Structure, Folding Dynamics, and Amyloidogenesis of D76N β2-Microglobulin

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.aggregation | conformational disease | electron tomography | protein assembly | prion

Section: Discussionmentioning

confidence: 99%

Electron tomography reveals the fibril structure and lipid interactions in amyloid deposits

Kollmer

Meinhardt

Haupt

et al. 2016

“…However, neither wild-type TTR nor the other TTR variants we tested (Val30Met, Leu55-Pro, and Val122Ile) were similarly cleaved in vitro, and thus they are notably more stable to proteolysis. Nevertheless, it is plausible that in in vivo conditions, in particular shear flow and exposure to hydrophobic surfaces in the dynamic environment of the interstitial space, may promote local structural destabilization (21,22) and enable proteolytic cleavage. Indeed focal mechanical destabilization of the polypeptide chain is a well-known mechanism for priming limited physiological proteolytic cleavage in the homeostasis of hemostatic proteins.…”

Section: Discussionmentioning

confidence: 99%

“…The first phase comprises cleavage of the 48-49 peptide bond but does not itself necessarily release the truncated residue 49-127 polypeptide from the intact native tetramer. In the second phase forces such as those generated by fluid agitation (22) are sufficient to release the fragments with consequent fibril formation. Thyroxine does not stabilize the tetramer sufficiently to prevent fibrillogenesis, whereas the binding of RBP is adequate to block the second phase, impeding release of the fibrillogenic free fragment.…”

Section: Discussionmentioning

confidence: 99%

“…Although this hydrogen bond might be sustained by the new N terminus generated by cleavage, the separation of this N-terminal end from the new carboxylate of Lys48 must widen from 1.2 to at least 2.8 Å and promote displacement of the CD turn. Departure of the 1-48 peptide caused by shear forces in solution would remove important dimer-dimer interaction regions (19)(20)(21)(22)(23) and promote dissociation of the remaining 49-127 fragment.…”

Section: Limited Proteolysis Of Recombinant Wild-type and Variant Ttrmentioning

confidence: 99%

See 1 more Smart Citation

Proteolytic cleavage of Ser52Pro variant transthyretin triggers its amyloid fibrillogenesis

Mangione

Porcari²,

Gillmore³

et al. 2014

Self Cite

142

The Ser52Pro variant of transthyretin (TTR) produces aggressive, highly penetrant, autosomal-dominant systemic amyloidosis in persons heterozygous for the causative mutation. Together with a minor quantity of full-length wild-type and variant TTR, the main component of the ex vivo fibrils was the residue 49-127 fragment of the TTR variant, the portion of the TTR sequence that previously has been reported to be the principal constituent of type A, cardiac amyloid fibrils formed from wild-type TTR and other TTR variants [Bergstrom J, et al. (2005) J Pathol 206(2):224-232]. This specific truncation of Ser52Pro TTR was generated readily in vitro by limited proteolysis. In physiological conditions and under agitation the residue 49-127 proteolytic fragment rapidly and completely self-aggregates into typical amyloid fibrils. The remarkable susceptibility to such cleavage is likely caused by localized destabilization of the β-turn linking strands C and D caused by loss of the wildtype hydrogen-bonding network between the side chains of residues Ser52, Glu54, Ser50, and a water molecule, as revealed by the high-resolution crystallographic structure of Ser52Pro TTR. We thus provide a structural basis for the recently hypothesized, crucial pathogenic role of proteolytic cleavage in TTR amyloid fibrillogenesis. Binding of the natural ligands thyroxine or retinol-binding protein (RBP) by Ser52Pro variant TTR stabilizes the native tetrameric assembly, but neither protected the variant from proteolysis. However, binding of RBP, but not thyroxine, inhibited subsequent fibrillogenesis.misfolding | protein aggregation

“…Each protein was subjected to 20 or 100 passes at 8 mm s −1 and the resulting aggregation quantified by UV spectrophotometry and the dispersity characterized by NTA and DLS. β 2 m (100 residues, R h = 2.3 nm) was selected as fluid flow has been implicated previously in the aggregation of the protein into amyloid fibrils in the joints of patients undergoing long-term dialysis (36). Five mg mL −1 β 2 m was found to be more sensitive than BSA to extensional flow as assessed by the pelleting assay (2% and 10% was insoluble after 20 or 100 passes, respectively, compared with 1% and 1.5% of BSA, Fig.…”

Section: Design and Computational Characterization Of Extensional Flowmentioning

confidence: 99%

Inducing protein aggregation by extensional flow

Dobson

Kumar

Willis

et al. 2017

127

Relative to other extrinsic factors, the effects of hydrodynamic flow fields on protein stability and conformation remain poorly understood. Flow-induced protein remodeling and/or aggregation is observed both in Nature and during the large-scale industrial manufacture of proteins. Despite its ubiquity, the relationships between the type and magnitude of hydrodynamic flow, a protein's structure and stability, and the resultant aggregation propensity are unclear. Here, we assess the effects of a defined and quantified flow field dominated by extensional flow on the aggregation of BSA, β 2 -microglobulin (β 2 m), granulocyte colony stimulating factor (G-CSF), and three monoclonal antibodies (mAbs). We show that the device induces protein aggregation after exposure to an extensional flow field for 0.36-1.8 ms, at concentrations as low as 0.5 mg mL −1 . In addition, we reveal that the extent of aggregation depends on the applied strain rate and the concentration, structural scaffold, and sequence of the protein. Finally we demonstrate the in situ labeling of a buried cysteine residue in BSA during extensional stress. Together, these data indicate that an extensional flow readily unfolds thermodynamically and kinetically stable proteins, exposing previously sequestered sequences whose aggregation propensity determines the probability and extent of aggregation.extensional flow | aggregation | unfolding | bioprocessing | antibody P roteins are dynamic and metastable and consequently have conformations that are highly sensitive to the environment (1). Over the last 50 y the effect of changes in temperature, pH, and the concentration of kosmatropic/chaotropic agents on the conformational energy landscape of proteins has become well understood (1). This, in turn, has allowed a link to be established between the partial or full unfolding of proteins and their propensity to aggregate (2). The force applied onto a protein as a consequence of hydrodynamic flow has also been observed to trigger protein aggregation and has fundamental (3), medical (4), and industrial relevance, especially in the manufacture of biopharmaceuticals (5-8). Although a wealth of studies have been performed (7,(9)(10)(11)(12)(13), no consensus has emerged on the ability of hydrodynamic flow to induce protein aggregation (7,14,15). This is due to the wide variety of proteins used (ranging from lysozyme, BSA, and alcohol dehydrogenase to IgGs), differences in the type of flow field generated (e.g., shear, extensional, or mixtures of these), and to the presence or absence of an interface (16). A shearing flow field (Fig. 1A, Top) is caused by a gradient in velocity perpendicular to the direction of travel and is characterized by the shear rate (s −1 ). This results in a weak rotating motion of a protein alongside translation in the direction of the flow. An extensional flow field (Fig. 1A, Bottom) is generated by a gradient in velocity in the direction of travel and is characterized by the strain rate (s −1 ). A protein in this type of flow would experien...