Nanobodies counteract the toxicity of an amyloidogenic light chain by stabilizing a partially open dimeric conformation.

Light chain amyloidosis (AL) is a systemic disease where fibrillar deposition of misfolded immunoglobulin light chains (LCs) severely affects organ function and results in poor prognosis for patients, especially when heart involvement is severe. Particularly relevant in this context is the cardiotoxicity exerted by still uncharacterized soluble LC species. Here, with the final goal of identifying alternative therapeutic strategies to tackle AL amyloidosis, we produced five llama-derived nanobodies (Nbs) specific against H3, a well-characterized amyloidogenic and cardiotoxic LC from an AL patient with severe cardiac involvement. We found that Nbs are specific and potent agents capable of abolishing H3 soluble toxicity in C. elegans in vivo model. Structural characterization of H3-Nb complexes revealed that the protective effect of Nbs is related to their ability to bind to the H3 VL domain and stabilise an unexpected partially open LC dimer in which the two VL domains no longer interact with each other. Thus, while identifying potent inhibitors of LC soluble toxicity, we also describe the first non-native structure of an amyloidogenic LC that may represent a crucial step in toxicity and aggregation mechanisms.

Amyloidogenic light chains impair plasma cell survival.

Systemic light chain amyloidosis (AL) is a clonal plasma cell disorder characterized by the deposition of misfolded immunoglobulin light chains (LC) as insoluble fibrils in organs. The lack of suitable models has hindered the investigation of the disease mechanisms. Our aim was to establish AL LC-producing plasma cell lines and use them to investigate the biology of the amyloidogenic clone. We used lentiviral vectors to generate cell lines expressing LC from patients suffering from AL amyloidosis. The AL LC-producing cell lines showed a significant decrease in proliferation, cell cycle arrest, and an increase in apoptosis and autophagy as compared with the multiple myeloma LC-producing cells. According to the results of RNA sequencing the AL LC-producing lines showed higher mitochondrial oxidative stress, and decreased activity of the Myc and cholesterol pathways. The neoplastic behavior of plasma cells is altered by the constitutive expression of amyloidogenic LC causing intracellular toxicity. This observation may explain the disparity in the malignant behavior of the amyloid clone compared to the myeloma clone. These findings should enable future in vitro studies and help delineate the unique cellular pathways of AL, thus expediting the development of specific treatments for patients with this disorder.

An N-glycosylation hotspot in immunoglobulin κ light chains is associated with AL amyloidosis.

Immunoglobulin light chain (AL) amyloidosis is caused by a small, minimally proliferating B-cell/plasma-cell clone secreting a patient-unique, aggregation-prone, toxic light chain (LC). The pathogenicity of LCs is encrypted in their sequence, yet molecular determinants of amyloidogenesis are poorly understood. Higher rates of N-glycosylation among clonal κ LCs from patients with AL amyloidosis compared to other monoclonal gammopathies indicate that this post-translational modification is associated with a higher risk of developing AL amyloidosis. Here, we exploited LC sequence information from previously published amyloidogenic and control clonal LCs and from a series of 220 patients with AL amyloidosis or multiple myeloma followed at our Institutions to define sequence and spatial features of N-glycosylation, combining bioinformatics, biochemical, proteomics, structural and genetic analyses. We found peculiar sequence and spatial pattern of N-glycosylation in amyloidogenic κ LCs, with most of the N-glycosylation sites laying in the framework region 3, particularly within the E strand, and consisting mainly of the NFT sequon, setting them apart with respect to non-amyloidogenic clonal LCs. Our data further support a potential role of N-glycosylation in determining the pathogenic behavior of a subset of amyloidogenic LCs and may help refine current N-glycosylation-based prognostic assessments for patients with monoclonal gammopathies.

Protease-sensitive regions in amyloid light chains: what a common pattern of fragmentation across organs suggests about aggregation.

Light-chain (AL) amyloidosis is characterized by deposition of immunoglobulin light chains (LC) as fibrils in target organs. Alongside the full-length protein, abundant LC fragments are always present in AL deposits. Herein, by combining gel-based and mass spectrometry analyses, we identified and compared the fragmentation sites of amyloid LCs from multiple organs of an AL λ amyloidosis patient (AL-55). The positions pinpointed here in kidney and subcutaneous fat, alongside those previously detected in heart of the same patient, were aligned and mapped on the LC's dimeric and fibrillar states. All tissues contain fragmented LCs along with the full-length protein; the fragment pattern is coincident across organs, although microheterogeneity exists. Multiple cleavage positions were detected; some are shared, whereas some are organ-specific, likely due to a complex of proteases. Cleavage sites are concentrated in 'proteolysis-prone' regions, common to all tissues. Several proteolytic sites are not accessible on native dimers, while they are compatible with fibrils. Overall, data suggest that the heterogeneous ensemble of LC fragments originates in tissues and is consistent with digestion of preformed fibrils, or with the hypothesis that initial proteolytic cleavage of the constant domain triggers the amyloidogenic potential of LCs, followed by subsequent proteolytic degradation. This work provides a unique set of molecular data on proteolysis from ex vivo amyloid, which allows discussing hypotheses on role and timing of proteolytic events occurring along amyloid formation and accumulation in AL patients.

