RESUMO
AL amyloidosis is a life-threatening disease caused by deposition of immunoglobulin light chains. While the mechanisms underlying light chains amyloidogenesis in vivo remain unclear, several studies have highlighted the role that tissue environment and structural amyloidogenicity of individual light chains have in the disease pathogenesis. AL natural deposits contain both full-length light chains and fragments encompassing the variable domain (VL) as well as different length segments of the constant region (CL), thus highlighting the relevance that proteolysis may have in the fibrillogenesis pathway. Here, we investigate the role of major truncated species of the disease-associated AL55 light chain that were previously identified in natural deposits. Specifically, we study structure, molecular dynamics, thermal stability, and capacity to form fibrils of a fragment containing both the VL and part of the CL (133-AL55), in comparison with the full-length protein and its variable domain alone, under shear stress and physiological conditions. Whereas the full-length light chain forms exclusively amorphous aggregates, both fragments generate fibrils, although, with different kinetics, aggregate structure, and interplay with the unfragmented protein. More specifically, the VL-CL 133-AL55 fragment entirely converts into amyloid fibrils microscopically and spectroscopically similar to their ex vivo counterpart and increases the amorphous aggregation of full-length AL55. Overall, our data support the idea that light chain structure and proteolysis are both relevant for amyloidogenesis in vivo and provide a novel biocompatible model of light chain fibrillogenesis suitable for future mechanistic studies.
Assuntos
Amiloide , Cadeias Leves de Imunoglobulina , Amiloide/metabolismo , Amiloide/química , Humanos , Cadeias Leves de Imunoglobulina/metabolismo , Cadeias Leves de Imunoglobulina/química , Cadeias Leves de Imunoglobulina/genética , Simulação de Dinâmica Molecular , Regiões Constantes de Imunoglobulina/metabolismo , Regiões Constantes de Imunoglobulina/genética , Regiões Constantes de Imunoglobulina/química , Amiloidose de Cadeia Leve de Imunoglobulina/metabolismo , Amiloidose de Cadeia Leve de Imunoglobulina/patologia , Cinética , Domínios ProteicosRESUMO
Mutations in the GBA gene, which encodes the lysosomal enzyme glucocerebrosidase (GCase), are the most important genetic risk factor for Parkinson disease (PD). GCase activity is also decreased in sporadic PD brains and with normal ageing. Loss of GCase activity impairs the autophagy lysosomal pathway resulting in increased α-synuclein (α-syn) levels. Furthermore, elevated α-syn results in decreased GCase activity. Although the role of α-syn in PD remains unclear, evidence indicates that aggregated α-syn fibrils are a pathogenic species in PD, passing between neurons and inducing endogenous native α-syn to aggregate; spreading pathology through the brain. We have investigated if preformed α-syn fibrils (PFFs) impair GCase activity in mouse cortical neurons and differentiated dopaminergic cells, and whether GCase deficiency in these models increased the transfer of α-syn pathology to naïve cells. Neurons treated with PFFs induced endogenous α-syn to become insoluble and phosphorylated at Ser129 to a greater extent than monomeric α-syn-treatment. PFFs, but not monomeric α-syn, inhibited lysosomal GCase activity in these cells and induced the unfolded protein response. Neurons in which GCase was inhibited by conduritol ß-epoxide did not increase the amount of insoluble monomeric α-syn or its phosphorylation status. Instead the release of α-syn fibrils from GCase deficient cells was significantly increased. Co-culture studies showed that the transfer of α-syn pathology to naïve cells was greater from GCase deficient cells. This study suggests that GCase deficiency increases the spread of α-syn pathology and likely contributes to the earlier age of onset and increased cognitive decline associated with GBA-PD.
Assuntos
Doença de Gaucher/genética , Glucosilceramidase/genética , Doença de Parkinson/genética , Sinucleinopatias/genética , alfa-Sinucleína/genética , Envelhecimento/genética , Envelhecimento/patologia , Animais , Autofagia/genética , Encéfalo/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Doença de Gaucher/metabolismo , Doença de Gaucher/patologia , Humanos , Lisossomos/genética , Camundongos , Mutação/genética , Neurônios/metabolismo , Neurônios/patologia , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Fosforilação/genética , Sinucleinopatias/metabolismo , Sinucleinopatias/patologiaRESUMO
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.
Assuntos
Amiloidose/patologia , Apolipoproteínas A , Sinais Direcionadores de Proteínas , Idoso , Feminino , Humanos , Masculino , Placa Amiloide/patologiaRESUMO
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.
