RESUMO
Systemic AL amyloidosis is one of the most frequently diagnosed forms of systemic amyloidosis. It arises from mutational changes in immunoglobulin light chains. To explore whether these mutations may affect the structure of the formed fibrils, we determine and compare the fibril structures from several patients with cardiac AL amyloidosis. All patients are affected by light chains that contain an IGLV3-19 gene segment, and the deposited fibrils differ by the mutations within this common germ line background. Using cryo-electron microscopy, we here find different fibril structures in each patient. These data establish that the mutations of amyloidogenic light chains contribute to defining the fibril architecture and hence the structure of the pathogenic agent.
Assuntos
Microscopia Crioeletrônica , Cadeias Leves de Imunoglobulina , Amiloidose de Cadeia Leve de Imunoglobulina , Mutação , Humanos , Amiloidose de Cadeia Leve de Imunoglobulina/genética , Amiloidose de Cadeia Leve de Imunoglobulina/patologia , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/metabolismo , Cadeias Leves de Imunoglobulina/química , Amiloide/metabolismo , Amiloide/genética , Amiloide/ultraestrutura , Masculino , Feminino , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Systemic AA amyloidosis is a world-wide occurring protein misfolding disease in humans and animals that arises from the formation of amyloid fibrils from serum amyloid A (SAA) protein and their deposition in multiple organs. OBJECTIVE: To identify new agents that prevent fibril formation from SAA protein and to determine their mode of action. MATERIALS AND METHODS: We used a cell model for the formation of amyloid deposits from SAA protein to screen a library of peptides and small proteins, which were purified from human hemofiltrate. To clarify the inhibitory mechanism the obtained inhibitors were characterised in cell-free fibril formation assays and other biochemical methods. RESULTS: We identified lysozyme as an inhibitor of SAA fibril formation. Lysozyme antagonised fibril formation both in the cell model as well as in cell-free fibril formation assays. The protein binds SAA with a dissociation constant of 16.5 ± 0.6 µM, while the binding site on SAA is formed by segments of positively charged amino acids. CONCLUSION: Our data imply that lysozyme acts in a chaperone-like fashion and prevents the aggregation of SAA protein through direct, physical interactions.
Assuntos
Amiloidose , Amiloidose de Cadeia Leve de Imunoglobulina , Animais , Humanos , Proteína Amiloide A Sérica/metabolismo , Muramidase , Amiloidose/metabolismo , Amiloide/metabolismoRESUMO
Antimicrobial peptides (AMPs) are major components of the innate immune defense. Accumulating evidence suggests that the antibacterial activity of many AMPs is dependent on the formation of amyloid-like fibrils. To identify novel fibril forming AMPs, we generated a spleen-derived peptide library and screened it for the presence of amyloidogenic peptides. This approach led to the identification of a C-terminal 32-mer fragment of alpha-hemoglobin, termed HBA(111-142). The non-fibrillar peptide has membranolytic activity against various bacterial species, while the HBA(111-142) fibrils aggregated bacteria to promote their phagocytotic clearance. Further, HBA(111-142) fibrils selectively inhibited measles and herpes viruses (HSV-1, HSV-2, HCMV), but not SARS-CoV-2, ZIKV and IAV. HBA(111-142) is released from its precursor by ubiquitous aspartic proteases under acidic conditions characteristic at sites of infection and inflammation. Thus, HBA(111-142) is an amyloidogenic AMP that may specifically be generated from a highly abundant precursor during bacterial or viral infection and may play an important role in innate antimicrobial immune responses.
