Unusual duplication mutation in a surface loop of human transthyretin leads to an aggressive drug-resistant amyloid disease.

Transthyretin (TTR) is a globular tetrameric transport protein in plasma. Nearly 140 single amino acid substitutions in TTR cause life-threatening amyloid disease. We report a one-of-a-kind pathological variant featuring a Glu51, Ser52 duplication mutation (Glu51_Ser52dup). The proband, heterozygous for the mutation, exhibited an unusually aggressive amyloidosis that was refractory to treatment with the small-molecule drug diflunisal. To understand the poor treatment response and expand therapeutic options, we explored the structure and stability of recombinant Glu51_Ser52dup. The duplication did not alter the protein secondary or tertiary structure but decreased the stability of the TTR monomer and tetramer. Diflunisal, which bound with near-micromolar affinity, partially restored tetramer stability. The duplication had no significant effect on the free energy and enthalpy of diflunisal binding, and hence on the drug-protein interactions. However, the duplication induced tryptic digestion of TTR at near-physiological conditions, releasing a C-terminal fragment 49-129 that formed amyloid fibrils under conditions in which the full-length protein did not. Such C-terminal fragments, along with the full-length TTR, comprise amyloid deposits in vivo. Bioinformatics and structural analyses suggested that increased disorder in the surface loop, which contains the Glu51_Ser52dup duplication, not only helped generate amyloid-forming fragments but also decreased structural protection in the amyloidogenic residue segment 25-34, promoting misfolding of the full-length protein. Our studies of a unique duplication mutation explain its diflunisal-resistant nature, identify misfolding pathways for amyloidogenic TTR variants, and provide therapeutic targets to inhibit amyloid fibril formation by variant TTR.

Glycosylation of Serum Clusterin in Wild-Type Transthyretin-Associated (ATTRwt) Amyloidosis: A Study of Disease-Associated Compositional Features Using Mass Spectrometry Analyses.

Wild-type transthyretin-associated (ATTRwt) amyloidosis is an age-related disease that causes heart failure in older adults. This disease frequently features cardiac amyloid fibril deposits that originate from dissociation of the tetrameric protein, transthyretin (TTR). Unlike hereditary TTR (ATTRm) amyloidosis, where amino acid replacements destabilize the native protein, in ATTRwt amyloidosis, amyloid-forming TTR lacks protein sequence alterations. The initiating cause of fibril formation in ATTRwt amyloidosis is unclear, and thus, it seems plausible that other factors are involved in TTR misfolding and unregulated accumulation of wild-type TTR fibrils. We believe that clusterin (CLU, UniProtKB P10909), a plasma circulating glycoprotein, plays a role in the pathobiology of ATTRwt amyloidosis. Previously, we have suggested a role for CLU in ATTRwt amyloidosis based on our studies showing that (1) CLU codeposits with non-native TTR in amyloid fibrils from ATTRwt cardiac tissue, (2) CLU interacts only with non-native (monomeric and aggregated) forms of TTR, and (3) CLU serum levels in patients with ATTRwt are significantly lower compared to healthy controls. In the present study, we provide comprehensive detail of compositional findings from mass spectrometry analyses of amino acid and glycan content of CLU purified from ATTRwt and control sera. The characterization of oligosaccharide content in serum CLU derived from patients with ATTRwt amyloidosis is novel data. Moreover, results comparing CLU oligosaccharide variations between patient and healthy controls are original and provide further evidence for the role of CLU in ATTRwt pathobiology, possibly linked to disease-specific structural features that limit the chaperoning capacity of CLU.

Structural Characterization of Cardiac Ex Vivo Transthyretin Amyloid: Insight into the Transthyretin Misfolding Pathway In Vivo.

Structural characterization of misfolded protein aggregates is essential to understanding the molecular mechanism of protein aggregation associated with various protein misfolding disorders. Here, we report structural analyses of ex vivo transthyretin aggregates extracted from human cardiac tissue. Comparative structural analyses of in vitro and ex vivo transthyretin aggregates using various biophysical techniques revealed that cardiac transthyretin amyloid has structural features similar to those of in vitro transthyretin amyloid. Our solid-state nuclear magnetic resonance studies showed that in vitro amyloid contains extensive nativelike ß-sheet structures, while other loop regions including helical structures are disrupted in the amyloid state. These results suggest that transthyretin undergoes a common misfolding and aggregation transition to nativelike aggregation-prone monomers that self-assemble into amyloid precipitates in vitro and in vivo.

