra Corazza
MDPI AG
The plasma protein transthyretin (TTR), a transporter for thyroid hormones and retinol in plasma and cerebrospinal fluid, is responsible for the second most common type of systemic (ATTR) amyloidosis either in its wild type form or as a result of destabilizing genetic mutations that increase its aggregation propensity. The association between free calcium ions (Ca2+) and TTR is still debated, although recent work seems to suggest that calcium induces structural destabilization of TTR and promotes its aggregation at non-physiological low pH in vitro. We apply high-resolution NMR spectroscopy to investigate calcium binding to TTR showing the formation of labile interactions, which leave the native structure of TTR substantially unaltered. The effect of calcium binding on TTR-enhanced aggregation is also assessed at physiological pH through the mechano-enzymatic mechanism. Our results indicate that, even if the binding is weak, about 7% of TTR is likely to be Ca2+-bound in vivo and therefore more aggregation prone as we have shown that this interaction is able to increase the protein susceptibility to the proteolytic cleavage that leads to aggregation at physiological pH. These events, even if involving a minority of circulating TTR, may be relevant for ATTR, a pathology that takes several decades to develop.
Giulia Faravelli, Valentina Mondani, P. Patrizia Mangione, Sara Raimondi, Loredana Marchese, Francesca Lavatelli, Monica Stoppini, Alessandra Corazza, Diana Canetti, Guglielmo Verona,et al.
Frontiers Media SA
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.
Ivana Slamova, Rozita Adib, Stephan Ellmerich, Michal R. Golos, Janet A. Gilbertson, Nicola Botcher, Diana Canetti, Graham W. Taylor, Nigel Rendell, Glenys A. Tennent,et al.
Springer Science and Business Media LLC
AbstractCardiac ATTR amyloidosis, a serious but much under-diagnosed form of cardiomyopathy, is caused by deposition of amyloid fibrils derived from the plasma protein transthyretin (TTR), but its pathogenesis is poorly understood and informative in vivo models have proved elusive. Here we report the generation of a mouse model of cardiac ATTR amyloidosis with transgenic expression of human TTRS52P. The model is characterised by substantial ATTR amyloid deposits in the heart and tongue. The amyloid fibrils contain both full-length human TTR protomers and the residue 49-127 cleavage fragment which are present in ATTR amyloidosis patients. Urokinase-type plasminogen activator (uPA) and plasmin are abundant within the cardiac and lingual amyloid deposits, which contain marked serine protease activity; knockout of α2-antiplasmin, the physiological inhibitor of plasmin, enhances amyloid formation. Together, these findings indicate that cardiac ATTR amyloid deposition involves local uPA-mediated generation of plasmin and cleavage of TTR, consistent with the previously described mechano-enzymatic hypothesis for cardiac ATTR amyloid formation. This experimental model of ATTR cardiomyopathy has potential to allow further investigations of the factors that influence human ATTR amyloid deposition and the development of new treatments.
Diana Canetti, Paola Nocerino, Nigel B Rendell, Nicola Botcher, Janet A Gilbertson, Angel Blanco, Dorota Rowczenio, Alessandra Morelli, P Patrizia Mangione, Alessandra Corazza,et al.
Wiley
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.
Sara Raimondi, P. Patrizia Mangione, Guglielmo Verona, Diana Canetti, Paola Nocerino, Loredana Marchese, Rebecca Piccarducci, Valentina Mondani, Giulia Faravelli, Graham W. Taylor,et al.
Elsevier BV
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.
Alexandra Moura, Paola Nocerino, Janet A. Gilbertson, Nigel B. Rendell, P. Patrizia Mangione, Guglielmo Verona, Dorota Rowczenio, Julian D. Gillmore, Graham W. Taylor, Vittorio Bellotti,et al.