Assuntos
COVID-19 , Infecção por Zika virus , Zika virus , Humanos , Peptídeos , Amiloide/química , Antibacterianos/farmacologia , HemoglobinasRESUMO
BACKGROUND: Systemic AL amyloidosis arises from the misfolding of patient-specific immunoglobulin light chains (LCs). Potential drivers of LC amyloid formation are mutational changes and post-translational modifications (PTMs). However, little information is available on the exact primary structure of the AL proteins and their precursor LCs. OBJECTIVE: We analyse the exact primary structure of AL proteins extracted from 10 λ AL amyloidosis patients and their corresponding precursor LCs. MATERIALS AND METHODS: By cDNA sequencing of the precursor LC genes in combination with mass spectrometry of the AL proteins, the exact primary structure and PTMs were determined. This information was used to analyse their biochemical properties. RESULTS: All AL proteins comprise the VL and a small part of the CL with a common C-terminal truncation region. While all AL proteins retain the conserved native disulphide bond of the VL, we found no evidence for presence of other common PTMs. The analysis of the biochemical properties revealed that the isoelectric point of the VL is significantly increased due to introduced mutations. CONCLUSION: Our data imply that mutational changes influence the surface charge properties of the VL and that common proteolytic processes are involved in the generation of the cleavage sites of AL proteins.
Assuntos
Amiloidose , Amiloidose de Cadeia Leve de Imunoglobulina , Humanos , Amiloidose de Cadeia Leve de Imunoglobulina/genética , Amiloidose/genética , Amiloidose/metabolismo , Cadeias Leves de Imunoglobulina/metabolismo , Amiloide/genética , Amiloide/metabolismo , Espectrometria de Massas , Gordura Abdominal/metabolismoRESUMO
Systemic AA amyloidosis is a debilitating protein misfolding disease in humans and animals. In humans, it occurs in two variants that are called 'vascular' and 'glomerular', depending on the main amyloid deposition site in the kidneys. Using cryo electron microscopy, we here show the amyloid fibril structure underlying the vascular disease variant. Fibrils purified from the tissue of such patients are mainly left-hand twisted and contain two non-equal stacks of fibril proteins. They contrast in these properties to the fibrils from the glomerular disease variant which are right-hand twisted and consist of two structurally equal stacks of fibril proteins. Our data demonstrate that the different disease variants in systemic AA amyloidosis are associated with different fibril morphologies.
Assuntos
Amiloidose , Amiloidose de Cadeia Leve de Imunoglobulina , Nefropatias , Animais , Humanos , Amiloide/metabolismo , Amiloidose/metabolismo , Amiloidose de Cadeia Leve de Imunoglobulina/metabolismo , Microscopia CrioeletrônicaRESUMO
AA amyloidosis is one of the most prevalent forms of systemic amyloidosis and affects both humans and other vertebrates. In this study, we compare MAS solid-state NMR data with a recent cryo-EM study of fibrils involving full-length murine SAA1.1. We address the question whether the specific requirements for the reconstitution of an amyloid fibril structure by cryo-EM can potentially yield a bias towards a particular fibril polymorph. We employ fibril seeds extracted from in to vivo material to imprint the fibril structure onto the biochemically produced protein. Sequential assignments yield the secondary structure elements in the fibril state. Long-range DARR and PAR experiments confirm largely the topology observed in the ex-vivo cryo-EM study. We find that the ß-sheets identified in the NMR experiments are similar to the ß-sheets found in the cryo-EM study, with the exception of amino acids 33-42. These residues cannot be assigned by solid-state NMR, while they adopt a stable ß-sheet in the cryo-EM structure. We suggest that the differences between MAS solid-state NMR and cryo-EM data are a consequence of a second conformer involving residues 33-42. Moreover, we were able to characterize the dynamic C-terminal tail of SAA in the fibril state. The C-terminus is flexible, remains detached from the fibrils, and does not affect the SAA fibril structure as confirmed further by molecular dynamics simulations. As the C-terminus can potentially interact with other cellular components, binding to cellular targets can affect its accessibility for protease digestion.
