Correction: Exploring exchange processes in proteins by paramagnetic perturbation of NMR spectra

Correction for 'Exploring exchange processes in proteins by paramagnetic perturbation of NMR spectra' by Yamanappa Hunashal et al., Phys. Chem. Chem. Phys., 2020, 22, 6247–6259, DOI: 10.1039/c9cp06950j

Properties of Some Variants of Human β2-Microglobulin and Amyloidogenesis

Three variants of human beta(2)-microglobulin (beta(2)-m) were compared with wild-type protein. For two variants, namely the mutant R3Abeta(2)-m and the form devoid of the N-terminal tripeptide (DeltaN3beta(2)-m), a reduced unfolding free energy was measured compared with wild-type beta(2)-m, whereas an increased stability was observed for the mutant H31Ybeta(2)-m. The solution structure could be determined by (1)H NMR spectroscopy and restrained modeling only for R3Abeta(2)-m that showed the same conformation as the parent species, except for deviations at the interstrand loops. Analogous conclusions were reached for H31Ybeta(2)-m and DeltaN3beta(2)-m. Precipitation and unfolding were observed over time periods shorter than 4-6 weeks with all the variants and, sometimes, with wild-type protein. The rate of structured protein loss from solution as a result of precipitation and unfolding always showed pseudo-zeroth order kinetics. This and the failure to observe an unfolded species without precipitation suggest that a nucleated conformational conversion scheme should apply for beta(2)-m fibrillogenesis. The mechanism is consistent with the previous and present results on beta(2)-m amyloid transition, provided a nucleated oligomeric species be considered the stable intermediate of fibrillogenesis, the monomeric intermediate being the necessary transition step along the pathway from the native protein to the nucleated oligomer

Molecular insights into cell toxicity of a novel familial amyloidogenic variant of β2-microglobulin

The first genetic variant of β(2)‐microglobulin (b2M) associated with a familial form of systemic amyloidosis has been recently described. The mutated protein, carrying a substitution of Asp at position 76 with an Asn (D76N b2M), exhibits a strongly enhanced amyloidogenic tendency to aggregate with respect to the wild‐type protein. In this study, we characterized the D76N b2M aggregation path and performed an unprecedented analysis of the biochemical mechanisms underlying aggregate cytotoxicity. We showed that, contrarily to what expected from other amyloid studies, early aggregates of the mutant are not the most toxic species, despite their higher surface hydrophobicity. By modulating ganglioside GM1 content in cell membrane or synthetic lipid bilayers, we confirmed the pivotal role of this lipid as aggregate recruiter favouring their cytotoxicity. We finally observed that the aggregates bind to the cell membrane inducing an alteration of its elasticity (with possible functional unbalance and cytotoxicity) in GM1‐enriched domains only, thus establishing a link between aggregate‐membrane contact and cell damage

Heparin Strongly Enhances the Formation of β2-Microglobulin Amyloid Fibrils in the Presence of Type I Collagen

The tissue specificity of fibrillar deposition in dialysis-related amyloidosis is most likely associated with the peculiar interaction of beta2-microglobulin (beta2-m) with collagen fibers. However, other co-factors such as glycosaminoglycans might facilitate amyloid formation. In this study we have investigated the role of heparin in the process of collagen-driven amyloidogenesis. In fact, heparin is a well known positive effector of fibrillogenesis, and the elucidation of its potential effect in this type of amyloidosis is particularly relevant because heparin is regularly given to patients subject to hemodialysis to prevent blood clotting. We have monitored by atomic force microscopy the formation of beta2-m amyloid fibrils in the presence of collagen fibers, and we have discovered that heparin strongly accelerates amyloid deposition. The mechanism of this effect is still largely unexplained. Using dynamic light scattering, we have found that heparin promotes beta2-m aggregation in solution at pH 6.4. Morphology and structure of fibrils obtained in the presence of collagen and heparin are highly similar to those of natural fibrils. The fibril surface topology, investigated by limited proteolysis, suggests that the general assembly of amyloid fibrils grown under these conditions and in vitro at low pH is similar. The exposure of these fibrils to trypsin generates a cleavage at the C-terminal of lysine 6 and creates the 7-99 truncated form of beta2-m (DeltaN6beta2-m) that is a ubiquitous constituent of the natural beta2-m fibrils. The formation of this beta2-m species, which has a strong propensity to aggregate, might play an important role in the acceleration of local amyloid deposition

Structural and Folding Dynamic Properties of the T70N Variant of Human Lysozyme

Definition of the transition mechanism from the native globular protein into fibrillar polymer was greatly improved by the biochemical and biophysical studies carried out on the two amyloidogenic variants of human lysozyme, I56T and D67H. Here we report thermodynamic and kinetic data on folding as well as structural features of a naturally occurring variant of human lysozyme, T70N, which is present in the British population at an allele frequency of 5% and, according to clinical and histopathological data, is not amyloidogenic. This variant is less stable than the wild-type protein by 3.7 kcal/mol, but more stable than the pathological, amyloidogenic variants. Unfolding kinetics in guanidine are six times faster than in the wild-type, but three and twenty times slower than in the amyloidogenic variants. Enzyme catalytic parameters, such as maximal velocity and affinity, are reduced in comparison to the wild-type. The solution structure, determined by 1H NMR and modeling calculations, exhibits a more compact arrangement at the interface between the beta-sheet domain and the subsequent loop on one side and part of the alpha domain on the other side, compared with the wild-type protein. This is the opposite of the conformational variation shown by the amyloidogenic variant D67H, but it accounts for the reduced stability and catalytic performance of T70N

Inhibition of the mechano-enzymatic amyloidogenesis of transthyretin: role of ligand affinity, binding cooperativity and occupancy of the inner channel

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

Topologically non-trivial metal-organic assemblies inhibit beta(2)-microglobulin amyloidogenesis

Inhibiting amyloid aggregation through high-turnover dynamic interactions could be an efficient strategy that is already used by small heat-shock proteins in different biological contexts. We report the interactions of three topologically non-trivial, zinc-templated metal-organic assemblies, a [2]catenane, a trefoil knot (TK), and Borromean rings, with two β2-microglobulin (β2m) variants responsible for amyloidotic pathologies. Fast exchange and similar patterns of preferred contact surface are observed by NMR, consistent with molecular dynamics simulations. In vitro fibrillation is inhibited by each complex, whereas the zinc-free TK induces protein aggregation and does not inhibit fibrillogenesis. The metal coordination imposes structural rigidity that determines the contact area on the β2m surface depending on the complex dimensions, ensuring in vitro prevention of fibrillogenesis. Administration of TK, the best protein-contacting species, to a disease-model organism, namely a Caenorhabditis elegans mutant expressing the D76N β2m variant, confirms the bioactivity potential of the knot topology and suggests new developments

Amyloid Formation by Globular Proteins: The Need to Narrow the Gap Between in Vitro and in Vivo Mechanisms

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