Informa UK Limited
Human lysozyme is a bacteriolytic enzyme synthesised by gastrointestinal (GI) tract macrophages and hepatocytes. It is found in many different tissues and body fluids including the liver, articular cartilage, saliva and plasma [1]. Lysozyme amyloidosis (ALys) is one of the rarest types of systemic amyloidosis [2]. It is a hereditary, autosomal dominant disease that is associated with a single point mutation in the lysozyme gene. To date, nine amyloid point mutations have been reported (http://amyloidosismutations.com/mut-alys.php). Proteomics represents a valuable tool for diagnosis and is complementary to immunohistochemistry and gene sequencing. The advantage of proteomics is the ability to directly characterise proteins in the amyloid deposits. In certain circumstances, this approach can be limited by the normal requirement to use trypsin as the proteolytic enzyme. Here, we report a case of a patient carrying the mutation W64R (p.W82R) in which we highlight the importance of the combined use of trypsin and Asp-N to identify the amyloid composition. The W64R lysozyme mutation has been originally described by Valleix et al. [3] in 2002. A similar case of W64R lysozyme amyloid has just been reported by Li et al. [4]. Although the genetic sequencing showed the presence of the mutation, the proteomics output confirmed the presence of lysozyme, but details on fibrils composition were not provided. A sample of Congo red positive formalin-fixed paraffinembedded (FFPE) GI tissue was collected by laser capture dissection and analysed by proteomics using trypsin as the digestion enzyme [5]. Data were then evaluated using MASCOT software to search the Swiss-Prot database to which was appended an additional lysozyme database of nine variants. A number of proteins were present in the sample (Supplementary Table 1) including lysozyme which was identified with high Mascot score (1403) and a protein coverage of 66%. Peptides containing the W64R mutation were not detected since the additional tryptic cleavage site generated by a W!R change resulted in the generation of small fragments outside the normal range of the instrument. A miscleaved variant peptide 51–64, (R)STDYGIFQINSRYR, was observed, however, the confidence in the assignment was low (Figure 1(A) and Supplementary Figure 1(A)). The wild type 63–69 and the miscleaved 63-97 peptides were not detected (Figure 1(B)). To determine whether this was due to the absence of wild type protein in the fibrils, a sample of wild type lysozyme from human neutrophils (Sigma-Aldrich, St. Louis, MO) was spiked into a Congo red negative FFPE GI tissue and analysed by proteomics. Lysozyme was identified with a sequence coverage of 84% and the wild type peptide 63–69 was detected (Figure 1(C)). This is in contrast with that observed in the natural amyloid fibrils of lysozyme where
Diana Canetti, Nigel B. Rendell, Janet A. Gilbertson, Nicola Botcher, Paola Nocerino, Angel Blanco, Lucia Di Vagno, Dorota Rowczenio, Guglielmo Verona, P. Patrizia Mangione,et al.
Walter de Gruyter GmbH
Abstract 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.
Giulia Faravelli, Sara Raimondi, Loredana Marchese, Frederick A. Partridge, Cristina Soria, P. Patrizia Mangione, Diana Canetti, Michele Perni, Francesco A. Aprile, Irene Zorzoli,et al.
Springer Science and Business Media LLC
AbstractThe availability of a genetic model organism with which to study key molecular events underlying amyloidogenesis is crucial for elucidating the mechanism of the disease and the exploration of new therapeutic avenues. The natural human variant of β2-microglobulin (D76N β2-m) is associated with a fatal familial form of systemic amyloidosis. Hitherto, no animal model has been available for studying in vivo the pathogenicity of this protein. We have established a transgenic C. elegans line, expressing the human D76N β2-m variant. Using the INVertebrate Automated Phenotyping Platform (INVAPP) and the algorithm Paragon, we were able to detect growth and motility impairment in D76N β2-m expressing worms. We also demonstrated the specificity of the β2-m variant in determining the pathological phenotype by rescuing the wild type phenotype when β2-m expression was inhibited by RNA interference (RNAi). Using this model, we have confirmed the efficacy of doxycycline, an inhibitor of the aggregation of amyloidogenic proteins, in rescuing the phenotype. In future, this C. elegans model, in conjunction with the INVAPP/Paragon system, offers the prospect of high-throughput chemical screening in the search for new drug candidates.
Alessandra Corazza, Guglielmo Verona, Christopher A. Waudby, P. Patrizia Mangione, Ryan Bingham, Iain Uings, Diana Canetti, Paola Nocerino, Graham W. Taylor, Mark B. Pepys,et al.
American Chemical Society (ACS)
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.
Graham W. Taylor, Janet A. Gilbertson, Rabya Sayed, Angel Blanco, Nigel B. Rendell, Dorota Rowczenio, Tamer Rezk, P. Patrizia Mangione, Diana Canetti, Paul Bass,et al.
