Systemic amyloidoses.

The amyloidoses are a group of protein misfolding diseases in which the precursor protein undergoes a conformational change that triggers the formation of amyloid fibrils in different tissues and organs, causing cell death and organ failure. Amyloidoses can be either localized or systemic. In localized amyloidosis, amyloid deposits form at the site of precursor protein synthesis, whereas in systemic amyloidosis, amyloid deposition occurs distant from the site of precursor protein secretion. We review the type of proteins and cells involved and what is known about the complex pathophysiology of these diseases. We focus on light chain amyloidosis to illustrate how biochemical and biophysical studies have led to a deeper understanding of the pathogenesis of this devastating disease. We also review current cellular, tissue, and animal models and discuss the challenges and opportunities for future studies of the systemic amyloidoses.

A proteomic atlas of kidney amyloidosis provides insights into disease pathogenesis.

The mechanisms of tissue damage in kidney amyloidosis are not well described. To investigate this further, we used laser microdissection-mass spectrometry to identify proteins deposited in amyloid plaques (expanded proteome) and proteins overexpressed in plaques compared to controls (plaque-specific proteome). This study encompassed 2650 cases of amyloidosis due to light chain (AL), heavy chain (AH), leukocyte chemotactic factor-2-type (ALECT2), secondary (AA), fibrinogen (AFib), apo AIV (AApoAIV), apo CII (AApoCII) and 14 normal/disease controls. We found that AFib, AA, and AApoCII have the most distinct proteomes predominantly driven by increased complement pathway proteins. Clustering of cases based on the expanded proteome identified two ALECT2 and seven AL subtypes. The main differences within the AL and ALECT2 subtypes were driven by complement proteins and, for AL only, 14-3-3 family proteins (a family of structurally similar phospho-binding proteins that regulate major cellular functions) widely implicated in kidney tissue dysfunction. The kidney AL plaque-specific proteome consisted of 24 proteins, including those implicated in kidney damage (α1 antitrypsin and heat shock protein ß1). Hierarchical clustering of AL cases based on their plaque-specific proteome identified four clusters, of which one was associated with improved kidney survival and was characterized by higher overall proteomic content and 14-3-3 proteins but lower levels of light chains and most signature proteins. Thus, our results suggest that there is significant heterogeneity across and within amyloid types, driven predominantly by complement proteins, and that the plaque protein burden does not correlate with amyloid toxicity.

Vesicular Stomatitis Virus Encoding a Destabilized Tumor Antigen Improves Activation of Anti-tumor T Cell Responses.

Enhancing the immunogenicity of tumor-associated antigens would represent a major advance for anti-tumor vaccination strategies. Here, we investigated structure-directed antigen destabilization as a strategy to improve the degradation, immunogenic epitope presentation, and T cell activation against a vesicular stomatitis virus (VSV)-encoded tumor antigen. We used the crystal structure of the model antigen ovalbumin to identify charge-disrupting amino acid mutations that were predicted to decrease the stability of the protein. One mutation, OVA-C12R, significantly reduced the half-life of the protein and was preferentially degraded in a 26-S proteasomal-dependent manner. The destabilized ovalbumin protein exhibited enhanced presentation of the major histocompatibility complex (MHC) class I immunogenic epitope, SIINFEKL, on the surface of B16F10 cells or murine bone marrow-derived dendritic cells (BMDCs) in vitro. Enhanced presentation correlated with better recognition by cognate CD8 OT-I T cells as measured by activation, proliferation, and effector cytokine production. Finally, VSV encoding the degradation-prone antigen was better able to prime an antigen ovalbumin-specific CD8 T cell response in vivo without altering the anti-viral CD8 T cell response. Our studies highlight that not only is the choice of antigen in cancer vaccines of importance, but that emphasis should be placed on modifying antigen quality to ensure optimal priming of anti-tumor responses.

Mechanistic Insights into the Early Events in the Aggregation of Immunoglobulin Light Chains.

