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

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.

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

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.

Morphological and primary structural consistency of fibrils from different AA patients (common variant).

Aims: To test the hypothesis that the fibril morphology and the fibril protein primary structure are conserved across different patients suffering from the common variant of systemic Amyloid A (AA) amyloidosis. Methods: Amyloid fibrils were extracted from the renal tissue of four patients. The fibril morphology was analysed in negatively stained samples with transmission electron microscopy (TEM). The fibril protein identity and fragment length were determined by using mass spectrometry. Results: The fibrils show a consistent morphology in all four patients and exhibit an average width of ∼9.6 nm and an average pitch of ∼112 nm. All fibrils are composed of polypeptide chains that can be assigned to human serum amyloid A (SAA) 1.1 protein. All fragments lack the N-terminal arginine residue and are C-terminally truncated. Differences exist concerning the exact C-terminal cleavage site. The most prominent cleavage site occurs at residues 64-67. Conclusions: Our data demonstrate that AA amyloid fibrils are consistent at the level of the protein primary structure and fibril morphology in the four analysed patients.

Cryo-EM fibril structures from systemic AA amyloidosis reveal the species complementarity of pathological amyloids.

Systemic AA amyloidosis is a worldwide occurring protein misfolding disease of humans and animals. It arises from the formation of amyloid fibrils from the acute phase protein serum amyloid A. Here, we report the purification and electron cryo-microscopy analysis of amyloid fibrils from a mouse and a human patient with systemic AA amyloidosis. The obtained resolutions are 3.0 Å and 2.7 Å for the murine and human fibril, respectively. The two fibrils differ in fundamental properties, such as presence of right-hand or left-hand twisted cross-ß sheets and overall fold of the fibril proteins. Yet, both proteins adopt highly similar ß-arch conformations within the N-terminal ~21 residues. Our data demonstrate the importance of the fibril protein N-terminus for the stability of the analyzed amyloid fibril morphologies and suggest strategies of combating this disease by interfering with specific fibril polymorphs.

Influence of C-terminal truncation of murine Serum amyloid A on fibril structure.

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.