Stabilization of Cardiac Function With Diflunisal in Transthyretin (ATTR) Cardiac Amyloidosis.

BACKGROUND: Transthyretin amyloidosis cardiomyopathy (ATTR-CM) is an underappreciated cause of heart failure that results from misfolded TTR (prealbumin) protein. Diflunisal is an approved non-steroidal anti-inflammatory drug that stabilizes TTR, with limited data available regarding effects on cardiac structure and function. METHODS AND RESULTS: ATTR-CM patients (n=81, 41% treated with 250 mg twice-daily diflunisal by clinical practice) were retrospectively identified with baseline and follow-up (median interval 1 year) serum biomarker and echocardiographic data compared, including global longitudinal strain (GLS). Chi-squared and Wilcoxon tests assessed differences between subjects, divided by treatment group, and univariable and multivariable linear regression was performed. At baseline, patients treated with diflunisal were younger (68 vs 77 years, P = .0001), with lower B-type natriuretic peptide (BNP; 249 vs 545 pg/mL, P = .009) and serum creatinine (1.1 vs 1.2 mg/dL, P = .04), but similar TTR concentration (P = .31), cardiac troponin I (P = .06), and GLS (P = .67). At follow-up, diflunisal untreated versus treated patients showed differences in TTR concentration (19 vs 33 mg/dL, P = .01) and favorable differences in left atrial volume index (+4.6 vs -1.4 mL/m2, P = .002) and cardiac troponin I (+0.03 vs -0.01 ng/mL, P = .01) for the entire cohort. Among the subset with wild-type ATTR (n=53), diflunisal treatment was associated with differences in GLS (+1.2% untreated vs +0.1% treated, P = .03). Changes in wall thickness (P = .2), left ventricular ejection fraction (P = .71), and BNP (P = .42) were similar between groups. CONCLUSIONS: In ATTR-CM, diflunisal treatment resulted in measurable differences in some parameters of cardiac structure and function after only 1 year of administration. Further longer-term analysis is warranted.

Patient outcomes in light chain (AL) amyloidosis: The clock is ticking from symptoms to diagnosis.

INTRODUCTION: Symptomology of AL amyloidosis can be vague, with a broad range of manifestations and potential etiologies. We sought to determine whether time from initial patient-reported symptom onset to diagnosis was associated with survival. METHODS: The Boston University Amyloidosis Patient Database was queried for patients with AL amyloidosis who presented to the Center for initial evaluation from 2010 to 2015. RESULTS: A total of 324 patients with AL amyloidosis were evaluated for initial evaluation. The median time to diagnosis from initial symptom onset was 7.1 months (range, 0-61). At data cutoff, 60.2% (n = 195) of patients were alive; of those, the majority were diagnosed <6 months from initial symptoms (52.3%, n = 102). In contrast, time to diagnosis from symptom onset was >6 months in 63.6% (n = 82) of patients who did not survive at the time of data cutoff (P = .0005). Survival analysis of time from diagnosis to death or data cutoff stratified by time from patient-reported symptom onset to diagnosis (<6, 6-12, and >12 months) showed significant differences among groups (P = .001). Additionally, multivariable regression demonstrated that an increase in time from self-reported symptom onset to diagnosis was significantly associated with an increased risk of death (HR = 1.02, 95% CI = 1.01-1.04, P = .002). CONCLUSION: These results support the importance of early diagnosis for patients with AL amyloidosis.

Serum Proteomic Variability Associated with Clinical Phenotype in Familial Transthyretin Amyloidosis (ATTRm).