Little is known about the mechanism of amyloid assembly in immunoglobulin light chain (AL) amyloidosis, in contrast to other amyloid diseases. Early events in the aggregation pathway are especially important, as these soluble species could be cytotoxic intermediates playing a critical role in the initiation of the amyloid assembly. In this work, we discuss the mechanism of the early events in in vitro fibril formation of immunoglobulin light chain AL-09 and AL-12 (involved in cardiac amyloidosis) and its germline (control) protein κI O18/O8. Previous work from our laboratory showed that AL-12 adopts a canonical dimer conformation (like the germline protein), whereas AL-09 presents an altered dimer interface as a result of somatic mutations. Both AL-12 and AL-09 aggregate with similar rates and significantly faster than the germline protein. AL-09 is the only protein in this study that forms stable oligomeric intermediates during the early stages of the aggregation reaction with some structural rearrangements that increase the thioflavin T fluorescence but maintain the same number of monomers in solution. The presence of the restorative mutation AL-09 H87Y changes the kinetics and the aggregation pathway compared to AL-09. The single restorative mutation AL-12 R65S slightly delayed the overall rate of aggregation as compared to AL-12. Collectively, our study provides a comprehensive analysis of species formed during amyloid nucleation in AL amyloidosis, shows a strong dependence between the altered dimer conformation and the formation of stable oligomeric intermediates, and sheds light on the structural features of amyloidogenic intermediates associated with cellular toxicity.

Clinical features, laboratory characteristics and outcomes of patients with renal versus cardiac light chain amyloidosis.

This study evaluated the differences in clinical features of 1077 newly diagnosed AL amyloidosis patients with renal involvement (n = 229, 21%), both cardiac and renal involvement (n = 443, 41%) and cardiac involvement (n = 405, 38%). Significant differences in dFLC (difference in involved and uninvolved light chains) were noted (renal, both, cardiac median: 83, 234 and 349 mg/l, P < 0.001). The proportion of patients with ≥ 10% bone marrow plasma cells (BMPCs) was lowest in renal only patients: 44%, 57%, 64%, respectively, P < 0.001. In a multivariate linear regression model incorporating organ involvement type and BMPCs ≥10%, organ involvement was a significant predictor of dFLC (P < 0.001). Median overall survival (OS) across the three groups was 83 vs. 19 vs. 16 months (P < 0.001) in patients not undergoing transplant and 5-year OS in patients undergoing transplant was 90% vs. 75% vs. 64% (P = 0.007), respectively. In conclusion, renal involvement alone or renal + cardiac involvement in AL amyloidosis is associated with lower circulating light chain burden, which cannot be fully explained by BMPC burden alone. Increased sensitivity of the kidney to light chains, given significant interactions with the renal tubular system and secretion of modified light chain products may play a role in pathogenesis of renal AL amyloidosis and warrants further investigation.

Clarifying immunoglobulin gene usage in systemic and localized immunoglobulin light-chain amyloidosis by mass spectrometry.

The goal of this study was to investigate the frequency of use of light-chain variable region (IGVL) genes among patients with systemic (ALS) and localized (ALL) amyloidosis and to assess for associations between IGVL gene usage and organ tropism. We evaluated clinic charts from 821 AL patients seen at the Mayo Clinic who had bone marrow, fat pad, and solid organ tissue samples typed by liquid chromatography tandem mass spectrometry (LC-MS). We identified 701 patients with ALS and 120 with ALL Overall, we were able to identify an IGVL gene in 87 (72%) patients with ALL and 573 (82%) patients with ALS When compared with ALL, LV6-57 was more common, whereas KV3-20 and heavy-chain codeposition were less common in ALS In this large series of ALS, characteristics particular to specific genotypes became apparent. LV6-57 patients were more likely to have renal involvement and to harbor a translocation 11;14. LV3-01 patients were less likely to have advanced cardiac disease and renal involvement. LV2-14 patients were more likely to have peripheral nerve involvement, an intact circulating immunoglobulin, and lower circulating dFLC. LV1-44 patients were more likely to have cardiac involvement. KV1-33 patients had more liver involvement and higher circulating dFLC. Finally, KV1-05 was associated with inferior overall survival but not independently of cardiac stage. IGVL gene usage appears to provide clues about disease pathophysiology and tissue tropism. LC-MS is a high-throughput and low-resource technique that can be used to identify IGVL gene from clinical tissue specimens.

Differences in Protein Concentration Dependence for Nucleation and Elongation in Light Chain Amyloid Formation.