RESUMO
Lysozyme-derived (ALys) amyloidosis is a rare type of hereditary amyloidosis. Nine amyloidogenic variants and â¼30 affected families have been described worldwide. The most common manifestations are renal dysfunction, gastrointestinal tract symptoms, and sicca syndrome. We report on the clinical course of ten patients from six families representing one of the largest cohorts published so far. Seven patients carried the W64R variant showing the whole spectrum of ALys-associated symptoms. Two patients-a mother-son pair-carried a novel lysozyme variant, which was associated with nephropathy and peripheral polyneuropathy. In accordance with previous findings, the phenotype resembled within these families but did not correlate with the genotype. To gain insights into the effect of the variants at the molecular level, we analysed the structure of lysozyme and performed comparative computational predictions on aggregation propensity and conformational stability. Our study supports that decreased conformational stability is a key factor for lysozyme variants to be prone to aggregation. In summary, ALys amyloidosis is a very rare, but still heterogeneous disease that can manifest at an early age. Our newly identified lysozyme variant is associated with nephropathy and peripheral polyneuropathy. Further research is needed to understand its pathogenesis and to enable the development of new treatments.
Assuntos
Amiloidose Familiar , Amiloidose , Gastroenteropatias , Nefropatias , Polineuropatias , Humanos , Muramidase/genética , Amiloidose/genética , Amiloidose/patologia , Amiloidose Familiar/genética , Amiloidose Familiar/patologia , Nefropatias/patologiaRESUMO
Several studies showed that seeding of solutions of monomeric fibril proteins with ex vivo amyloid fibrils accelerated the kinetics of fibril formation in vitro but did not necessarily replicate the seed structure. In this research we use cryo-electron microscopy and other methods to analyze the ability of serum amyloid A (SAA)1.1-derived amyloid fibrils, purified from systemic AA amyloidosis tissue, to seed solutions of recombinant SAA1.1 protein. We show that 98% of the seeded fibrils remodel the full fibril structure of the main ex vivo fibril morphology, which we used for seeding, while they are notably different from unseeded in vitro fibrils. The seeded fibrils show a similar proteinase K resistance as ex vivo fibrils and are substantially more stable to proteolytic digestion than unseeded in vitro fibrils. Our data support the view that the fibril morphology contributes to determining proteolytic stability and that pathogenic amyloid fibrils arise from proteolytic selection.
Assuntos
Amiloide/química , Proteína Amiloide A Sérica/química , Amiloidose/genética , Amiloidose/patologia , Animais , Clonagem Molecular , Microscopia Crioeletrônica , Endopeptidase K/química , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Simulação de Dinâmica Molecular , Conformação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estabilidade Proteica , Proteólise , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína Amiloide A Sérica/genética , Proteína Amiloide A Sérica/metabolismoRESUMO
Systemic AL amyloidosis is a rare disease that is caused by the misfolding of immunoglobulin light chains (LCs). Potential drivers of amyloid formation in this disease are post-translational modifications (PTMs) and the mutational changes that are inserted into the LCs by somatic hypermutation. Here we present the cryo electron microscopy (cryo-EM) structure of an ex vivo λ1-AL amyloid fibril whose deposits disrupt the ordered cardiomyocyte structure in the heart. The fibril protein contains six mutational changes compared to the germ line and three PTMs (disulfide bond, N-glycosylation and pyroglutamylation). Our data imply that the disulfide bond, glycosylation and mutational changes contribute to determining the fibril protein fold and help to generate a fibril morphology that is able to withstand proteolytic degradation inside the body.
Assuntos
Amiloidose de Cadeia Leve de Imunoglobulina/metabolismo , Microscopia Crioeletrônica , Glicosilação , Amiloidose de Cadeia Leve de Imunoglobulina/genética , Mutação , Conformação Proteica , Dobramento de ProteínaRESUMO
Several studies recently showed that ex vivo fibrils from patient or animal tissue were structurally different from in vitro formed fibrils from the same polypeptide chain. Analysis of serum amyloid A (SAA) and Aß-derived amyloid fibrils additionally revealed that ex vivo fibrils were more protease stable than in vitro fibrils. These observations gave rise to the proteolytic selection hypothesis that suggested that disease-associated amyloid fibrils were selected inside the body by their ability to resist endogenous clearance mechanisms. We here show, for more than twenty different fibril samples, that ex vivo fibrils are more protease stable than in vitro fibrils. These data support the idea of a proteolytic selection of pathogenic amyloid fibril morphologies and help to explain why only few amino acid sequences lead to amyloid diseases, although many, if not all, polypeptide chains can form amyloid fibrils in vitro.