Dissecting the Molecular Features of Systemic Light Chain (AL) Amyloidosis: Contributions from Proteomics.

Amyloidoses are characterized by aggregation of proteins into highly ordered amyloid fibrils, which deposit in the extracellular space of tissues, leading to organ dysfunction. In AL (amyloid light chain) amyloidosis, the most common form in Western countries, the amyloidogenic precursor is a misfolding-prone immunoglobulin light chain (LC), which, in the systemic form, is produced in excess by a plasma cell clone and transported to target organs though blood. Due to the primary role that proteins play in the pathogenesis of amyloidoses, mass spectrometry (MS)-based proteomic studies have gained an established position in the clinical management and research of these diseases. In AL amyloidosis, in particular, proteomics has provided important contributions for characterizing the precursor light chain, the composition of the amyloid deposits and the mechanisms of proteotoxicity in target organ cells and experimental models of disease. This review will provide an overview of the major achievements of proteomic studies in AL amyloidosis, with a presentation of the most recent acquisitions and a critical discussion of open issues and ongoing trends.

Mass spectrometry characterization of light chain fragmentation sites in cardiac AL amyloidosis: insights into the timing of proteolysis.

Amyloid fibrils are polymeric structures originating from aggregation of misfolded proteins. In vivo, proteolysis may modulate amyloidogenesis and fibril stability. In light chain (AL) amyloidosis, fragmented light chains (LCs) are abundant components of amyloid deposits; however, site and timing of proteolysis are debated. Identification of the N and C termini of LC fragments is instrumental to understanding involved processes and enzymes. We investigated the N and C terminome of the LC proteoforms in fibrils extracted from the hearts of two AL cardiomyopathy patients, using a proteomic approach based on derivatization of N- and C-terminal residues, followed by mapping of fragmentation sites on the structures of native and fibrillar relevant LCs. We provide the first high-specificity map of proteolytic cleavages in natural AL amyloid. Proteolysis occurs both on the LC variable and constant domains, generating a complex fragmentation pattern. The structural analysis indicates extensive remodeling by multiple proteases, largely taking place on poorly folded regions of the fibril surfaces. This study adds novel important knowledge on amyloid LC processing: although our data do not exclude that proteolysis of native LC dimers may destabilize their structure and favor fibril formation, the data show that LC deposition largely precedes the proteolytic events documentable in mature AL fibrils.

Inherent Biophysical Properties Modulate the Toxicity of Soluble Amyloidogenic Light Chains.

In light chain amyloidosis (AL), fibrillar deposition of monoclonal immunoglobulin light chains (LCs) in vital organs, such as heart, is associated with their severe dysfunction. In addition to the cellular damage caused by fibril deposition, direct toxicity of soluble prefibrillar amyloidogenic proteins has been reported, in particular, for cardiotoxicity. However, the molecular bases of proteotoxicity by soluble LCs have not been clarified. Here, to address this issue, we rationally engineered the amino acid sequence of the highly cardiotoxic LC H6 by introducing three residue mutations, designed to reduce the dynamics of its native state. The resulting mutant (mH6) is less toxic than its parent H6 to human cardiac fibroblasts and C. elegans. The high sequence and structural similarity, together with the different toxicity, make H6 and its non-toxic designed variant mH6 a test case to shed light on the molecular properties underlying soluble toxicity. Our comparative structural and biochemical study of H6 and mH6 shows closely matching crystal structures, whereas spectroscopic data and limited proteolysis indicate that H6 displays poorly cooperative fold, higher flexibility, and kinetic instability, and a higher dynamic state in its native fold. Taken together, the results of this study show a strong correlation between the overall conformational properties of the native fold and the proteotoxicity of cardiotropic LCs.

Cryo-EM structure of cardiac amyloid fibrils from an immunoglobulin light chain AL amyloidosis patient.