Light chain (AL) amyloidosis is a lethal disease characterized by the deposition of the immunoglobulin light chain into amyloid fibrils, resulting in organ dysfunction and failure. Amyloid fibrils have the ability to self-propagate, recruiting soluble protein into the fibril by a nucleation-polymerization mechanism, characteristic of autocatalytic reactions. Experimental data suggest the existence of a critical concentration for initiation of fibril formation. As such, the initial concentration of soluble amyloidogenic protein is expected to have a profound effect on the rate of fibril formation. In this work, we present in vitro evidence that fibril formation rates for AL light chains are affected by the protein concentration in a differential manner. De novo reactions of the proteins with the fastest amyloid kinetics (AL-09, AL-T05, and AL-103) do not present protein concentration dependence. Seeded reactions, however, exhibited weak protein concentration dependence. For AL-12, seeded and protein concentration dependence data suggest a synergistic effect for recruitment and elongation at low protein concentrations, while reactions of κI exhibited poor efficiency in nucleating and elongating preformed fibrils. Additionally, co-aggregation and cross seeding of κI variable domain (VL) and the κI full length (FL) light chain indicate that the presence of the constant domain in κI FL modulates fibril formation, facilitating the recruitment of κI VL. Together, these results indicate that the dominant process in fibril formation varies among the AL proteins tested with a differential dependence of the protein concentration.

Cell Damage in Light Chain Amyloidosis: FIBRIL INTERNALIZATION, TOXICITY AND CELL-MEDIATED SEEDING.

Light chain (AL) amyloidosis is an incurable human disease characterized by the misfolding, aggregation, and systemic deposition of amyloid composed of immunoglobulin light chains (LC). This work describes our studies on potential mechanisms of AL cytotoxicity. We have studied the internalization of AL soluble proteins and amyloid fibrils into human AC16 cardiomyocytes by using real time live cell image analysis. Our results show how external amyloid aggregates rapidly surround the cells and act as a recruitment point for soluble protein, triggering the amyloid fibril elongation. Soluble protein and external aggregates are internalized into AC16 cells via macropinocytosis. AL amyloid fibrils are shown to be highly cytotoxic at low concentrations. Additionally, caspase assays revealed soluble protein induces apoptosis, demonstrating different cytotoxic mechanisms between soluble protein and amyloid aggregates. This study emphasizes the complex immunoglobulin light chain-cell interactions that result in fibril internalization, protein recruitment, and cytotoxicity that may occur in AL amyloidosis.

B-type natriuretic peptide overexpression ameliorates hepatorenal fibrocystic disease in a rat model of polycystic kidney disease.

Polycystic kidney disease (PKD) involves progressive hepatorenal cyst expansion and fibrosis, frequently leading to end-stage renal disease. Increased vasopressin and cAMP signaling, dysregulated calcium homeostasis, and hypertension play major roles in PKD progression. The guanylyl cyclase A agonist, B-type natriuretic peptide (BNP), stimulates cGMP and shows anti-fibrotic, anti-hypertensive, and vasopressin-suppressive effects, potentially counteracting PKD pathogenesis. Here, we assessed the impacts of guanylyl cyclase A activation on PKD progression in a rat model of PKD. Sustained BNP production significantly reduced kidney weight, renal cystic indexes and fibrosis, in concert with suppressed hepatic cystogenesis in vivo. In vitro, BNP decreased cystic epithelial cell proliferation, suppressed fibrotic gene expression, and increased intracellular calcium. Together, our data demonstrate multifaceted effects of sustained activation of guanylyl cyclase A on polycystic kidney and liver disease. Thus, targeting the guanylyl cyclase A-cGMP axis may provide a novel therapeutic strategy for hepatorenal fibrocystic diseases.

Mesenchymal stromal cells protect human cardiomyocytes from amyloid fibril damage.

BACKGROUND AIMS: Light chain (AL) amyloidosis is a protein misfolding disease characterized by extracellular deposition of immunoglobulin light chains (LC) as amyloid fibrils. Patients with LC amyloid involvement of the heart have the worst morbidity and mortality. Current treatments target the plasma cells to reduce further production of amyloid proteins. There is dire need to understand the mechanisms of cardiac tissue damage from amyloid to develop novel therapies. We recently reported that LC soluble and fibrillar species cause apoptosis and inhibit cell growth in human cardiomyocytes. Mesenchymal stromal cells (MSCs) can promote wound healing and tissue remodeling. The objective of this study was to evaluate MSCs to protect cardiomyocytes affected by AL amyloid fibrils. METHODS: We used live cell imaging and proteomics to analyze the effect of MSCs in the growth arrest caused by AL amyloid fibrils. RESULTS: We evaluated the growth of human cardiomyocytes (RFP-AC16 cells) in the presence of cytotoxic LC amyloid fibrils. MSCs reversed the cell growth arrest caused by LC fibrils. We also demonstrated that this effect requires cell contact and may be mediated through paracrine factors modulating cell adhesion and extracellular matrix remodeling. To our knowledge, this is the first report of MSC protection of human cardiomyocytes in amyloid disease. CONCLUSIONS: This important proof of concept study will inform future rational development of MSC therapy in cardiac LC amyloid.