Elsevier BV
Tamer Rezk, Janet A Gilbertson, P Patrizia Mangione, Dorota Rowczenio, Nigel B Rendell, Diana Canetti, Helen J Lachmann, Ashutosh D Wechalekar, Paul Bass, Philip N Hawkins,et al.
Wiley
The tissue diagnosis of amyloidosis and confirmation of fibril protein type, which are crucial for clinical management, have traditionally relied on Congo red (CR) staining followed by immunohistochemistry (IHC) using fibril protein specific antibodies. However, amyloid IHC is qualitative, non‐standardised, requires operator expertise, and not infrequently fails to produce definitive results. More recently, laser dissection mass spectrometry (LDMS) has been developed as an alternative method to characterise amyloid in tissue sections. We sought to compare these techniques in a real world setting. During 2017, we performed LDMS on 640 formalin‐fixed biopsies containing amyloid (CR+ve) comprising all 320 cases that could not be typed by IHC (IHC−ve) and 320 randomly selected CR+ve samples that had been typed (IHC+ve). In addition, we studied 60 biopsies from patients in whom there was a strong suspicion of amyloidosis, but in whom histology was non‐diagnostic (CR–ve). Comprehensive clinical assessments were conducted in 532 (76%) of cases. Among the 640 CR+ve samples, 602 (94%) contained ≥2 of 3 amyloid signature proteins (ASPs) on LDMS (ASP+ve) supporting the presence of amyloid. A total of 49 of the 60 CR‐ve samples were ASP–ve; 7 of 11 that were ASP+ve were glomerular. The amyloid fibril protein was identified by LDMS in 255 of 320 (80%) of the IHC–ve samples and in a total of 545 of 640 (85%) cases overall. The LDMS and IHC techniques yielded discordant results in only 7 of 320 (2%) cases. CR histology and LDMS are corroborative for diagnosis of amyloid, but LDMS is superior to IHC for confirming amyloid type.
P. Patrizia Mangione, Guglielmo Verona, Alessandra Corazza, Julien Marcoux, Diana Canetti, Sofia Giorgetti, Sara Raimondi, Monica Stoppini, Marilena Esposito, Annalisa Relini,et al.
Elsevier BV
Systemic amyloidosis is a usually fatal disease caused by extracellular accumulation of abnormal protein fibers, amyloid fibrils, derived by misfolding and aggregation of soluble globular plasma protein precursors. Both WT and genetic variants of the normal plasma protein transthyretin (TTR) form amyloid, but neither the misfolding leading to fibrillogenesis nor the anatomical localization of TTR amyloid deposition are understood. We have previously shown that, under physiological conditions, trypsin cleaves human TTR in a mechano-enzymatic mechanism that generates abundant amyloid fibrils in vitro. In sharp contrast, the widely used in vitro model of denaturation and aggregation of TTR by prolonged exposure to pH 4.0 yields almost no clearly defined amyloid fibrils. However, the exclusive duodenal location of trypsin means that this enzyme cannot contribute to systemic extracellular TTR amyloid deposition in vivo. Here, we therefore conducted a bioinformatics search for systemically active tryptic proteases with appropriate tissue distribution, which unexpectedly identified plasmin as the leading candidate. We confirmed that plasmin, just as trypsin, selectively cleaves human TTR between residues 48 and 49 under physiological conditions in vitro. Truncated and full-length protomers are then released from the native homotetramer and rapidly aggregate into abundant fibrils indistinguishable from ex vivo TTR amyloid. Our findings suggest that physiological fibrinolysis is likely to play a critical role in TTR amyloid formation in vivo. Identification of this surprising intersection between two hitherto unrelated pathways opens new avenues for elucidating the mechanisms of TTR amyloidosis, for seeking susceptibility risk factors, and for therapeutic innovation.