Systemic light chain amyloidosis (AL)  is a life-threatening disease caused by aggregation and deposition of monoclonal immunoglobulin light chains (LC) in target organs. Severity of heart involvement is the most important factor determining prognosis. Here, we report the 4.0 Å resolution cryo-electron microscopy map and molecular model of amyloid fibrils extracted from the heart of an AL amyloidosis patient with severe amyloid cardiomyopathy. The helical fibrils are composed of a single protofilament, showing typical 4.9 Å stacking and cross-ß architecture. Two distinct polypeptide stretches (total of 77 residues) from the LC variable domain (Vl) fit the fibril density. Despite Vl high sequence variability, residues stabilizing the fibril core are conserved through different cardiotoxic Vl, highlighting structural motifs that may be common to misfolding-prone LCs. Our data shed light on the architecture of LC amyloids, correlate amino acid sequences with fibril assembly, providing the grounds for development of innovative medicines.

Concurrent structural and biophysical traits link with immunoglobulin light chains amyloid propensity.

Light chain amyloidosis (AL), the most common systemic amyloidosis, is caused by the overproduction and the aggregation of monoclonal immunoglobulin light chains (LC) in target organs. Due to genetic rearrangement and somatic hypermutation, virtually, each AL patient presents a different amyloidogenic LC. Because of such complexity, the fine molecular determinants of LC aggregation propensity and proteotoxicity are, to date, unclear; significantly, their decoding requires investigating large sets of cases. Aiming to achieve generalizable observations, we systematically characterised a pool of thirteen sequence-diverse full length LCs. Eight amyloidogenic LCs were selected as responsible for severe cardiac symptoms in patients; five non-amyloidogenic LCs were isolated from patients affected by multiple myeloma. Our comprehensive approach (consisting of spectroscopic techniques, limited proteolysis, and X-ray crystallography) shows that low fold stability and high protein dynamics correlate with amyloidogenic LCs, while hydrophobicity, structural rearrangements and nature of the LC dimeric association interface (as observed in seven crystal structures here presented) do not appear to play a significant role in defining amyloid propensity. Based on the structural and biophysical data, our results highlight shared properties driving LC amyloid propensity, and these data will be instrumental for the design of synthetic inhibitors of LC aggregation.

Proteotoxicity in cardiac amyloidosis: amyloidogenic light chains affect the levels of intracellular proteins in human heart cells.

AL amyloidosis is characterized by widespread deposition of immunoglobulin light chains (LCs) as amyloid fibrils. Cardiac involvement is frequent and leads to life-threatening cardiomyopathy. Besides the tissue alteration caused by fibrils, clinical and experimental evidence indicates that cardiac damage is also caused by proteotoxicity of prefibrillar amyloidogenic species. As in other amyloidoses, the damage mechanisms at cellular level are complex and largely undefined. We have characterized the molecular changes in primary human cardiac fibroblasts (hCFs) exposed in vitro to soluble amyloidogenic cardiotoxic LCs from AL cardiomyopathy patients. To evaluate proteome alterations caused by a representative cardiotropic LC, we combined gel-based with label-free shotgun analysis and performed bioinformatics and data validation studies. To assess the generalizability of our results we explored the effects of multiple LCs on hCF viability and on levels of a subset of cellular proteins. Our results indicate that exposure of hCFs to cardiotropic LCs translates into proteome remodeling, associated with apoptosis activation and oxidative stress. The proteome alterations affect proteins involved in cytoskeletal organization, protein synthesis and quality control, mitochondrial activity and metabolism, signal transduction and molecular trafficking. These results support and expand the concept that soluble amyloidogenic cardiotropic LCs exert toxic effects on cardiac cells.

Cardiac Light Chain Amyloidosis: The Role of Metal Ions in Oxidative Stress and Mitochondrial Damage.

AIMS: The knowledge of the mechanism underlying the cardiac damage in immunoglobulin light chain (LC) amyloidosis (AL) is essential to develop novel therapies and improve patients' outcome. Although an active role of reactive oxygen species (ROS) in LC-induced cardiotoxicity has already been envisaged, the actual mechanisms behind their generation remain elusive. This study was aimed at further dissecting the action of ROS generated by cardiotoxic LC in vivo and investigating whether transition metal ions are involved in this process. In the absence of reliable vertebrate model of AL, we used the nematode Caenorhabditis elegans, whose pharynx is an "ancestral heart." RESULTS: LC purified from patients with severe cardiac involvement intrinsically generated high levels of ROS and when administered to C. elegans induced ROS production, activation of the DAF-16/forkhead transcription factor (FOXO) pathway, and expression of proteins involved in stress resistance and survival. Profound functional and structural ROS-mediated mitochondrial damage, similar to that observed in amyloid-affected hearts from AL patients, was observed. All these effects were entirely dependent on the presence of metal ions since addition of metal chelator or metal-binding 8-hydroxyquinoline compounds (chelex, PBT2, and clioquinol) permanently blocked the ROS production and prevented the cardiotoxic effects of amyloid LC. Innovation and Conclusion: Our findings identify the key role of metal ions in driving the ROS-mediated toxic effects of LC. This is a novel conceptual advance that paves the way for new pharmacological strategies aimed at not only counteracting but also totally inhibiting the vicious cycle of redox damage. Antioxid. Redox Signal. 27, 567-582.