Assessment of renal response with urinary exosomes in patients with AL amyloidosis: A proof of concept.

Immunoglobulin light chain (AL) amyloidosis is a fatal complication of B-cell proliferation secondary to deposition of amyloid fibrils in various organs. Urinary exosomes (UEX) are the smallest of the microvesicles excreted in the urine. Previously, we found UEX of patients with AL amyloidosis contained immunoglobulin light chain (LC) oligomers that patients with multiple myeloma did not have. To further explore the role of the LC oligomers, UEX was isolated from an AL amyloidosis patient with progressive renal disease despite achieving a complete response. LC oligomers were identified. Mass spectrometry (MS) of the UEX and serum identified two monoclonal lambda LCs. Proteomics of the trypsin digested amyloid fragments in the kidney by laser microdissection and MS analysis identified a λ6 LC. The cDNA from plasma cell clone was from the IGLV- 6-57 family and it matched the amino acid sequences of the amyloid peptides. The predicted mass of the peptide product of the cDNA matched the mass of one of the two LCs identified in the UEX and serum. UEX combined with MS were able to identify 2 monoclonal lambda LCs that current clinical methods could not. It also identified the amyloidogenic LC which holds potential for response assessment in the future.

Recruitment of Light Chains by Homologous and Heterologous Fibrils Shows Distinctive Kinetic and Conformational Specificity.

Light chain amyloidosis is a protein misfolding disease in which immunoglobulin light chains aggregate as insoluble fibrils that accumulate in extracellular deposits. Amyloid fibril formation in vitro has been described as a nucleation-polymerization, autocatalytic reaction in which nascent fibrils catalyze formation of new fibrils, recruiting soluble protein into the fibril. In this context, it is also established that preformed fibrils or "seeds" accelerate fibril formation. In some cases, seeds with a substantially different sequence are able to accelerate the reaction, albeit with a lower efficiency. In this work, we studied the recruitment and addition of monomers in the presence of seeds of five immunoglobulin light chain proteins, covering a broad range of protein stabilities and amyloidogenic properties. Our data reveal that in the presence of homologous or heterologous seeds, the fibril formation reactions become less stochastic than de novo reactions. The kinetics of the most amyloidogenic proteins (AL-T05 and AL-09) do not present significant changes in the presence of seeds. Amyloidogenic protein AL-103 presented fairly consistent acceleration with all seeds. In contrast, the less amyloidogenic proteins (AL-12 and κI) presented dramatic differential effects that are dependent on the kind of seed used. κI had a poor efficiency to elongate preformed fibrils. Together, these results indicate that fibril formation is kinetically determined by the conformation of the amyloidogenic precursor and modulated by the differential ability of each protein to either nucleate or elongate fibrils. We observe morphological and conformational properties of some seeds that do not favor elongation with some proteins, resulting in a delay in the reaction.

Differential effects on light chain amyloid formation depend on mutations and type of glycosaminoglycans.

Amyloid light chain (AL) amyloidosis is a protein misfolding disease where immunoglobulin light chains sample partially folded states that lead to misfolding and amyloid formation, resulting in organ dysfunction and death. In vivo, amyloid deposits are found in the extracellular space and involve a variety of accessory molecules, such as glycosaminoglycans, one of the main components of the extracellular matrix. Glycosaminoglycans are a group of negatively charged heteropolysaccharides composed of repeating disaccharide units. In this study, we investigated the effect of glycosaminoglycans on the kinetics of amyloid fibril formation of three AL cardiac amyloidosis light chains. These proteins have similar thermodynamic stability but exhibit different kinetics of fibril formation. We also studied single restorative and reciprocal mutants and wild type germ line control protein. We found that the type of glycosaminoglycan has a different effect on the kinetics of fibril formation, and this effect seems to be associated with the natural propensity of each AL protein to form fibrils. Heparan sulfate accelerated AL-12, AL-09, κI Y87H, and AL-103 H92D fibril formation; delayed fibril formation for AL-103; and did not promote any fibril formation for AL-12 R65S, AL-103 delP95aIns, or κI O18/O8. Chondroitin sulfate A, on the other hand, showed a strong fibril formation inhibition for all proteins. We propose that heparan sulfate facilitates the formation of transient amyloidogenic conformations of AL light chains, thereby promoting amyloid formation, whereas chondroitin sulfate A kinetically traps partially unfolded intermediates, and further fibril elongation into fibrils is inhibited, resulting in formation/accumulation of oligomeric/protofibrillar aggregates.