Cristina Cantarutti, Paolo Bertoncin, Alessandra Corazza, Sofia Giorgetti, P. Mangione, Vittorio Bellotti, Federico Fogolari, and Gennaro Esposito
MDPI AG
The application of gold nanoparticles (AuNPs) is emerging in many fields, raising the need for a systematic investigation on their safety. In particular, for biomedical purposes, a relevant issue are certainly AuNP interactions with biomolecules, among which proteins are the most abundant ones. Elucidating the effects of those interactions on protein structure and on nanoparticle stability is a major task towards understanding their mechanisms at a molecular level. We investigated the interaction of the 3-mercaptopropionic acid coating of AuNPs (MPA-AuNPs) with β2-microglobulin (β2m), which is a paradigmatic amyloidogenic protein. To this aim, we prepared and characterized MPA-AuNPs with an average diameter of 3.6 nm and we employed NMR spectroscopy and fluorescence spectroscopy to probe protein structure perturbations. We found that β2m interacts with MPA-AuNPs through a highly localized patch maintaining its overall native structure with minor conformational changes. The interaction causes the reversible precipitation of clusters that can be easily re-dispersed through brief sonication.
Diana Canetti, Nigel Brian Rendell, Lucia Di Vagno, Janet A. Gilbertson, Dorota Rowczenio, Tamar Rezk, Julian D. Gillmore, Phillip N. Hawkins, Guglielmo Verona, Palma Patrizia Mangione,et al.
Informa UK Limited
Abstract Proteomics is becoming the de facto gold standard for identifying amyloid proteins and is now used routinely in a number of centres. The technique is compound class independent and offers the added ability to identify variant and modified proteins. We re-examined proteomics results from a number of formalin-fixed paraffin-embedded amyloid samples, which were positive for transthyretin (TTR) by immunohistochemistry and proteomics, using the UniProt human protein database modified to include TTR variants. The amyloidogenic variant, V122I TTR, was incorrectly identified in 26/27 wild-type and non-V122I variant samples due to its close mass spectral similarity with the methyl lysine-modified WT peptide [126KMe]105–127 (p.[146 KMe]125–147) generated during formalin fixation. Similarly, the methyl lysine peptide, [50KMe]43–59, from immunoglobulin lambda light chain constant region was also misidentified as arising from a rare myeloma-derived lambda variant V49I. These processing-derived modifications are not present in fresh cardiac tissue, non-fixed fat nor serum and do not materially affect the identification of amyloid proteins. They could result in the incorrect assignment of a variant, and this may have consequences for the immediate family who will require genetic counselling and potentially early clinical intervention. As proteomics becomes a routine clinical test for amyloidosis, it becomes important to be aware of potentially confounding issues such as formalin-mediated lysine methylation, and how these may influence diagnosis and possibly treatment.
P. Patrizia Mangione, Giuseppe Mazza, Janet A. Gilbertson, Nigel B. Rendell, Diana Canetti, Sofia Giorgetti, Luca Frenguelli, Marco Curti, Tamer Rezk, Sara Raimondi,et al.
Elsevier BV
Cristina Cantarutti, Sara Raimondi, Giorgia Brancolini, Alessandra Corazza, Sofia Giorgetti, Maurizio Ballico, Stefano Zanini, Giovanni Palmisano, Paolo Bertoncin, Loredana Marchese,et al.
Royal Society of Chemistry (RSC)
Nanoparticles have repeatedly been shown to enhance fibril formation when assayed with amyloidogenic proteins. Recently, however, evidence casting some doubt about the generality of this conclusion started to emerge. Therefore, to investigate further the influence of nanoparticles on the fibrillation process, we used a naturally occurring variant of the paradigmatic amyloidogenic protein β2-microglobulin (β2m), namely D76N β2m where asparagine replaces aspartate at position 76. This variant is responsible for aggressive systemic amyloidosis. After characterizing the interaction of the variant with citrate-stabilized gold nanoparticles (Cit-AuNPs) by NMR and modeling, we analyzed the fibril formation by three different methods: thioflavin T fluorescence, native agarose gel electrophoresis and transmission electron microscopy. The NMR evidence indicated a fast-exchange interaction involving preferentially specific regions of the protein that proved, by subsequent modeling, to be consistent with a dimeric adduct interacting with Cit-AuNPs. The fibril detection assays showed that AuNPs are able to hamper D76N β2m fibrillogenesis through an effective interaction that competes with protofibril formation or recruitment. These findings open promising perspectives for the optimization of the nanoparticle surface to design tunable interactions with proteins.