Transthyretin (TTR), normally a plasma circulating protein, can become misfolded and aggregated, ultimately leading to extracellular deposition of amyloid fibrils usually targeted to heart or nerve tissues. Referred to as TTR-associated amyloidoses (ATTR), this group of diseases is frequently life threatening and fatal if untreated. ATTR, caused by amyloid-forming variant TTR proteins (ATTRm) that arise from point mutations in the TTR gene, were classically referred to as familial amyloid cardiomyopathy (FAC) or familial amyloid polyneuropathy (FAP), reflecting the clinical phenotype. FAC and FAP are pathologies that can be challenging to diagnose as there are no definitive biomarkers of disease; moreover, disease-specific measures of progression are lacking, and treatment options are limited. Thus, the discovery of sensitive and specific indicators of disease has the potential to improve recognition, enable accurate measurement of amyloid progression and response to treatment, and reveal key information regarding FAC and FAP pathobiological mechanisms. In this study, the goal was to investigate serum proteomic features unique to FAC and FAP types of ATTRm. Multiple-reaction monitoring mass spectrometry (MRM-MS), a powerful technique in profiling proteomes, was used to measure the serum concentrations of 160 proteins in samples from FAC and FAP patients. Results were compared to data from healthy control sera obtained from individuals matched to age (≥60 years), gender (male), and race (Caucasian). Proteomic analyses of ATTRm (FAC and FAP) and control samples showed significant concentration differences in 107 of 192 (56%) of the serum proteins that were studied. In comparing FAC to FAP, differences in concentrations as well as interactions and functions of several proteins were identified as unique to each disease; significantly lower levels of TTR were specific to FAC, but not to FAP. Annotated functional clustering identified extracellular region, signal, and signal peptide as terms common to FAC and FAP. Conversely, disulfide bond was unique to FAC; secreted, glycosylation site: N-linked, glycosylation, glycoprotein, polymorphism, and sequence variant were associated solely with FAP. Predicted protein-protein associations in FAC were seen for reaction, binding, and activation processes; no associations were found in FAP. This study demonstrates significant proteomic differences between ATTRm patient and control sera, as well as ATTRm phenotype-associated variations in the circulating levels of several proteins including TTR. The identification of serum proteins unique to FAC and FAP may have diagnostic and prognostic utility and could possibly provide important clues about disease mechanisms.

Blood Proteomic Profiling in Inherited (ATTRm) and Acquired (ATTRwt) Forms of Transthyretin-Associated Cardiac Amyloidosis.

Transthyretin-associated forms of cardiac amyloidosis are fatal protein misfolding diseases that can be inherited (ATTRm) or acquired (ATTRwt). An accurate diagnosis of ATTR amyloidosis can be challenging as biopsy evidence, usually from the affected organ, is required. Precise biomarkers for ATTR disease identification and monitoring are undiscovered, disease-specific therapeutic options are needed, and the current understanding of ATTR molecular pathogenesis is limited. The aim of this study was to investigate and compare the serum proteomes in ATTRm and ATTRwt cardiac amyloidosis to identify differentially expressed blood proteins that were disease-specific. Using multiple-reaction monitoring mass spectrometry (MRM-MS), the concentrations of 160 proteins were analyzed in serum samples from ATTRm and ATTRwt patients, and a healthy control group. Patient and control sera were matched to age (≥60 years), gender (male), and race (Caucasian). The circulating concentrations of 123/160 proteins were significantly different in patient vs control sera; TTR and retinol-binding protein (RBP4) levels were significantly decreased (p < 0.03) in ATTRm compared to controls. In ATTRm, 14/123 proteins were identified as unique to that group and found generally to be lower than controls; moreover, the concentrations of RBP4 and 6 other proteins in this group were significantly different (p < 0.04) compared to ATTRwt. Predicted interactions among the 14 proteins unique to ATTRm were categorized as reaction and binding associations. Alternatively, 27 proteins were found to be unique to ATTRwt with associated interactions defined as activation, catalysis, and inhibition, in addition to reaction and binding. This study demonstrates significant proteomic differences between ATTR patient and control sera, and disease-associated variations in circulating levels of several proteins including TTR and RBP4. The identification of serum proteins unique to ATTRm and ATTRwt cardiac amyloidosis may have diagnostic and prognostic utility, and may provide important clues about disease mechanisms.

Heart Failure Resulting From Age-Related Cardiac Amyloid Disease Associated With Wild-Type Transthyretin: A Prospective, Observational Cohort Study.

