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Amyloid fibrils derived from antibody light chains are key pathogenic agents in systemic AL amyloidosis. They can be deposited in multiple organs but cardiac amyloid is the major risk factor of mortality. Here we report the structure of a λ1 AL amyloid fibril from an explanted human heart at a resolution of 3.3 Å which we determined using cryo-electron microscopy. The fibril core consists of a 91-residue segment presenting an all-beta fold with ten mutagenic changes compared to the germ line. The conformation differs substantially from natively folded light chains: a rotational switch around the intramolecular disulphide bond being the crucial structural rearrangement underlying fibril formation. Our structure provides insight into the mechanism of protein misfolding and the role of patient-specific mutations in pathogenicity. Systemic AL amyloidosis is caused by misfolding of immunoglobulin light chains and is one of the most frequently occurring forms of systemic amyloidosis. Here the authors present the 3.3 Å cryo-EM structure of a λ1 AL amyloid fibril that was isolated from an explanted human heart.

Systemic AL amyloidosis is a debilitating and potentially fatal disease that arises from the misfolding and fibrillation of immunoglobulin light chains (LCs). The disease is patient-specific with essentially each patient possessing a unique LC sequence. In this study, we present two ex vivo fibril structures of a λ3 LC. The fibrils were extracted from the explanted heart of a patient (FOR005) and consist of 115-residue fibril proteins, mainly from the LC variable domain. The fibril structures imply that a 180° rotation around the disulfide bond and a major unfolding step are necessary for fibrils to form. The two fibril structures show highly similar fibril protein folds, differing in only a 12-residue segment. Remarkably, the two structures do not represent separate fibril morphologies, as they can co-exist at different z-axial positions within the same fibril. Our data imply the presence of structural breaks at the interface of the two structural forms. Systemic AL amyloidosis is a protein misfolding disease caused by the aggregation and fibrillation of immunoglobulin light chains (LCs). Here, the authors present the cryo-EM structures of λ3 LC-derived amyloid fibrils that were isolated from patient tissue and they observe structural breaks, where the two different fibril structures co-exist at different z-axial positions within the same fibril.

Systemic AL amyloidosis is a rare disease that is caused by the misfolding of immunoglobulin light chains (LCs). Potential drivers of amyloid formation in this disease are post-translational modifications (PTMs) and the mutational changes that are inserted into the LCs by somatic hypermutation. Here we present the cryo electron microscopy (cryo-EM) structure of an ex vivo λ1-AL amyloid fibril whose deposits disrupt the ordered cardiomyocyte structure in the heart. The fibril protein contains six mutational changes compared to the germ line and three PTMs (disulfide bond, N-glycosylation and pyroglutamylation). Our data imply that the disulfide bond, glycosylation and mutational changes contribute to determining the fibril protein fold and help to generate a fibril morphology that is able to withstand proteolytic degradation inside the body. Systemic AL amyloidosis is caused by misfolding of immunoglobulin light chains (LCs) but how post-translational modifications (PTMs) of LCs influence amyloid formation is not well understood. Here, the authors present the cryo-EM structure of an AL amyloid fibril derived from the heart tissue of a patient that is partially pyroglutamylated, N-glycosylated and contains an intramolecular disulfide bond. Based on their structure and biochemical experiments the authors conclude that the mutational changes, disulfide bond and glycosylation determine the fibril protein fold and that glycosylation protects the fibril core from proteolytic degradation.