Daisaku Ozawa, Ryo Nomura, P. Patrizia Mangione, Kazuhiro Hasegawa, Tadakazu Okoshi, Riccardo Porcari, Vittorio Bellotti, and Hironobu Naiki
Informa UK Limited
C-reactive protein (CRP) and serum amyloid P component (SAP), two major classical pentraxins in humans, are soluble pattern recognition molecules that regulate the innate immune system. They have a unique pentameric structure and bind to their ligands calcium-dependently with their B faces. Pentameric CRP binds calcium-independently to various proteins, including amyloid b (Ab), at acidic pH in vitro [1]. It is hypothesized that pentameric CRP protects against toxic conditions caused by protein misfolding and aggregation in acidic inflammatory environments, but the chaperone activity of CRP remain poorly understood. SAP is present universally in all extracellular amyloid deposits [2]. Its primary role in amyloidogenesis is thought to enhance the formation and deposition of amyloid fibrils by binding to the surface of amyloid fibrils calcium-dependently with the B face. On the other hand, SAP inhibits the amyloid fibril formation of Ab [3] and enhances the refolding yield of denatured lactate dehydrogenase in vitro [4]. No convincing data or models have been published thus far to explain the discrepancy between the proand anti-amyloidogenic activities of SAP. In this study, we investigated the effects of CRP and SAP on amyloid fibril formation and amorphous protein aggregation in vitro [5].
Sara Raimondi, Riccardo Porcari, P. Patrizia Mangione, Guglielmo Verona, Julien Marcoux, Sofia Giorgetti, Graham W. Taylor, Stephan Ellmerich, Maurizio Ballico, Stefano Zanini,et al.
Springer Science and Business Media LLC
AbstractSystemic amyloidosis is caused by misfolding and aggregation of globular proteins in vivo for which effective treatments are urgently needed. Inhibition of protein self-aggregation represents an attractive therapeutic strategy. Studies on the amyloidogenic variant of β2-microglobulin, D76N, causing hereditary systemic amyloidosis, have become particularly relevant since fibrils are formed in vitro in physiologically relevant conditions. Here we compare the potency of two previously described inhibitors of wild type β2-microglobulin fibrillogenesis, doxycycline and single domain antibodies (nanobodies). The β2-microglobulin -binding nanobody, Nb24, more potently inhibits D76N β2-microglobulin fibrillogenesis than doxycycline with complete abrogation of fibril formation. In β2-microglobulin knock out mice, the D76N β2-microglobulin/ Nb24 pre-formed complex, is cleared from the circulation at the same rate as the uncomplexed protein; however, the analysis of tissue distribution reveals that the interaction with the antibody reduces the concentration of the variant protein in the heart but does not modify the tissue distribution of wild type β2-microglobulin. These findings strongly support the potential therapeutic use of this antibody in the treatment of systemic amyloidosis.
Guglielmo Verona, P. Patrizia Mangione, Sara Raimondi, Sofia Giorgetti, Giulia Faravelli, Riccardo Porcari, Alessandra Corazza, Julian D. Gillmore, Philip N. Hawkins, Mark B. Pepys,et al.
Springer Science and Business Media LLC
AbstractDissociation of the native transthyretin (TTR) tetramer is widely accepted as the critical step in TTR amyloid fibrillogenesis. It is modelled by exposure of the protein to non-physiological low pH in vitro and is inhibited by small molecule compounds, such as the drug tafamidis. We have recently identified a new mechano-enzymatic pathway of TTR fibrillogenesis in vitro, catalysed by selective proteolytic cleavage, which produces a high yield of genuine amyloid fibrils. This pathway is efficiently inhibited only by ligands that occupy both binding sites in TTR. Tolcapone, which is bound with similar high affinity in both TTR binding sites without the usual negative cooperativity, is therefore of interest. Here we show that TTR fibrillogenesis by the mechano-enzymatic pathway is indeed more potently inhibited by tolcapone than by tafamidis but neither, even in large molar excess, completely prevents amyloid fibril formation. In contrast, mds84, the prototype of our previously reported bivalent ligand TTR ‘superstabiliser’ family, is notably more potent than the monovalent ligands and we show here that this apparently reflects the critical additional interactions of its linker within the TTR central channel. Our findings have major implications for therapeutic approaches in TTR amyloidosis.
