scholarly journals Cryo-EM demonstrates the in vitro proliferation of an ex vivo amyloid fibril morphology by seeding

2022 ◽  
Vol 13 (1) ◽  
Author(s):  
Thomas Heerde ◽  
Matthies Rennegarbe ◽  
Alexander Biedermann ◽  
Dilan Savran ◽  
Peter B. Pfeiffer ◽  
...  
Keyword(s):  
Amyloid Fibrils ◽  
Ex Vivo ◽  
Proteinase K ◽  
Seed Structure ◽  
In Vitro Proliferation ◽  
Amyloid A ◽  
Fibril Structure ◽  
Proteolytic Stability ◽  
Fibril Morphology

AbstractSeveral 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.

Methods to study the structure of misfolded protein states in systemic amyloidosis

2021 ◽  
Vol 49 (2) ◽  
pp. 977-985
Author(s):  
Marcus Fändrich ◽  
Matthias Schmidt
Keyword(s):  
Protein Misfolding ◽  
Amyloid Fibrils ◽  
Ex Vivo ◽  
Systemic Amyloidosis ◽  
Structural Basis ◽  
Amyloid A ◽  
Serum Amyloid A Protein ◽  
Proteolytic Stability

Systemic amyloidosis is defined as a protein misfolding disease in which the amyloid is not necessarily deposited within the same organ that produces the fibril precursor protein. There are different types of systemic amyloidosis, depending on the protein constructing the fibrils. This review will focus on recent advances made in the understanding of the structural basis of three major forms of systemic amyloidosis: systemic AA, AL and ATTR amyloidosis. The three diseases arise from the misfolding of serum amyloid A protein, immunoglobulin light chains or transthyretin. The presented advances in understanding were enabled by recent progress in the methodology available to study amyloid structures and protein misfolding, in particular concerning cryo-electron microscopy (cryo-EM) and nuclear magnetic resonance (NMR) spectroscopy. An important observation made with these techniques is that the structures of previously described in vitro formed amyloid fibrils did not correlate with the structures of amyloid fibrils extracted from diseased tissue, and that in vitro fibrils were typically more protease sensitive. It is thus possible that ex vivo fibrils were selected in vivo by their proteolytic stability.

scholarly journals AA amyloid fibrils from diseased tissue are structurally different from in vitro formed SAA fibrils

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Akanksha Bansal ◽  
Matthias Schmidt ◽  
Matthies Rennegarbe ◽  
Christian Haupt ◽  
Falk Liberta ◽  
...  
Keyword(s):  
Protein Misfolding ◽  
Serum Amyloid A ◽  
Amyloid Fibrils ◽  
Ex Vivo ◽  
Surrounding Tissue ◽  
Amyloid A ◽  
Cryo Electron Microscopy ◽  
Β Sheet ◽  
Misfolding Disease

AbstractSystemic 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 β-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.

scholarly journals Protease Resistance of ex vivo Amyloid Fibrils implies the proteolytic Selection of disease-associated Fibril Morphologies

2021 ◽  
Author(s):  
Jonathan Schoenfelder ◽  
Peter Benedikt Pfeiffer ◽  
Tejaswini Pradhan ◽  
Johan Bijzet ◽  
Bouke P.C. Hazenberg ◽  
...  
Keyword(s):  
Amyloid Fibrils ◽  
Ex Vivo ◽  
Amino Acid Sequences ◽  
The Body ◽  
Amyloid A ◽  
Chain Analysis ◽  
Polypeptide Chains ◽  
Amyloid Diseases ◽  
Selection Of

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 Aβ-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.

scholarly journals Ex Vivo and In Vitro Effect of Serum Amyloid A in the Induction of Macrophage M2 Markers and Efferocytosis of Apoptotic Neutrophils

2015 ◽  
Vol 194 (10) ◽  
pp. 4891-4900 ◽  
Author(s):  
Lei Sun ◽  
Huibin Zhou ◽  
Ziyan Zhu ◽  
Qian Yan ◽  
Lili Wang ◽  
...  
Keyword(s):  
Serum Amyloid A ◽  
Ex Vivo ◽  
Serum Amyloid ◽  
Amyloid A ◽  
Vitro Effect

scholarly journals Serum amyloid A induces G-CSF expression and neutrophilia via Toll-like receptor 2

Blood ◽  
2009 ◽  
Vol 113 (2) ◽  
pp. 429-437 ◽  
Author(s):  
Rong L. He ◽  
Jian Zhou ◽  
Crystal Z. Hanson ◽  
Jia Chen ◽  
Ni Cheng ◽  
...  
Keyword(s):  
Serum Amyloid A ◽  
Inflammatory Diseases ◽  
Serum Amyloid ◽  
Proteinase K ◽  
In Vivo Studies ◽  
Toll Like Receptor ◽  
Amyloid A ◽  
Toll Like Receptor 2

