The role of the IT-state in D76N β2-microglobulin amyloid assembly: a crucial intermediate or an innocuous bystander?

The D76N variant of human β2-microglobulin (β2m) is the causative agent of a hereditary amyloid disease. Interestingly, D76N-associated amyloidosis has a distinctive pathology compared with aggregation of wild-type (WT) β2m which occurs in dialysis-related amyloidosis. A folding intermediate of WT-β2m, known as the IT-state, which contains a non-native trans Pro32, has been shown to be a key precursor of WT-β2m aggregation in vitro. However, how a single amino acid substitution enhances the rate of aggregation of D76N-β2m and gives rise to a different amyloid disease remained unclear. Using real-time refolding experiments monitored by CD and NMR, we show that the folding mechanisms of WT- and D76N-β2m are conserved in that both proteins fold slowly via an IT-state that has similar structural properties. Surprisingly, however, direct measurement of the equilibrium population of IT using NMR showed no evidence for an increased population of the IT-state for D76N-β2m, ruling out previous models suggesting that this could explain its enhanced aggregation propensity. Producing a kinetically trapped analogue of IT by deleting the N-terminal six amino acids increases the aggregation rate of WT-β2m, but slows aggregation of D76N-β2m, supporting the view that while the folding mechanisms of the two proteins are conserved, their aggregation mechanisms differ. The results exclude the IT-state as the cause of the rapid aggregation of D76N-β2m, suggesting that other non-native states must cause its high aggregation rate. The results highlight how a single substitution at a solvent-exposed site can affect the mechanism of aggregation and the resulting disease

Half a century of amyloids: past, present and future

Amyloid diseases are global epidemics with profound health, social and economic implications and yet remain without a cure. This dire situation calls for research into the origin and pathological manifestations of amyloidosis to stimulate continued development of new therapeutics. In basic science and engineering, the cross-ß architecture has been a constant thread underlying the structural characteristics of pathological and functional amyloids, and realizing that amyloid structures can be both pathological and functional in nature has fuelled innovations in artificial amyloids, whose use today ranges from water purification to 3D printing. At the conclusion of a half century since Eanes and Glenner's seminal study of amyloids in humans, this review commemorates the occasion by documenting the major milestones in amyloid research to date, from the perspectives of structural biology, biophysics, medicine, microbiology, engineering and nanotechnology. We also discuss new challenges and opportunities to drive this interdisciplinary field moving forward. This journal i

Structural analysis of ex vivo amyloid fibrils from AL amyloidosis

The disease AL amyloidosis is caused by the misfolding of immunoglobulin light chains (LC). Due to an underlying plasma cell dyscrasia, amyloidogenic LCs are overproduced and their high serum concentrations lead to aggregation and amyloid fibril formation. The ensuing organ damage can be fatal, in particular in case of cardiac involvement. Current treatment options target the plasma cell clone, thereby reducing LC production and amyloid formation. However, no amyloid-targeting therapies are available and high-risk patients with advanced cardiac damage often die within months of diagnosis as the available treatments are unable to rapidly restore organ function. Being a patient-specific disease, the molecular mechanisms determining pathogenesis in AL amyloidosis are poorly understood. In particular, it is unclear how different factors influence amyloid fibril formation by the pathogenic LCs. AL amyloidosis is a member of the group of clinical disorders termed the amyloidoses, which are based on the misfolding of different proteins into amyloid fibrils. Amyloid fibrils formed in vivo or in vitro from different proteins show similar characteristics: a long and straight appearance under an electron microscope, binding to amyloid-specific dyes, as well as a cross-β X-ray diffraction pattern. Although in vitro formed fibrils are generally easier to study than fibrils that are directly extracted from amyloidotic tissue, it remains to be established whether in vitro fibril structures are relevant to disease. Therefore, in order to shed light on disease processes, it is important to study ex vivo fibrils. LCs are highly variable proteins due their natural function as antibody constituents. The LC sequence variability is a result of genetic recombination, junctional diversity and somatic hypermutation. These processes take place during the production and maturation of B cells. Previous studies have shown that AL fibril proteins contain several post-translational modifications (PTMs). AL fibril proteins have generally been proteolyzed and contain a disulfide bond. Also, AL 6LCs are more often glycosylated than non-amyloidogenic LCs and sometimes pyroglutamylated. The contributions of sequence variability and PTMs to the structure and stability of AL amyloid fibrils are unclear. Structural information on AL amyloid fibrils is required to elucidate the influence of these factors. Previous studies have investigated the three-dimensional (3D) structure of AL amyloid fibrils using X-ray crystallography, solid-state nuclear magnetic resonance (ssNMR), negative-stain electron microscopy and cryo-electron microscopy (cryo-EM). However, these models were based on (short) fragments of LCs that were fibrillated in vitro and it is unclear whether they correspond to pathogenic, in vivo formed amyloid fibril structures. In this thesis, 3D structures of pathogenic AL fibrils from three patient cases were obtained using cryo-EM. By imaging the AL fibrils directly in vitreous ice, their structures could be obtained by 3D reconstruction and molecular modeling. The models revealed that a rotational switch around the LC disulfide bond takes place during misfolding. Two similar amyloid folds, differing only in a short segment of the polypeptide chain, were found to occur within a single amyloid fibril, forming structural breaks at their interface. These structural breaks could play a role in, e.g., fibril fragmentation or interactions with other molecules. Furthermore, the structure of a glycosylated AL fibril showed that the glycosylation is present on the surface of the fibril where it protects the fibril from proteolytic degradation. By comparing the available ex vivo AL fibril structures, it was shown that mutations enable the LC fragments to adopt a proteolytically stable morphology that is able to withstand the degradation mechanisms in the body. The same LC fragments may adopt different amyloid folds in vitro, as demonstrated by an ssNMR study. In conclusion, this thesis has expanded the understanding of the molecular basis of AL amyloidosis by elucidating the influence of mutations and PTMs on the structure and stability of AL fibrils, as well as on the misfolding process. These insights could aid in the development of novel diagnostic tools and therapeutic approaches