Assuntos
Amiloide , Amiloidose , Sequência de Aminoácidos , Animais , Humanos , Peptídeo Hidrolases , Proteína Amiloide A SéricaRESUMO
Systemic AA amyloidosis is a world-wide occurring protein misfolding disease of humans and animals. It arises from the formation of amyloid fibrils from serum amyloid A (SAA) protein. Using cryo electron microscopy we here show that amyloid fibrils which were purified from AA amyloidotic mice are structurally different from fibrils formed from recombinant SAA protein in vitro. Ex vivo amyloid fibrils consist of fibril proteins that contain more residues within their ordered parts and possess a higher ß-sheet content than in vitro fibril proteins. They are also more resistant to proteolysis than their in vitro formed counterparts. These data suggest that pathogenic amyloid fibrils may originate from proteolytic selection, allowing specific fibril morphologies to proliferate and to cause damage to the surrounding tissue.
Assuntos
Amiloide/metabolismo , Amiloidose/metabolismo , Proteína Amiloide A Sérica/metabolismo , Animais , Microscopia Crioeletrônica , Camundongos , Modelos Moleculares , Conformação Proteica em Folha beta , Proteínas Recombinantes , Proteína Amiloide A Sérica/genéticaRESUMO
Systemic AL amyloidosis is a debilitating and potentially fatal disease that arises from the misfolding and fibrillation of immunoglobulin light chains (LCs). The disease is patient-specific with essentially each patient possessing a unique LC sequence. In this study, we present two ex vivo fibril structures of a λ3 LC. The fibrils were extracted from the explanted heart of a patient (FOR005) and consist of 115-residue fibril proteins, mainly from the LC variable domain. The fibril structures imply that a 180° rotation around the disulfide bond and a major unfolding step are necessary for fibrils to form. The two fibril structures show highly similar fibril protein folds, differing in only a 12-residue segment. Remarkably, the two structures do not represent separate fibril morphologies, as they can co-exist at different z-axial positions within the same fibril. Our data imply the presence of structural breaks at the interface of the two structural forms.
Assuntos
Amiloide/ultraestrutura , Microscopia Crioeletrônica , Amiloidose de Cadeia Leve de Imunoglobulina/metabolismo , Sequência de Aminoácidos , Feminino , Humanos , Cadeias Leves de Imunoglobulina/metabolismo , Pessoa de Meia-Idade , Mutação/genética , Agregados Proteicos , Conformação ProteicaRESUMO
Serum amyloid A action in immune response and deposition in inflammation-linked amyloidosis involve SAA-lipid interactions. We show that SAA sequesters neutral and anionic phospholipids and their hydrolytic products to form nanoparticles, suggesting a synergy with phospholipase A2. The lipid charge and shape affect SAA protection from proteolysis, aggregation and fibrillogenesis.
Assuntos
Ésteres/química , Fosfolipídeos/química , Proteólise , Proteína Amiloide A Sérica/química , HidróliseRESUMO
Serum amyloid A (SAA) is an acute-phase plasma protein that functions in innate immunity and lipid homeostasis. SAA is a protein precursor of reactive AA amyloidosis, the major complication of chronic inflammation and one of the most common human systemic amyloid diseases worldwide. Most circulating SAA is protected from proteolysis and misfolding by binding to plasma high-density lipoproteins. However, unbound soluble SAA is intrinsically disordered and is either rapidly degraded or forms amyloid in a lysosome-initiated process. Although acidic pH promotes amyloid fibril formation by this and many other proteins, the molecular underpinnings are unclear. We used an array of spectroscopic, biochemical, and structural methods to uncover that at pH 3.5-4.5, murine SAA1 forms stable soluble oligomers that are maximally folded at pH 4.3 with â¼35% α-helix and are unusually resistant to proteolysis. In solution, these oligomers neither readily convert into mature fibrils nor bind lipid surfaces via their amphipathic α-helices in a manner typical of apolipoproteins. Rather, these oligomers undergo an α-helix to ß-sheet conversion catalyzed by lipid vesicles and disrupt these vesicles, suggesting a membranolytic potential. Our results provide an explanation for the lysosomal origin of AA amyloidosis. They suggest that high structural stability and resistance to proteolysis of SAA oligomers at pH 3.5-4.5 help them escape lysosomal degradation, promote SAA accumulation in lysosomes, and ultimately damage cellular membranes and liberate intracellular amyloid. We posit that these soluble prefibrillar oligomers provide a missing link in our understanding of the development of AA amyloidosis.