Assuntos
Neuropatias Amiloides Familiares/patologia , Amiloide/química , Pré-Albumina/química , Amiloide/genética , Amiloide/ultraestrutura , Neuropatias Amiloides Familiares/genética , Humanos , Mutação , Pré-Albumina/genética , Agregados Proteicos , Agregação Patológica de Proteínas/genética , Agregação Patológica de Proteínas/patologia , Estabilidade Proteica , TermodinâmicaAssuntos
Neuropatias Amiloides Familiares , Amiloidose , Cardiomiopatias , Humanos , Neuropatias Amiloides Familiares/complicações , Neuropatias Amiloides Familiares/tratamento farmacológico , Anticorpos/imunologia , Anticorpos/uso terapêutico , Cardiomiopatias/tratamento farmacológico , Cardiomiopatias/etiologia , Cardiomiopatias/imunologia , Pré-AlbuminaRESUMO
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.
Assuntos
Proteínas Amiloidogênicas/análise , Amiloidose/diagnóstico , Proteômica/métodos , Algoritmos , Sequência de Aminoácidos , Humanos , Reino UnidoRESUMO
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.
Assuntos
Amiloidose/metabolismo , Plasminogênio/metabolismo , Pré-Albumina/metabolismo , Amiloide/metabolismo , Bases de Dados de Proteínas , Fibrinolisina/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Desnaturação Proteica , Dobramento de Proteína , Proteólise , Tripsina/metabolismoRESUMO
The conversion of α-synuclein from its intrinsically disordered monomeric state into the fibrillar cross-ß aggregates characteristically present in Lewy bodies is largely unknown. The investigation of α-synuclein variants causative of familial forms of Parkinson disease can provide unique insights into the conditions that promote or inhibit aggregate formation. It has been shown recently that a newly identified pathogenic mutation of α-synuclein, H50Q, aggregates faster than the wild-type. We investigate here its aggregation propensity by using a sequence-based prediction algorithm, NMR chemical shift analysis of secondary structure populations in the monomeric state, and determination of thermodynamic stability of the fibrils. Our data show that the H50Q mutation induces only a small increment in polyproline II structure around the site of the mutation and a slight increase in the overall aggregation propensity. We also find, however, that the H50Q mutation strongly stabilizes α-synuclein fibrils by 5.0 ± 1.0 kJ mol(-1), thus increasing the supersaturation of monomeric α-synuclein within the cell, and strongly favors its aggregation process. We further show that wild-type α-synuclein can decelerate the aggregation kinetics of the H50Q variant in a dose-dependent manner when coaggregating with it. These last findings suggest that the precise balance of α-synuclein synthesized from the wild-type and mutant alleles may influence the natural history and heterogeneous clinical phenotype of Parkinson disease.
Assuntos
Mutação , alfa-Sinucleína/genética , Amiloide/química , Sítios de Ligação , Humanos , Corpos de Lewy/metabolismo , Espectroscopia de Ressonância Magnética , Microscopia de Força Atômica , Doença de Parkinson/metabolismo , Peptídeos/química , Fenótipo , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Solubilidade , Termodinâmica , alfa-Sinucleína/químicaRESUMO
The 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.
Assuntos
Amiloidose , Glicosaminoglicanos , Humanos , Pré-Albumina/genética , Amiloidose/genética , Amiloidose/metabolismo , Amiloide/metabolismo , HeparinaRESUMO
The development of effective disease-modifying therapies to halt Parkinson's disease (PD) progression is required. In a subtype of PD patients, alpha-synuclein pathology may start in the enteric nervous system (ENS) or autonomic peripheral nervous system. Consequently, strategies to decrease the expression of alpha-synuclein in the ENS will be an approach to prevent PD progression at pre-clinical stages in these patients. In the present study, we aimed to assess if anti-alpha-synuclein shRNA-minicircles (MC) delivered by RVG-extracellular vesicles (RVG-EV) could downregulate alpha-synuclein expression in the intestine and spinal cord. RVG-EV containing shRNA-MC were injected intravenously in a PD mouse model, and alpha-synuclein downregulation was evaluated by qPCR and Western blot in the cord and distal intestine. Our results confirmed the downregulation of alpha-synuclein in the intestine and spinal cord of mice treated with the therapy. We demonstrated that the treatment with anti-alpha-synuclein shRNA-MC RVG-EV after the development of pathology is effective to downregulate alpha-synuclein expression in the brain as well as in the intestine and spinal cord. Moreover, we confirmed that a multidose treatment is necessary to maintain downregulation for long-term treatments. Our results support the potential use of anti-alpha-synuclein shRNA-MC RVG-EV as a therapy to delay or halt PD pathology progression.
RESUMO
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.
RESUMO
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.
Assuntos
Amiloidose , Pré-Albumina , Amiloidose/metabolismo , Cálcio/metabolismo , Humanos , Pré-Albumina/química , ProteóliseRESUMO
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.
