Thermodynamic and fibril formation studies of full length immunoglobulin light chain AL-09 and its germline protein using scan rate dependent thermal unfolding

Blancas‐Mejia, Luis M.; Horn, Timothy; Argany, Meysam; Auton, Matthew; Tischer, Alexander; Ramı́rez-Alvarado, Marina

doi:10.1016/j.bpc.2015.07.005

Cited by 46 publications

(92 citation statements)

References 29 publications

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“…Whereas the denaturation of most LC was reversible, all -MM LC denatured irreversibly after heating. We found that -and -isotype LC had different stabilities in thermal denaturation, with an average T m of 53 Ϯ 5 and 59 Ϯ 2°C, respectively, which corresponds to previously published data (28,30,44,64). We did not find strong systematic differences in stability between AL-and MM-LC.…”

Section: Discussionsupporting

confidence: 57%

“…The independent unfolding of the two protein domains fails to explain the observed unfolding dynamics (28,29,63). Furthermore, thermodynamic instability is not sufficient for amyloid formation, suggesting the importance of kinetic factors (27,30). All studies suggest that the enhanced aggregation of the full-length protein results not only from the combined aggregating propensities of its variable and constant domain but also from the intramolecular interactions between these regions.…”

Section: Discussionmentioning

confidence: 87%

“…The significance of full-length LC for amyloid formation was only recently addressed (27)(28)(29)(30). These studies find that interactions between the constant and variable domain of LC alter the unfolding/folding pathways and that the unfolding of native state LC is a complex, often irreversible, process.…”

Section: Discussionmentioning

confidence: 99%

“…The significance of full-length LC for amyloid formation is not yet fully understood (27)(28)(29)(30).…”

Section: Systemic Light Chain Amyloidosis (Al)mentioning

confidence: 99%

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Aggregation of Full-length Immunoglobulin Light Chains from Systemic Light Chain Amyloidosis (AL) Patients Is Remodeled by Epigallocatechin-3-gallate

Andrich

Hegenbart

Kimmich

et al. 2017

Journal of Biological Chemistry

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Edited by Paul E. FraserIntervention into amyloid deposition with anti-amyloid agents like the polyphenol epigallocatechin-3-gallate (EGCG) is emerging as an experimental secondary treatment strategy in systemic light chain amyloidosis (AL). In both AL and multiple myeloma (MM), soluble immunoglobulin light chains (LC) are produced by clonal plasma cells, but only in AL do they form amyloid deposits in vivo. We investigated the amyloid formation of patient-derived LC and their susceptibility to EGCG in vitro to probe commonalities and systematic differences in their assembly mechanisms. We isolated nine LC from the urine of AL and MM patients. We quantified their thermodynamic stabilities and monitored their aggregation under physiological conditions by thioflavin T fluorescence, light scattering, SDS stability, and atomic force microscopy. LC from all patients formed amyloid-like aggregates, albeit with individually different kinetics. LC existed as dimers, ϳ50% of which were linked by disulfide bridges. Our results suggest that cleavage into LC monomers is required for efficient amyloid formation. The kinetics of AL LC displayed a transition point in concentration dependence, which MM LC lacked. The lack of concentration dependence of MM LC aggregation kinetics suggests that conformational change of the light chain is rate-limiting for these proteins. Aggregation kinetics displayed two distinct phases, which corresponded to the formation of oligomers and amyloid fibrils, respectively. EGCG specifically inhibited the second aggregation phase and induced the formation of SDS-stable, non-amyloid LC aggregates. Our data suggest that EGCG intervention does not depend on the individual LC sequence and is similar to the mechanism observed for amyloid-␤ and ␣-synuclein. Systemic light chain amyloidosis (AL)2 is the most common form of systemic amyloidosis (1), but it is still a rare disease with an incidence of 6 -7 in 1,000,000 people (2). Underlying AL pathology is a clonal plasma cell disorder, which produces an immunoglobulin light chain in the bone marrow with a unique sequence. These light chains are released into the blood (monoclonal gammopathy). When elevated LC levels in serum overwhelm renal absorption, LC is also found in the urine. Fulllength LC can be isolated from the urine of these patients (3-5).Clonal plasma cell disorders can present different types of pathologies; light chains form amyloid deposits in AL patients. Here, LC fibrils adopt a conformation characterized by highly stable, intramolecular cross-␤-sheets that stain with Congo red (6) and thioflavin T (ThT) (7-9).In contrast, amyloid deposits are not observed in patients suffering from multiple myeloma (MM), a malignant disease characterized by bone marrow failure and bone destruction. The two contrasting pathologies of LC gammopathies raise the question of which factors determine amyloid formation. Amyloid formation could be initiated (a) by the sequences of the individual light chains, (b) by high levels of light chain expression, (c) by p...