Assuntos
Amiloidose , Membranas Intracelulares , Lisossomos , Multimerização Proteica , Proteína Amiloide A Sérica , Amiloidose/metabolismo , Amiloidose/patologia , Animais , Concentração de Íons de Hidrogênio , Membranas Intracelulares/metabolismo , Membranas Intracelulares/patologia , Lisossomos/química , Lisossomos/metabolismo , Lisossomos/patologia , Camundongos , Estrutura Secundária de Proteína , Proteína Amiloide A Sérica/química , Proteína Amiloide A Sérica/metabolismoRESUMO
Amyloid A (AA) amyloidosis is a systemic protein misfolding disease affecting humans and other vertebrates. While the protein precursor in humans and mice is the acute-phase reactant serum amyloid A (SAA) 1.1, the deposited fibrils consist mainly of C-terminally truncated SAA fragments, termed AA proteins. For yet unknown reasons, phenotypic variations in the AA amyloid distribution pattern are clearly associated with specific AA proteins. Here we describe a bacterial expression system and chromatographic strategies to obtain significant amounts of C-terminally truncated fragments of murine SAA1.1 that correspond in truncation position to relevant pathological AA proteins found in humans. This enables us to investigate systematically structural features of derived fibrils. All fragments form fibrils under nearly physiological conditions that show similar morphological appearance and amyloid-like properties as evident from amyloid-specific dye binding, transmission electron microscopy and infrared spectroscopy. However, infrared spectroscopy suggests variations in the structural organization of the amyloid fibrils that might be derived from a modulating role of the C-terminus for the fibril structure. These results provide insights, which can help to get a better understanding of the molecular mechanisms underlying the different clinical phenotypes of AA amyloidosis.
Assuntos
Amiloide/química , Proteína Amiloide A Sérica/química , Proteína Amiloide A Sérica/genética , Amiloide/genética , Animais , Camundongos , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Proteínas Recombinantes/químicaRESUMO
Serum amyloid A1 (SAA1) is an apolipoprotein that binds to the high-density lipoprotein (HDL) fraction of the serum and constitutes the fibril precursor protein in systemic AA amyloidosis. We here show that HDL binding blocks fibril formation from soluble SAA1 protein, whereas internalization into mononuclear phagocytes leads to the formation of amyloid. SAA1 aggregation in the cell model disturbs the integrity of vesicular membranes and leads to lysosomal leakage and apoptotic death. The formed amyloid becomes deposited outside the cell where it can seed the fibrillation of extracellular SAA1. Our data imply that cells are transiently required in the amyloidogenic cascade and promote the initial nucleation of the deposits. This mechanism reconciles previous evidence for the extracellular location of deposits and amyloid precursor protein with observations the cells are crucial for the formation of amyloid.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Amiloide/metabolismo , Proteína Amiloide A Sérica/metabolismo , Amiloidose , Animais , Linhagem Celular , Clatrina/fisiologia , Endocitose , Humanos , Macrófagos/metabolismo , Camundongos , Modelos Biológicos , Agregados ProteicosRESUMO
Systemic amyloidosis is caused by the misfolding of a circulating amyloid precursor protein and the deposition of amyloid fibrils in multiple organs. Chemical and biophysical analysis of amyloid fibrils from human AL and murine AA amyloidosis reveal the same fibril morphologies in different tissues or organs of one patient or diseased animal. The observed structural similarities concerned the fibril morphology, the fibril protein primary and secondary structures, the presence of post-translational modifications and, in case of the AL fibrils, the partially folded characteristics of the polypeptide chain within the fibril. Our data imply for both analyzed forms of amyloidosis that the pathways of protein misfolding are systemically conserved; that is, they follow the same rules irrespective of where inside one body fibrils are formed or accumulated.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Amiloidose/metabolismo , Dobramento de Proteína , Tecido Adiposo/metabolismo , Sequência de Aminoácidos , Animais , Eletroforese em Gel de Poliacrilamida , Humanos , Espectrometria de Massas , Camundongos , Microscopia Eletrônica de Transmissão , Miocárdio/metabolismo , Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Estrutura Secundária de Proteína , Baço/metabolismo , Difração de Raios XRESUMO