Abstract The acute-phase protein serum amyloid A (SAA) is commonly considered a marker for inflammatory diseases; however, its precise role in inflammation and infection, which often result in neutrophilia, remains ambiguous. In this study, we demonstrate that SAA is a potent endogenous stimulator of granulocyte colony-stimulated factor (G-CSF), a principal cytokine-regulating granulocytosis. This effect of SAA is dependent on Toll-like receptor 2 (TLR2). Our data demonstrate that, in mouse macrophages, both G-CSF mRNA and protein were significantly increased after SAA stimulation. The induction of G-CSF was blocked by an anti-TLR2 antibody and markedly decreased in the TLR2-deficient macrophages. SAA stimulation results in the activation of nuclear factor–κB and binding activity to the CK-1 element of the G-CSF promoter region. In vitro reconstitution experiments also support that TLR2 mediates SAA-induced G-CSF expression. In addition, SAA-induced secretion of G-CSF was sensitive to heat and proteinase K treatment, yet insensitive to polymyxin B treatment, indicating that the induction is a direct effect of SAA. Finally, our in vivo studies confirmed that SAA treatment results in a significant increase in plasma G-CSF and neutrophilia, whereas these responses are ablated in G-CSF– or TLR2-deficient mice.

scholarly journals Comparative study of the stabilities of synthetic in vitro and natural ex vivo transthyretin amyloid fibrils

2020 ◽  
Vol 295 (33) ◽  
pp. 11379-11387 ◽  
Author(s):  
Sara Raimondi ◽  
P. Patrizia Mangione ◽  
Guglielmo Verona ◽  
Diana Canetti ◽  
Paola Nocerino ◽  
...  
Keyword(s):  
Thermodynamic Stability ◽  
Amyloid Fibrils ◽  
Current Knowledge ◽  
Ex Vivo ◽  
Biochemical Properties ◽  
Amyloid Formation ◽  
Free Energies

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.

scholarly journals Seeded fibrils of the germline variant of human λ-III immunoglobulin light chain FOR005 have a similar core as patient fibrils with reduced stability

2020 ◽  
Vol 295 (52) ◽  
pp. 18474-18484
Author(s):  
Tejaswini Pradhan ◽  
Karthikeyan Annamalai ◽  
Riddhiman Sarkar ◽  
Stefanie Huhn ◽  
Ute Hegenbart ◽  
...  
Keyword(s):  
Solid State ◽  
Light Chain ◽  
Solid State Nmr ◽  
Amyloid Fibrils ◽  
Ex Vivo ◽  
Chemical Shifts ◽  
Single Point ◽  
Single Point Mutation ◽  
Al Amyloidosis ◽  
Fibril Structure

Systemic antibody light chains (AL) amyloidosis is characterized by deposition of amyloid fibrils derived from a particular antibody light chain. Cardiac involvement is a major risk factor for mortality. Using MAS solid-state NMR, we studied the fibril structure of a recombinant light chain fragment corresponding to the fibril protein from patient FOR005, together with fibrils formed by protein sequence variants that are derived from the closest germline (GL) sequence. Both analyzed fibril structures were seeded with ex-vivo amyloid fibrils purified from the explanted heart of this patient. We find that residues 11-42 and 69-102 adopt β-sheet conformation in patient protein fibrils. We identify arginine-49 as a key residue that forms a salt bridge to aspartate-25 in the patient protein fibril structure. In the germline sequence, this residue is replaced by a glycine. Fibrils from the GL protein and from the patient protein harboring the single point mutation R49G can be both heterologously seeded using patient ex-vivo fibrils. Seeded R49G fibrils show an increased heterogeneity in the C-terminal residues 80-102, which is reflected by the disappearance of all resonances of these residues. By contrast, residues 11-42 and 69-77, which are visible in the MAS solid-state NMR spectra, show 13Cα chemical shifts that are highly like patient fibrils. The mutation R49G thus induces a conformational heterogeneity at the C terminus in the fibril state, whereas the overall fibril topology is retained. These findings imply that patient mutations in FOR005 can stabilize the fibril structure.

scholarly journals Recent Insights into the Pathogenesis of Type AA Amyloidosis

2011 ◽  
Vol 11 ◽  
pp. 641-650 ◽  
Author(s):  
J. C. H. van der Hilst
Keyword(s):  
Amino Acids ◽  
Amyloid Fibrils ◽  
Rare Event ◽  
Helical Structure ◽  
Amyloid A ◽  
Acid Protein ◽  
Life Threatening ◽  
Shock Proteins ◽  
Β Sheet