Clonotypic Light Chain Peptides Identified for Monitoring Minimal Residual Disease in Multiple Myeloma without Bone Marrow Aspiration.

BACKGROUND: Analytically sensitive techniques for measuring minimal residual disease (MRD) in multiple myeloma (MM) currently require invasive and costly bone marrow aspiration. These methods include immunohistochemistry (IHC), flow cytometry, quantitative PCR, and next-generation sequencing. An ideal MM MRD test would be a serum-based test sensitive enough to detect low concentrations of Ig secreted from multifocal lesions. METHODS: Patient serum with abundant M-protein before treatment was separated on a 1-dimensional SDS-PAGE gel, and the Ig light-chain (LC) band was excised, trypsin digested, and analyzed on a Q Exactive mass spectrometer by LC-MS/MS. We used the peptide's abundance and sequence to identify tryptic peptides that mapped to complementary determining regions of Ig LCs. The clonotypic target tryptic peptides were used to monitor MRD in subsequent serum samples with prior affinity enrichment. RESULTS: Sixty-two patients were tested, 20 with no detectable disease by IHC and 42 with no detectable disease by 6-color flow cytometry. A target peptide that could be monitored was identified in 57 patients (91%). Of these 57, detectable disease by LC-MS/MS was found in 52 (91%). CONCLUSIONS: The ability to use LC-MS/MS to measure disease in patients who are negative by bone marrow-based methodologies indicates that a serum-based approach has more analytical sensitivity and may be useful for measuring deeper responses to MM treatment. The method requires no bone marrow aspiration.

Evaluation of the BH3-only protein Puma as a direct Bak activator.

Interactions among Bcl-2 family proteins play critical roles in cellular life and death decisions. Previous studies have established the BH3-only proteins Bim, tBid, and Noxa as "direct activators" that are able to directly initiate the oligomerization and activation of Bak and/or Bax. Earlier studies of Puma have yielded equivocal results, with some concluding that it also acts as a direct activator and other studies suggesting that it acts solely as a sensitizer BH3-only protein. In the present study we examined the interaction of Puma BH3 domain or full-length protein with Bak by surface plasmon resonance, assessed Bak oligomerization status by cross-linking followed by immunoblotting, evaluated the ability of the Puma BH3 domain to induce Bak-mediated permeabilization of liposomes and mitochondria, and determined the effect of wild type and mutant Puma on cell viability in a variety of cellular contexts. Results of this analysis demonstrate high affinity (KD = 26 ± 5 nM) binding of the Puma BH3 domain to purified Bak ex vivo, leading to Bak homo-oligomerization and membrane permeabilization. Mutations in Puma that inhibit (L141E/M144E/L148E) or enhance (M144I/A145G) Puma BH3 binding to Bak also produce corresponding alterations in Bak oligomerization, Bak-mediated membrane permeabilization and, in a cellular context, Bak-mediated killing. Collectively, these results provide strong evidence that Puma, like Bim, Noxa, and tBid, is able to act as a direct Bak activator.

Phenotyping polyclonal kappa and lambda light chain molecular mass distributions in patient serum using mass spectrometry.

We previously described a microLC-ESI-Q-TOF MS method for identifying monoclonal immunoglobulins in serum and then tracking them over time using their accurate molecular mass. Here we demonstrate how the same methodology can be used to identify and characterize polyclonal immunoglobulins in serum. We establish that two molecular mass distributions observed by microLC-ESI-Q-TOF MS are from polyclonal kappa and lambda light chains using a combination of theoretical molecular masses from gene sequence data and the analysis of commercially available purified polyclonal IgG kappa and IgG lambda from normal human serum. A linear regression comparison of kappa/lambda ratios for 74 serum samples (25 hypergammaglobulinemia, 24 hypogammaglobulinemia, 25 normal) determined by microflowLC-ESI-Q-TOF MS and immunonephelometry had a slope of 1.37 and a correlation coefficient of 0.639. In addition to providing kappa/lambda ratios, the same microLC-ESI-Q-TOF MS analysis can determine the molecular mass for oligoclonal light chains observed above the polyclonal background in patient samples. In 2 patients with immune disorders and hypergammaglobulinemia, we observed a skewed polyclonal molecular mass distribution which translated into biased kappa/lambda ratios. Mass spectrometry provides a rapid and simple way to combine the polyclonal kappa/lambda light chain abundance ratios with the identification of dominant monoclonal as well as oligoclonal light chain immunoglobulins. We anticipate that this approach to evaluating immunoglobulin light chains will lead to improved understanding of immune deficiencies, autoimmune diseases, and antibody responses.