BACKGROUND: Heart failure caused by wild-type transthyretin amyloidosis (ATTRwt) is an underappreciated cause of morbidity and mortality in the aging population. The aims of this study were to examine features of disease and to characterize outcomes in a large ATTRwt cohort. METHODS AND RESULTS: Over 20 years, 121 patients with ATTRwt were enrolled in a prospective, observational study. Median age at enrollment was 75.6 years (range, 62.6-87.8 years); 97% of patients were white. The median survival, measured from biopsy diagnosis, was 46.69 months (95% confidence interval, 41.95-56.77); 78% of deaths were attributable to cardiac causes. By Kaplan-Meier analysis, 5-year survival was 35.7% (95% confidence interval, 25-46). Impaired functional capacity (mean Vo2max, 13.5 mL·kg(-1)·min(-1)) and atrial fibrillation (67%) were common clinical features. Multivariate predictors of reduced survival were elevated serum brain natriuretic peptide (482 ± 337 pg/mL) and uric acid (8.2 ± 2.6 mg/dL), decreased left ventricular ejection fraction (50% median; range, 10%-70%), and increased relative wall thickness (0.75 ± 0.19). CONCLUSIONS: In this series of patients with biopsy-proven ATTRwt, poor functional capacity and atrial arrhythmias were common clinical features. Elevated brain natriuretic peptide and uric acid, decreased left ventricular ejection fraction, and increased relative wall thickness were associated with limited survival of only 35.7% at 5 years for the group as a whole. These data establish the natural history of ATTRwt, provide statistical basis for the design of future interventional clinical trials, and highlight the need for more sensitive diagnostic tests and disease-specific treatments for this disease.

Oxidative Post-Translational Modifications of an Amyloidogenic Immunoglobulin Light Chain Protein.

Immunoglobulin light chain amyloidosis (AL) is a plasma cell disorder characterized by overproduction and deposition of monoclonal immunoglobulin (Ig) light chains (LC) or variable region fragments as amyloid fibrils in various organs and tissues. Much clinical evidence indicates that patients with AL amyloidosis sustain cardiomyocyte impairment and suffer from oxidative stress. We seek to understand the underlying biochemical pathways whose disruption or amplification during sporadic or sustained disease states leads to harmful physiological consequences and to determine the detailed structures of intermediates and products that serve as signposts for the biochemical changes and represent potential biomarkers. In this study, matrix-assisted laser desorption/ionization mass spectrometry provided extensive evidence for oxidative post-translational modifications (PTMs) of an amyloidogenic Ig LC protein from a patient with AL amyloidosis. Some of the tyrosine residues were heavily mono- or di-chlorinated. In addition, a novel oxidative conversion to a nitrile moiety was observed for many of the terminal aminomethyl groups on lysine side chains. In vitro experiments using model peptides, in-solution oxidation, and click chemistry demonstrated that hypochlorous acid produced by the myeloperoxidase - hydrogen peroxide - chloride system could be responsible for these and other, more commonly observed modifications.

Cooperative stabilization of transthyretin by clusterin and diflunisal.

The circulating protein transthyretin (TTR) can unfold, oligomerize, and form highly structured amyloid fibrils that are deposited in tissues, causing organ damage and disease. This pathogenic process is caused by a heritable TTR point mutation in cases of familial TTR-related amyloidosis or wild-type TTR in cases of age-associated amyloidosis (previously called senile systemic amyloidosis). The TTR amyloid cascade is hypothesized to begin with the dissociation of the TTR native tetrameric structure into folded but unstable monomeric TTR subunits. Unfolding of monomeric TTR initiates an oligomerization process leading to aggregation and fibril formation. Numerous proteostatic mechanisms for regulating the TTR amyloid cascade exist. Extracellular chaperones provide an innate defense against misfolded proteins. Clusterin (CLU), a plasma protein, has the capacity to recognize exposed hydrophobic regions of misfolded proteins, shielding them from aggregation. We have previously demonstrated that CLU is associated with the amyloid fibrils in cardiac tissues from patients with TTR amyloidosis. In this study, we have used tetrameric and monomeric TTR structural variants to determine the ability of CLU to inhibit TTR amyloid fibril formation. Using circular dichroism spectroscopy, we determined that CLU preferentially stabilizes monomeric TTR and generates increasingly stable conformations under acid stress. Moreover, studies using surface plasmon resonance showed a direct interaction of CLU with high-molecular weight TTR oligomers. The interactions of CLU with monomeric and aggregated TTR proceed in a cooperative manner in the presence of diflunisal, a small molecule drug used to stabilize TTR tetramers.

Genetic variation of the transthyretin gene in wild-type transthyretin amyloidosis (ATTRwt).