The amyloidoses are a group of life-threatening diseases in which fibrils made of misfolded proteins are deposited in organs and tissues. The fibrils are stable, insoluble aggregates of precursor proteins that have adopted an antiparallel β-sheet structure. In type AA, or reactive, amyloidosis, the precursor protein of the fibrils is serum amyloid A (SAA). SAA is a 104-amino-acid protein that is produced in the liver in response to proinflammatory cytokines. Although the protein that is produced by the liver contains 104 amino acids, only the N-terminal 66–76 amino acids are found in amyloid fibrils. Furthermore, SAA has been shown to have an α-helical structure primarily. Thus, for SAA to be incorporated into an amyloid fibril, two processes have to occur: C-terminal cleavage and conversion into a β-sheet. Only a minority of patients with elevated SAA levels develop amyloidosis. Factors that contribute to the risk of amyloidosis include the duration and degree of SAA elevation, polymorphisms in SAA, and the type of autoinflammatory syndrome. In the Hyper-IgD syndrome, amyloidosis is less prevalent than in the other autoinflammatory diseases.In vitrowork has shown that the isoprenoid pathway influences amyloidogenesis by farnesylated proteins. Although many proteins contain domains that have a potential for self-aggregation, amyloidosis is only a very rare event. Heat shock proteins (HSPs) are chaperones that assist other proteins to attain, maintain, and regain a functional conformation. In this review, recent insights into the pathogenesis of amyloidosis are discussed, in addition to a new hypothesis for a role of HSPs in the pathogenesis of type AA.

scholarly journals Identification of a Unique Amyloid Sequence in AA Amyloidosis of a Pig Associated With Streptococcus Suis Infection

2016 ◽  
Vol 54 (1) ◽  
pp. 111-118 ◽  
Author(s):  
J. Kamiie ◽  
G. Sugahara ◽  
S. Yoshimoto ◽  
N. Aihara ◽  
T. Mineshige ◽  
...  
Keyword(s):  
Amyloid Fibrils ◽  
Streptococcus Suis ◽  
Peptide Sequence ◽  
Spectrometric Analysis ◽  
Aa Amyloidosis ◽  
Amyloid A ◽  
Amyloid Deposits ◽  
N Terminus ◽  
Seeding Polymerization

Here we report a pig with amyloid A (AA) amyloidosis associated with Streptococcus suis infection and identification of a unique amyloid sequence in the amyloid deposits in the tissue. Tissues from the 180-day-old underdeveloped pig contained foci of necrosis and suppurative inflammation associated with S. suis infection. Congo red stain, immunohistochemistry, and electron microscopy revealed intense AA deposition in the spleen and renal glomeruli. Mass spectrometric analysis of amyloid material extracted from the spleen showed serum AA 2 (SAA2) peptide as well as a unique peptide sequence previously reported in a pig with AA amyloidosis. The common detection of the unique amyloid sequence in the current and past cases of AA amyloidosis in pigs suggests that this amyloid sequence might play a key role in the development of porcine AA amyloidosis. An in vitro fibrillation assay demonstrated that the unique AA peptide formed typically rigid, long amyloid fibrils (10 nm wide) and the N-terminus peptide of SAA2 formed zigzagged, short fibers (7 nm wide). Moreover, the SAA2 peptide formed long, rigid amyloid fibrils in the presence of sonicated amyloid fibrils formed by the unique AA peptide. These findings indicate that the N-terminus of SAA2 as well as the AA peptide mediate the development of AA amyloidosis in pigs via cross-seeding polymerization.

scholarly journals Amyloid seeding of transthyretin by ex vivo cardiac fibrils and its inhibition

2018 ◽  
Vol 115 (29) ◽  
pp. E6741-E6750 ◽  
Author(s):  
Lorena Saelices ◽  
Kevin Chung ◽  
Ji H. Lee ◽  
Whitaker Cohn ◽  
Julian P. Whitelegge ◽  
...  
Keyword(s):  
Amyloid Fibrils ◽  
Ex Vivo ◽  
Standard Of Care ◽  
Wild Type ◽  
Current Standard ◽  
Transthyretin Amyloidosis ◽  
Physiological Conditions ◽  
Type Gene ◽  
Amyloid Diseases

Each of the 30 human amyloid diseases is associated with the aggregation of a particular precursor protein into amyloid fibrils. In transthyretin amyloidosis (ATTR), mutant or wild-type forms of the serum carrier protein transthyretin (TTR), synthesized and secreted by the liver, convert to amyloid fibrils deposited in the heart and other organs. The current standard of care for hereditary ATTR is liver transplantation, which replaces the mutantTTRgene with the wild-type gene. However, the procedure is often followed by cardiac deposition of wild-type TTR secreted by the new liver. Here we find that amyloid fibrils extracted from autopsied and explanted hearts of ATTR patients robustly seed wild-type TTR into amyloid fibrils in vitro. Cardiac-derived ATTR seeds can accelerate fibril formation of wild-type and monomeric TTR at acidic pH and under physiological conditions, respectively. We show that this seeding is inhibited by peptides designed to complement structures of TTR fibrils. These inhibitors cap fibril growth, suggesting an approach for halting progression of ATTR.

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