Nanoliposomes protect against AL amyloid light chain protein-induced endothelial injury.

CONTEXT: A newly-recognized pathogenic mechanism underlying light chain amyloidosis (AL) involves endothelial dysfunction and cell injury caused by misfolded light chain proteins (LC). Nanoliposomes (NL) are artificial phospholipid vesicles that could attach to misfolded proteins and reduce tissue injury. OBJECTIVE: To test whether co-treatment with NL reduces LC-induced endothelial dysfunction and cell death. METHODS: Abdominal subcutaneous adipose arterioles from 14 non-AL subjects were cannulated; dilator response to acetylcholine and papaverine were measured at baseline and following 1-hour exposure to LC (20 µg/mL, 2 purified from AL subjects' urine, 1 from human recombinant LC [AL-09]) ± NL (phosphatidylcholine/cholesterol/phosphatidic acid 70/25/5 molar ratio) or NL alone. Human aortic artery endothelial cells (HAEC) were exposed to Oregon Green-labeled LC ± NL for 24 hours and intracellular LC and apoptosis (Hoechst stain) were measured. Circular dichroism spectroscopy was performed on AL-09 LC ± NL to follow changes in secondary structure and protein thermal stability. RESULTS: LC caused impaired dilation to acetylcholine that was restored by NL (control - 94.0 ± 1.8%, LC - 65.0 ± 7.1%, LC + NL - 95.3 ± 1.8%, p ≤ 0.001 LC versus control or LC + NL). NL protection was inhibited by L-NG-nitroarginine methyl ester. NL increased the beta sheet structure of LC, reduced endothelial cell internalization of LC and protected against LC-induced endothelial cell death. CONCLUSIONS: LC induced human adipose arteriole endothelial dysfunction and endothelial cell death, which were reversed by co-treatment with NL. This protection may partly be due to enhancing LC protein structure and reducing LC internalization. Nanoliposomes represent a promising new class of agents to ameliorate tissue injury from protein misfolding diseases such as AL.

Biophysical characterization of human-cell-expressed, full-length κI O18/O8, AL-09, λ6a, and Wil immunoglobulin light chains.

Immunoglobulin light chain (AL) amyloidosis involves the deposition of insoluble monoclonal AL protein fibrils in the extracellular space of different organs leading to dysfunction and death. Development of methods to efficiently express and purify AL proteins with acceptable standards of homogeneity and structural integrity has become critical to understand the in vitro and in vivo aspects of AL protein aggregation, and thus the disease progression. In this study, we report the biophysical characterization of His-tagged and untagged versions of AL full-length (FL) κI and λ6 subgroup proteins and their mutants expressed from the Expi293F human cell line. We used an array of biophysical and biochemical methods to analyze the structure and stability of the monomers, oligomerization states, and thermodynamic characteristics of the purified FL proteins and how they compare with the bacterially expressed FL proteins. Our results demonstrate that the tagged and untagged versions of FL proteins have comparable stability to proteins expressed in bacterial cells but exhibit multiple unfolding transitions and reversibility. Non-reducing SDS-PAGE and analytical ultracentrifugation analysis showed presence of monomers and dimers, with an insignificant amount of higher-order oligomers, in the purified fraction of all proteins. Overall, the FL proteins were expressed with sufficient yields for biophysical studies and can replace bacterial expression systems.

Tyrosine residues mediate fibril formation in a dynamic light chain dimer interface.

Light chain amyloidosis is an incurable protein misfolding disease where monoclonal immunoglobulin light chains misfold and deposit as amyloid fibrils, causing organ failure and death. Previously, we determined that amyloidogenic light chains AL-09 and AL-103 do not form fibrils at pH 10 (tyrosine pK(a)). There are three tyrosine residues (32, 91, and 96) clustered in the dimer interface, interacting differently in the two light chain proteins due to their two different dimer conformations. These tyrosines may be ionized at pH 10, causing repulsion and inhibiting fibril formation. Here, we characterize single and double Tyr-to-Phe mutations in AL-09 and AL-103. All AL-09 Tyr-to-Phe mutants form fibrils at pH 10, whereas none of the AL-103 mutants form fibrils at pH 10. NMR studies suggest that although both AL-09 and AL-103 present conformational heterogeneity, only AL-09 favors dimer conformations where tyrosine residues mediate crucial interactions for amyloid formation.