The amyloidogenic light chain is a stressor that sensitizes plasma cells to proteasome inhibitor toxicity.

Systemic light chain (AL) amyloidosis is caused by the clonal production of an unstable immunoglobulin light chain (LC), which affects organ function systemically. Although pathogenic LCs have been characterized biochemically, little is known about the biology of amyloidogenic plasma cells (PCs). Intrigued by the unique response rates of AL amyloidosis patients to the first-in-class proteasome inhibitor (PI) bortezomib, we purified and investigated patient-derived AL PCs, in comparison with primary multiple myeloma (MM) PCs, the prototypical PI-responsive cells. Functional, biochemical, and morphological characterization revealed an unprecedented intrinsic sensitivity of AL PCs to PIs, even higher than that of MM PCs, associated with distinctive organellar features and expression patterns indicative of cellular stress. These consisted of expanded endoplasmic reticulum (ER), perinuclear mitochondria, and a higher abundance of stress-related transcripts, and were consistent with reduced autophagic control of organelle homeostasis. To test whether PI sensitivity stems from AL LC production, we engineered PC lines that can be induced to express amyloidogenic and nonamyloidogenic LCs, and found that AL LC expression alters cell growth and proteostasis and confers PI sensitivity. Our study discloses amyloidogenic LC production as an intrinsic PC stressor, and identifies stress-responsive pathways as novel potential therapeutic targets. Moreover, we contribute a cellular disease model to dissect the biology of AL PCs.

In situ characterization of protein aggregates in human tissues affected by light chain amyloidosis: a FTIR microspectroscopy study.

Light chain (AL) amyloidosis, caused by deposition of amyloidogenic immunoglobulin light chains (LCs), is the most common systemic form in industrialized countries. Still open questions, and premises for developing targeted therapies, concern the mechanisms of amyloid formation in vivo and the bases of organ targeting and dysfunction. Investigating amyloid material in its natural environment is crucial to obtain new insights on the molecular features of fibrillar deposits at individual level. To this aim, we used Fourier transform infrared (FTIR) microspectroscopy for studying in situ unfixed tissues (heart and subcutaneous abdominal fat) from patients affected by AL amyloidosis. We compared the infrared response of affected tissues with that of ex vivo and in vitro fibrils obtained from the pathogenic LC derived from one patient, as well as with that of non amyloid-affected tissues. We demonstrated that the IR marker band of intermolecular ß-sheets, typical of protein aggregates, can be detected in situ in LC amyloid-affected tissues, and that FTIR microspectroscopy allows exploring the inter- and intra-sample heterogeneity. We extended the infrared analysis to the characterization of other biomolecules embedded within the amyloid deposits, finding an IR pattern that discloses a possible role of lipids, collagen and glycosaminoglycans in amyloid deposition in vivo.

Novel mitochondrial protein interactors of immunoglobulin light chains causing heart amyloidosis.

In immunoglobulin (Ig) light-chain (LC) (AL) amyloidosis, AL deposition translates into life-threatening cardiomyopathy. Clinical and experimental evidence indicates that soluble cardiotoxic LCs are themselves harmful for cells, by which they are internalized. Hypothesizing that interaction of soluble cardiotoxic LCs with cellular proteins contributes to damage, we characterized their interactome in cardiac cells. LCs were purified from patients with AL amyloidosis cardiomyopathy or multiple myeloma without amyloidosis (the nonamyloidogenic/noncardiotoxic LCs served as controls) and employed at concentrations in the range observed in AL patients' sera. A functional proteomic approach, based on direct and inverse coimmunoprecipitation and mass spectrometry, allowed identifying LC-protein complexes. Findings were validated by colocalization, fluorescence lifetime imaging microscopy (FLIM)-fluorescence resonance energy transfer (FRET), and ultrastructural studies, using human primary cardiac fibroblasts (hCFs) and stem cell-derived cardiomyocytes. Amyloidogenic cardiotoxic LCs interact in vitro with specific intracellular proteins involved in viability and metabolism. Imaging confirmed that, especially in hCFs, cardiotoxic LCs (not controls) colocalize with mitochondria and spatially associate with selected interactors: mitochondrial optic atrophy 1-like protein and peroxisomal acyl-coenzyme A oxidase 1 (FLIM-FRET efficiencies 11 and 6%, respectively). Cardiotoxic LC-treated hCFs display mitochondrial ultrastructural changes, supporting mitochondrial involvement. We show that cardiotoxic LCs establish nonphysiologic protein-protein contacts in human cardiac cells, offering new clues on the pathogenesis of AL cardiomyopathy.