RESUMO
Neuroinflammation is increasingly recognized as an important feature in the pathogenesis of Parkinson's disease (PD). However, it remains unclear whether neuroinflammation contributes to nigral degeneration in PD or is merely a secondary marker of neurodegeneration. We aimed to investigate the temporal relationship between synucleopathy, neuroinflammation and nigrostriatal degeneration in a mouse model of PD. Mice received unilateral intrastriatal injection of alpha-synuclein pre-formed fibrils, alpha-synuclein monomer or vehicle and were sacrificed at 15, 30 and 90 days post-injection. Intrastriatal inoculation of alpha-synuclein fibrils led to significant alpha-synuclein aggregation in the substantia nigra peaking at 30 days after injection while the significant increase in Iba-1 cells, GFAP cells and IL-1ß expression peaked earlier at 15 days. At 90 days, the striatal dopaminergic denervation was associated with astroglial activation. Alpha-synuclein monomer did not result in long-term glia activation or increase in inflammatory markers. The spread of alpha-synuclein aggregates into the cortex was not associated with any changes to neuroinflammatory markers. Our results demonstrate that in the substantia nigra glial activation is an early event that precedes alpha-synuclein inclusion formation, suggesting neuroinflammation could play an important early role in the pathogenesis of PD.
Assuntos
Doença de Parkinson , alfa-Sinucleína , Animais , Corpo Estriado/metabolismo , Modelos Animais de Doenças , Camundongos , Substância Negra/metabolismo , alfa-Sinucleína/metabolismoRESUMO
Cardiac 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.
Assuntos
Neuropatias Amiloides Familiares/metabolismo , Amiloide/metabolismo , Fibrinolisina/genética , Fibrinolisina/metabolismo , Placa Amiloide/metabolismo , Animais , Cardiomiopatias , Humanos , Camundongos Transgênicos , Pré-Albumina/metabolismo , Dobramento de Proteína , ProteóliseRESUMO
Parkinson disease is the most common neurodegenerative movement disorder, estimated to affect one in twenty-five individuals over the age of 80. Mutations in glucocerebrosidase 1 (GBA1) represent the most common genetic risk factor for Parkinson disease. The link between GBA1 mutations and α-synuclein accumulation, a hallmark of Parkinson disease, is not fully understood. Following our recent finding that Gba1 mutations lead to increased α-synuclein accumulation in mice, we have studied the effects of a single injection of mouse α-synuclein pre-formed fibrils into the striatum of Gba1 mice that carry a L444P knock-in mutation. We found significantly greater formation and spread of α-synuclein inclusions in Gba1-transgenic mice compared to wild-type controls. This indicates that the Gba1 L444P mutation accelerates α-synuclein pathology and spread.
Assuntos
Técnicas de Introdução de Genes , Glucosilceramidase/genética , Mutação , Agregados Proteicos/genética , alfa-Sinucleína/química , Animais , Humanos , Injeções , Camundongos , Neostriado/metabolismo , alfa-Sinucleína/metabolismoRESUMO
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.
Assuntos
Fenamatos/química , Pré-Albumina/química , Sítios de Ligação , Fenamatos/síntese química , Humanos , Ligantes , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Pré-Albumina/síntese química , Relação Estrutura-AtividadeRESUMO
Dissociation 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.
Assuntos
Amiloide/metabolismo , Benzofenonas/farmacologia , Benzoxazóis/farmacologia , Nitrofenóis/farmacologia , Pré-Albumina/química , Pré-Albumina/metabolismo , Sítios de Ligação/efeitos dos fármacos , Fenamatos/farmacologia , Humanos , Modelos Moleculares , Estrutura Molecular , Pré-Albumina/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Multimerização Proteica , Proteólise , TolcaponaRESUMO
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.
Assuntos
Amiloide , Amiloidose , Cadeias lambda de Imunoglobulina , Mutação de Sentido Incorreto , Pré-Albumina , Proteômica , Adulto , Idoso , Idoso de 80 Anos ou mais , Substituição de Aminoácidos , Amiloide/genética , Amiloide/metabolismo , Amiloidose/genética , Amiloidose/metabolismo , Amiloidose/patologia , Feminino , Formaldeído , Humanos , Cadeias lambda de Imunoglobulina/genética , Cadeias lambda de Imunoglobulina/metabolismo , Lisina/genética , Lisina/metabolismo , Masculino , Pessoa de Meia-Idade , Inclusão em Parafina , Pré-Albumina/genética , Pré-Albumina/metabolismoRESUMO
Systemic 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 ß2-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 ß2-microglobulin fibrillogenesis, doxycycline and single domain antibodies (nanobodies). The ß2-microglobulin -binding nanobody, Nb24, more potently inhibits D76N ß2-microglobulin fibrillogenesis than doxycycline with complete abrogation of fibril formation. In ß2-microglobulin knock out mice, the D76N ß2-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 ß2-microglobulin. These findings strongly support the potential therapeutic use of this antibody in the treatment of systemic amyloidosis.