Wild-type transthyretin amyloidosis (ATTRwt), typically diagnosed as congestive heart failure in elderly Caucasian men, features myocardial amyloid deposits of wild-type plasma protein transthyretin (TTR). ATTRwt is sporadic, its pathogenesis is poorly understood, and currently there are no biomarkers for diagnosis or prognosis. Genetic studies of variant-associated transthyretin amyloidosis have suggested that non-coding TTR gene variants modulate disease. We hypothesized that cis-acting regulatory elements in the TTR gene non-coding regions may modify expression, affecting ATTRwt onset and progression. We studied an ATTRwt cohort consisting of 108 Caucasian males ranging in age from 59 to 87 years with cardiomyopathy due to wild-type TTR deposition; results were compared to 118 anonymous controls matched by age, sex, and race. Four predicted non-coding regulatory regions and all exons in the TTR gene were sequenced using the Sanger method. Eleven common variants were identified; three variants were significantly associated with ATTRwt (p < 0.05), though only one, rs72922940, remained near significance (p corrected = 0.083) after multiple testing correction. Exon analyses demonstrated the occurrence of the p.G26S (G6S) polymorphism in 7 % of ATTRwt subjects and 12 % of controls; this variant was predicted to be a protective factor (p = 0.051). Four variants were significantly associated with age at onset and survival. In this first genetic study of a large, well-characterized cohort of ATTRwt, non-coding and coding variants associated with disease, age at onset, and survival were identified. Further investigation is warranted to determine the prevalence of these variants in ATTRwt, their regulatory function, and potential role in assessing disease risk.

Human amyloidogenic light chain proteins result in cardiac dysfunction, cell death, and early mortality in zebrafish.

Systemic amyloid light-chain (AL) amyloidosis is associated with rapidly progressive and fatal cardiomyopathy resulting from the direct cardiotoxic effects of circulating AL light chain (AL-LC) proteins and the indirect effects of AL fibril tissue infiltration. Cardiac amyloidosis is resistant to standard heart failure therapies, and, to date, there are limited treatment options for these patients. The mechanisms underlying the development of cardiac amyloidosis and AL-LC cardiotoxicity are largely unknown, and their study has been limited by the lack of a suitable in vivo model system. Here, we establish an in vivo zebrafish model of human AL-LC-induced cardiotoxicity. AL-LC isolated from AL cardiomyopathy patients or control nonamyloidogenic LC protein isolated from multiple myeloma patients (Con-LC) was directly injected into the circulation of zebrafish at 48 h postfertilization. AL-LC injection resulted in impaired cardiac function, pericardial edema, and increased cell death relative to Con-LC, culminating in compromised survival with 100% mortality within 2 wk, independent of AL fibril deposition. Prior work has implicated noncanonical p38 MAPK activation in the pathogenesis of AL-LC-induced cardiotoxicity, and p38 MAPK inhibition via SB-203580 rescued AL-LC-induced cardiac dysfunction and cell death and attenuated mortality in zebrafish. This in vivo zebrafish model of AL-LC cardiotoxicity demonstrates that antagonism of p38 MAPK within the AL-LC cardiotoxic signaling response may serve to improve cardiac function and mortality in AL cardiomyopathy. Furthermore, this in vivo model system will allow for further study of the molecular underpinnings of AL cardiotoxicity and identification of novel therapeutic strategies.

Stanniocalcin1 is a key mediator of amyloidogenic light chain induced cardiotoxicity.