Increasing evidence suggests that amyloid polymorphism gives rise to different strains of amyloids with distinct toxicities and pathologyspreading properties. Validating this hypothesis is challenging due to a lack of tools and methods that allow for the direct characterization of amyloid polymorphism in hydrated and complex biological samples. Here, we report on the development of 11- mercapto-1-undecanesulfonate-coated gold nanoparticles (NPs) that efficiently label the edges of synthetic, recombinant, and native amyloid fibrils derived from different amyloidogenic proteins. We demonstrate that these NPs represent powerful tools for assessing amyloid morphological polymorphism, using cryogenic transmission electron microscopy (cryo-EM). The NPs allowed for the visualization of morphological features that are not directly observed using standard imaging techniques, including transmission electron microscopy with use of the negative stain or cryo-EM imaging. The use of these NPs to label native paired helical filaments (PHFs) from the postmortem brain of a patient with Alzheimer’s disease, as well as amyloid fibrils extracted from the heart tissue of a patient suffering from systemic amyloid light-chain amyloidosis, revealed a high degree of homogeneity across the fibrils derived from human tissue in comparison with fibrils aggregated in vitro. These findings are consistent with, and strongly support, the emerging view that the physiologic milieu is a key determinant of amyloid fibril strains. Together, these advances should not only facilitate the profiling and characterization of amyloids for structural studies by cryo-EM, but also pave the way to elucidate the structural basis of amyloid strains and toxicity, and possibly the correlation between the pathological and clinical heterogeneity of amyloid diseases.

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 LCs 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.

Abstract Systemic AL amyloidosis is a debilitating and potentially fatal disease that arises from the misfolding and fibrillation of immunoglobulin light chains (LCs). The disease is patient-specific with essentially each patient possessing a unique LC sequence. In this study, we present the first ex vivo fibril structures of a λ3 LC. The fibrils were extracted from the explanted heart of a patient (FOR005) and consist of 115 residues, mainly from the LC variable domain. The fibril structures imply that a 180° rotation around the disulfide bond and a major unfolding step are necessary for fibrils to form. The two fibril structures show highly similar fibril protein folds, differing in only a 12-residue segment. Remarkably, the two structures do not represent separate fibril morphologies, as they can co-exist at different z-axial positions within the same fibril. Our data imply the presence of structural breaks at the interface of the two structural forms. Competing Interest Statement The authors have declared no competing interest.

The misfolding and self-assembly of proteins into β-sheet-rich amyloid fibrils of various structures and morphologies is a hallmark of several neurodegenerative and systemic diseases. Increasing evidence suggests that amyloid polymorphism gives rise to different strains of amyloids with distinct toxicity and pathology-spreading properties. Validating this hypothesis is challenging due to a lack of tools and methods that allow for the direct characterization of amyloid polymorphism in hydrated and complex biological samples. Here, we report on the use of 11-mercapto-1-undecanesulfonate-coated gold nanoparticles (NPs) to label the edges of synthetic, recombinant and native amyloid fibrils to assess amyloid morphological polymorphism using cryogenic transmission electron microscopy (cryo TEM). The fibrils studied were derived from amyloid proteins involved in disorders of the central nervous system (amyloid-β, tau, α-synuclein) and in systemic amyloidosis (a fragment of an immunoglobulin light chain). The labeling efficiency enabled imaging and characterization of amyloid fibrils of different morphologies under hydrated conditions using cryo TEM. These NPs allowed for the visualization of morphological features that are not directly observed using standard imaging techniques, including TEM with use of the negative stain or cryo TEM imaging. We also demonstrate the use of these NPs to label native paired helical filaments (PHFs) from the postmortem brain of an Alzheimer's disease patient, as well as amyloid fibrils extracted from the heart tissue of a patient suffering from systemic amyloid light-chain (AL) amyloidosis. Analysis of the cryo TEM images of amyloids decorated with NPs shows exceptional homogeneity across the fibrils derived from human tissue in comparison to fibrils aggregated in vitro. The use of these NPs enabled us to gain novel insight into the structural features that distinguish amyloid fibrils formed in vivo from those formed in cell-free in vitro systems. Our findings demonstrate that these NPs represent a potent tool for rapid imaging and profiling of amyloid morphological polymorphism in different types of samples, including those derived from complex biological aggregates found in human tissue and animal models of amyloid diseases. This study should not only facilitate the profiling and characterization of amyloids for structural studies by cryo TEM but also pave the way to elucidate the structural basis of amyloid strains and toxicity and possibly the correlation between the pathological and clinical heterogeneity of amyloid diseases.