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Section: Discussionsupporting

confidence: 57%

Section: Discussionmentioning

confidence: 87%

Section: Discussionmentioning

confidence: 99%

“…The significance of full-length LC for amyloid formation is not yet fully understood (27)(28)(29)(30).…”

Section: Systemic Light Chain Amyloidosis (Al)mentioning

confidence: 99%

See 2 more Smart Citations

Aggregation of Full-length Immunoglobulin Light Chains from Systemic Light Chain Amyloidosis (AL) Patients Is Remodeled by Epigallocatechin-3-gallate

Andrich

Hegenbart

Kimmich

et al. 2017

Journal of Biological Chemistry

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“…Thermodynamic studies proposed a stabilizing role for the 3C L domain in the stability and a modulating effect on fibril formation (9). Recently, our laboratory has demonstrated that the C L domain modulates the amyloid formation reaction but has no effect on the stability of the protein (10).…”

Section: Light Chain (Al)mentioning

confidence: 99%

Cell Damage in Light Chain Amyloidosis

Argany¹,

Lin²,

Misra³

et al. 2016

Journal of Biological Chemistry

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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. Light chain (AL)2 amyloidosis is a protein misfolding disease characterized by extracellular deposition of immunoglobulin light chains (LC) as amyloid fibrils. LC proteins are comprised of two distinct domains: the variable (V L ) and constant (C L ) domains (also called LC full-length (FL) protein to differentiate with the V L domain). In patients with AL amyloidosis, the LC aggregate and deposit in vital organs, causing organ failure and death (1). The factors governing deposition in individual tissues are unknown. Patients with cardiac AL amyloidosis have the worst prognosis, with a median survival of less than a year (2, 3).The finding that the V L was the primary component of amyloid fibrils influenced previous biophysical studies (4,5). Recent proteomic studies have demonstrated that amyloid deposits are likely heterogeneous in nature and can be formed by FL, V L , C L , or mixtures of all types of LC fragments (6 -8). Thermodynamic studies proposed a stabilizing role for the 3C L domain in the stability and a modulating effect on fibril formation (9). Recently, our laboratory has demonstrated that the C L domain modulates the amyloid formation reaction but has no effect on the stability of the protein (10).Soluble monoclonal LC, isolated from patients with amyloidosis, can impair rat cardiomyocyte function (11) and induce apoptotic events in mouse cardiomyocytes (12, 13). Also, urinederived LC can be internalized into primary rat cardiac fibroblasts (14) and primary human renal mesangial cells (15) through a pinocytic pathway (16) or via receptor, clathrin-mediated mechanisms, respectively (15).Within the amyloid field, it is widely accepted that oligomeric species are potentially more toxic than mature fibrils (17-20). However, toxicity associated with amyloid fibrils may also be pathologically relevant. Engel et al. (21) described a mechanism in which growth of islet amyloid associated polypeptides fibrils is re...

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A kinetic coupling between protein unfolding and aggregation controls time‐dependent solubility of the human myeloma antibody light chain

Džupponová

Huntošová

2020

Protein Science

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Protein aggregation is one of the most critical processes affecting protein solubility in various contexts-from protein therapeutics formulation to protein diseases. In general, time-dependent changes in protein solubility are complex kinetically driven processes that often involve a triggering event that consists of a protein unfolding/misfolding followed by the assembling of aggregationcompetent protein species. In this study, we have examined the relation between stability and time-dependent solubility of the recombinant human antibody light chain, hLC, which was found to form renal tubular casts in the multiple myeloma patient. To analyze the aggregation quantitatively, the hLC stability and protein solubility assays were performed in vitro at elevated temperatures. A differential acceleration of the processes at high temperatures enabled us to dissect observed kinetics of irreversible hLC unfolding and aggregation. We find that for hLC these processes have different molecularity and activation energy barriers. While the irreversible unfolding of hLC is a unimolecular step with a substantial activation energy barrier of 260 kJ/mol, the aggregation is rate-limited by the bimolecular reaction, which is characterized by a lower activation energy barrier of 40 kJ/mol. By the combination of experimental assays at different temperatures, different protein concentrations and kinetic modeling using ordinary differential equations, we were able to extrapolate time-dependent protein solubility to temperatures where both unfolding and aggregation processes are strongly kinetically coupled. Our study enables mechanism-based evaluation and interpretation of different physico-chemical factors contributing to the hLC unfolding and aggregation and their effect on the formation of extracellular protein deposits.

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Thermodynamic and fibril formation studies of full length immunoglobulin light chain AL-09 and its germline protein using scan rate dependent thermal unfolding

Cited by 46 publications

References 29 publications

Aggregation of Full-length Immunoglobulin Light Chains from Systemic Light Chain Amyloidosis (AL) Patients Is Remodeled by Epigallocatechin-3-gallate

Aggregation of Full-length Immunoglobulin Light Chains from Systemic Light Chain Amyloidosis (AL) Patients Is Remodeled by Epigallocatechin-3-gallate

Cell Damage in Light Chain Amyloidosis

A kinetic coupling between protein unfolding and aggregation controls time‐dependent solubility of the human myeloma antibody light chain

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