A Caenorhabditis elegans-based assay recognizes immunoglobulin light chains causing heart amyloidosis.

Poor prognosis and limited therapeutic options characterize immunoglobulin light-chain (AL) amyloidosis with major heart involvement. Reliable experimental models are needed to study light-chain (LC)/heart interactions and to explore strategies for prevention of cardiac damage. We have exploited the nematode Caenorhabditis elegans as a novel tool, because its pharynx is evolutionarily related to the vertebrate heart. Our data demonstrate that the pharyngeal pumping of C elegans is significantly and selectively reduced by LCs from AL patients suffering from cardiomyopathy, but not by amyloid LCs with different organ tropism or nonamyloidogenic LCs from multiple myeloma. This functional alteration is dependent on the LC concentration and results in persistent pharyngeal dysfunction and in a significant reduction of the worms' lifespan. These manifestations are paralleled by an increase of mitochondrial reactive oxygen species and can be prevented by treatment with antioxidant agents. In conclusion, these data indicate that this nematode-based assay is a promising surrogate model for investigating the heart-specific toxicity of amyloidogenic LCs and for a rapid screening of new therapeutic strategies.

Investigating heart-specific toxicity of amyloidogenic immunoglobulin light chains: A lesson from C. elegans.

Abnormalities in protein folding are involved in many localized and systemic diseases, all of which are characterized by insoluble amyloid formation and deposition. In immunoglobulin light chain (LC) amyloidosis, the most frequent systemic form of amyloidosis, the amyloid involvement of the heart dictates the prognosis and the elucidation of the mechanism of heart targeting and toxicity is essential for designing and testing new effective treatments. To this end, the availability of an appropriate animal model is crucial. We recently described the use of C. elegans as an innovative experimental system to investigate in vivo the pathogenic effects of monoclonal LC. This idea stems from the knowledge that the worm's pharynx is an "ancestral heart" with the additional ability to recognize stressor compounds. The feeding of worms with LC purified from patients suffering from cardiomyopathy, selectively and permanently impaired the pharyngeal function. This irreversible damage resulted in time, in a significant reduction in the lifespan of worms. We also reported that the ability of LC to generate reactive oxygen species was associated with their toxic effects and was counteracted by anti-oxidant compounds. This new nematode-based assay represents a promising model for elucidating the heart-specific toxicity of LC and for a rapid screening of new therapeutic strategies.

A strategy for synthesis of pathogenic human immunoglobulin free light chains in E. coli.

Monoclonal immunoglobulin light chains are normally synthesized in excess compared to the heavy chain partners and can be detected in serum and urine ("free" LC). Occasionally free LC are per se cause of organ toxicity, as in free LC-related disorders. In AL amyloidosis, the most common of these conditions, free LC with peculiar biophysical properties related to their primary structure damage target organs and organize in amyloid fibrils. Unlimited availability of well-characterized free LC is instrumental to investigate the toxic effect of these proteins and to study their interactions with targets. We present a straightforward strategy to obtain recombinant monoclonal free LC by using a bacterial system. These proteins, expressed as inclusion bodies, were subjected to solubilization and refolding procedures to recover them in native form. To minimize differences from the circulating natural LC, full-length recombinant LC were expressed, i.e. complete of variable and constant regions, with the original amino acid sequence along the entire protein, and with no purification tags. The strategy was exploited to generate free LC from three AL amyloidosis patients. After purification, recombinant proteins were biochemically characterized and compared to the natural Bence Jones protein isolated from one of the patients. Results showed that the recombinant free LC were properly folded and formed homodimers in solution, similar to the natural Bence Jones protein used for comparison. Furthermore, as proof of pathogenicity, recombinant proteins formed amyloid fibrils in vitro. We believe that the present strategy represents a valuable tool to speed research in free LC-related disorders.