Daisaku Ozawa, Ryo Nomura, P. Patrizia Mangione, Kazuhiro Hasegawa, Tadakazu Okoshi, Riccardo Porcari, Vittorio Bellotti, and Hironobu Naiki
Springer Science and Business Media LLC
AbstractC-reactive protein (CRP) and serum amyloid P component (SAP), two major classical pentraxins in humans, are soluble pattern recognition molecules that regulate the innate immune system, but their chaperone activities remain poorly understood. Here, we examined their effects on the amyloid fibril formation from Alzheimer’s amyloid β (Aβ) (1-40) and on that from D76N β2-microglobulin (β2-m) which is related to hereditary systemic amyloidosis. CRP and SAP dose-dependently and substoichiometrically inhibited both Aβ(1-40) and D76N β2-m fibril formation in a Ca2+-independent manner. CRP and SAP interacted with fresh and aggregated Aβ(1-40) and D76N β2-m on the fibril-forming pathway. Interestingly, in the presence of Ca2+, SAP first inhibited, then significantly accelerated D76N β2-m fibril formation. Electron microscopically, the surface of the D76N β2-m fibril was coated with pentameric SAP. These data suggest that SAP first exhibits anti-amyloidogenic activity possibly via A face, followed by pro-amyloidogenic activity via B face, proposing a model that the pro- and anti-amyloidogenic activities of SAP are not mutually exclusive, but reflect two sides of the same coin, i.e., the B and A faces, respectively. Finally, SAP inhibits the heat-induced amorphous aggregation of human glutathione S-transferase. A possible role of pentraxins to maintain extracellular proteostasis is discussed.
Antonino Natalello, P. Patrizia Mangione, Sofia Giorgetti, Riccardo Porcari, Loredana Marchese, Irene Zorzoli, Annalisa Relini, Diletta Ami, Giulia Faravelli, Maurizia Valli,et al.
Elsevier BV
The amyloidogenic variant of β2-microglobulin, D76N, can readily convert into genuine fibrils under physiological conditions and primes in vitro the fibrillogenesis of the wild-type β2-microglobulin. By Fourier transformed infrared spectroscopy, we have demonstrated that the amyloid transformation of wild-type β2-microglobulin can be induced by the variant only after its complete fibrillar conversion. Our current findings are consistent with preliminary data in which we have shown a seeding effect of fibrils formed from D76N or the natural truncated form of β2-microglobulin lacking the first six N-terminal residues. Interestingly, the hybrid wild-type/variant fibrillar material acquired a thermodynamic stability similar to that of homogenous D76N β2-microglobulin fibrils and significantly higher than the wild-type homogeneous fibrils prepared at neutral pH in the presence of 20% trifluoroethanol. These results suggest that the surface of D76N β2-microglobulin fibrils can favor the transition of the wild-type protein into an amyloid conformation leading to a rapid integration into fibrils. The chaperone crystallin, which is a mild modulator of the lag phase of the variant fibrillogenesis, potently inhibits fibril elongation of the wild-type even once it is absorbed on D76N β2-microglobulin fibrils.
Sophie Valleix, Guglielmo Verona, Noémie Jourde-Chiche, Brigitte Nédelec, P. Patrizia Mangione, Frank Bridoux, Alain Mangé, Ahmet Dogan, Jean-Michel Goujon, Marie Lhomme,et al.
Springer Science and Business Media LLC
AbstractApolipoprotein C-III deficiency provides cardiovascular protection, but apolipoprotein C-III is not known to be associated with human amyloidosis. Here we report a form of amyloidosis characterized by renal insufficiency caused by a new apolipoprotein C-III variant, D25V. Despite their uremic state, the D25V-carriers exhibit low triglyceride (TG) and apolipoprotein C-III levels, and low very-low-density lipoprotein (VLDL)/high high-density lipoprotein (HDL) profile. Amyloid fibrils comprise the D25V-variant only, showing that wild-type apolipoprotein C-III does not contribute to amyloid deposition in vivo. The mutation profoundly impacts helical structure stability of D25V-variant, which is remarkably fibrillogenic under physiological conditions in vitro producing typical amyloid fibrils in its lipid-free form. D25V apolipoprotein C-III is a new human amyloidogenic protein and the first conferring cardioprotection even in the unfavourable context of renal failure, extending the evidence for an important cardiovascular protective role of apolipoprotein C-III deficiency. Thus, fibrate therapy, which reduces hepatic APOC3 transcription, may delay amyloid deposition in affected patients.