Systemic AA amyloidosis arises from the misfolding of serum amyloid A1 (SAA1) protein and the deposition of AA amyloid fibrils at multiple sites within the body. Previous research already established that mononuclear phagocytes are crucial for the formation of the deposits in vivo and exposure of cultures of such cells to SAA1 protein induces the formation of amyloid deposits within the culture dish. In this study we show that both non-fibrillar and fibrillar SAA1 protein can be readily transferred between cultured J774A.1 cells, a widely used model of mononuclear phagocytes. We find that the exchange is generally faster with non-fibrillar SAA1 protein than with fibrils. Exchange is blocked if cells are separated by a membrane, while increasing the volume of cell culture medium had only small effects on the observed exchange efficiency. Taken together with scanning electron microscopy showing the presence of the respective types of physical interactions between the cultured cells, we conclude that the transfer of SAA1 protein depends on direct cell-to-cell contacts or tunneling nanotubes.
Assuntos
Amiloidose/metabolismo , Comunicação Celular , Proteína Amiloide A Sérica/metabolismo , Amiloide/metabolismo , Animais , Células Cultivadas , Camundongos , Fagócitos/metabolismoRESUMO
Oxidative stress and inflammation, which involve a dramatic increase in serum amyloid A (SAA) levels, are critical in the development of atherosclerosis. Most SAA circulates on plasma HDL particles, altering their cardioprotective properties. SAA-enriched HDL has diminished anti-oxidant effects on LDL, which may contribute to atherogenesis. We determined combined effects of SAA enrichment and oxidation on biochemical changes in HDL. Normal human HDLs were incubated with SAA, oxidized by various factors (Cu2+, myeloperoxidase, H2O2, OCl-), and analyzed for lipid and protein modifications and biophysical remodeling. Three novel findings are reported: addition of SAA reduces oxidation of HDL and LDL lipids; oxidation of SAA-containing HDL in the presence of OCl- generates a covalent heterodimer of SAA and apoA-I that resists the release from HDL; and mild oxidation promotes spontaneous release of proteins (SAA and apoA-I) from SAA-enriched HDL. We show that the anti-oxidant effects of SAA extend to various oxidants and are mediated mainly by the unbound protein. We propose that free SAA sequesters lipid hydroperoxides and delays lipoprotein oxidation, though much less efficiently than other anti-oxidant proteins, such as apoA-I, that SAA displaces from HDL. These findings prompt us to reconsider the role of SAA in lipid oxidation in vivo.
Assuntos
Antioxidantes/química , Lipoproteínas HDL/química , Lipoproteínas LDL/química , Proteína Amiloide A Sérica/química , Animais , Antioxidantes/fisiologia , Apolipoproteína A-I/química , Cobre/química , Humanos , Peroxidação de Lipídeos , Camundongos , Peroxidase/química , Proteína Amiloide A Sérica/fisiologiaRESUMO
Electron tomography is an increasingly powerful method to study the detailed architecture of macromolecular complexes or cellular structures. Applied to amyloid deposits formed in a cell culture model of systemic amyloid A amyloidosis, we could determine the structural morphology of the fibrils directly in the deposit. The deposited fibrils are arranged in different networks, and depending on the relative fibril orientation, we can distinguish between fibril meshworks, fibril bundles, and amyloid stars. These networks are frequently infiltrated by vesicular lipid inclusions that may originate from the death of the amyloid-forming cells. Our data support the role of nonfibril components for constructing fibril deposits and provide structural views of different types of lipid-fibril interactions.