Immunoglobulin light chain (LC) amyloidosis (AL) results from overproduction of circulating amyloidogenic LC proteins and subsequent amyloid fibril deposition in organs. Mortality in AL amyloidosis patients is highly associated with a rapidly progressive AL cardiomyopathy, marked by profound impairment of diastolic and systolic cardiac function and significant early mortality. While myocardial fibril deposition contributes to the severe diastolic dysfunction seen in AL cardiomyopathy patients, the degree of fibril deposition has not been found to correlate with prognosis. Previously, we and others showed a direct cardiotoxic effect of amyloidogenic LC proteins (AL-LC), which may contribute to the pathophysiology and mortality observed in AL cardiomyopathy patients. However, the mechanisms underlying AL-LC related cardiotoxicity remain unknown. Mammalian stanniocalcin1 (STC1) is associated with a number of cellular processes including oxidative stress and cell death. Herein, we find that STC1 expression is elevated in cardiac tissue from AL cardiomyopathy patients, and is induced in isolated cardiomyocytes in response to AL-LC, but not non-amyloidogenic LC. STC1 overexpression in vitro recapitulates the pathophysiology of AL-LC mediated cardiotoxicity, with increased ROS production, contractile dysfunction and cell death. Overexpression of STC1 in vivo results in significant cardiac dysfunction and cell death. Genetic silencing of STC1 prevents AL-LC induced cardiotoxicity in cardiomyocytes and protects against AL-LC induced cell death and early mortality in zebrafish. The cardiotoxic effects of STC1 appears to be mediated via mitochondrial dysfunction as indicated by loss of mitochondrial membrane potential, ROS production and increased mitochondrial calcium levels. Collectively, this work identifies STC1 as a critical determinant of AL-LC cardiotoxicity.

Doxycycline reduces fibril formation in a transgenic mouse model of AL amyloidosis.

Systemic AL amyloidosis results from the aggregation of an amyloidogenic immunoglobulin (Ig) light chain (LC) usually produced by a plasma cell clone in the bone marrow. AL is the most rapidly fatal of the systemic amyloidoses, as amyloid fibrils can rapidly accumulate in tissues including the heart, kidneys, autonomic or peripheral nervous systems, gastrointestinal tract, and liver. Chemotherapy is used to eradicate the cellular source of the amyloidogenic precursor. Currently, there are no therapies that target the process of LC aggregation, fibril formation, or organ damage. We developed transgenic mice expressing an amyloidogenic λ6 LC using the cytomegalovirus (CMV) promoter to circumvent the disruption of B cell development by premature expression of recombined LC. The CMV-λ6 transgenic mice develop neurologic dysfunction and Congophilic amyloid deposits in the stomach. Amyloid deposition was inhibited in vivo by the antibiotic doxycycline. In vitro studies demonstrated that doxycycline directly disrupted the formation of recombinant LC fibrils. Furthermore, treatment of ex vivo LC amyloid fibrils with doxycycline reduced the number of intact fibrils and led to the formation of large disordered aggregates. The CMV-λ6 transgenic model replicates the process of AL amyloidosis and is useful for testing the antifibril potential of orally available agents.

Amyloidogenic light chains induce cardiomyocyte contractile dysfunction and apoptosis via a non-canonical p38alpha MAPK pathway.

Patients with primary (AL) cardiac amyloidosis suffer from progressive cardiomyopathy with a median survival of less than 8 months and a 5-year survival of <10%. Contributing to this poor prognosis is the fact that these patients generally do not tolerate standard heart failure therapies. The molecular mechanisms underlying this deadly form of heart disease remain unclear. Although interstitial amyloid fibril deposition of Ig light chain proteins is a major cause of cardiac dysfunction in AL cardiac amyloidosis, we have previously shown that amyloid precursor proteins directly impair cardiac function at the cellular and isolated organ levels, independent of fibril formation. In this study, we report that amyloidogenic light chain (AL-LC) proteins provoke oxidative stress, cellular dysfunction, and apoptosis in isolated adult cardiomyocytes through activation of p38 mitogen-activated protein kinase (MAPK). AL-LC-induced p38 activation was found to be independent of the upstream MAPK kinase, MKK3/6, and instead depends upon transforming growth factor-beta-activated protein kinase-1 binding protein-1 (TAB1)-mediated p38alpha MAPK autophosphorylation. Treatment of cardiomyocytes with SB203580, a selective p38 MAPK inhibitor, significantly attenuated AL-LC-induced oxidative stress, cellular dysfunction, and apoptosis. Our data provide a unique mechanistic insight into the pathogenesis of AL-LC cardiac toxicity and suggest that TAB1-mediated p38alpha MAPK autophosphorylation may serve as an important event leading to cardiac dysfunction and subsequent heart failure.

Role of Complementarity-Determining Regions 1 and 3 in Pathologic Amyloid Formation by Human Immunoglobulin κ1 Light Chains.

Immunoglobulin light chain (LC) amyloidosis is a life-threatening disease whose understanding and treatment is complicated by vast numbers of patient-specific mutations. To address molecular origins of the disease, we explored 14 patient-derived and engineered proteins related to κ1-family germline genes IGKVLD-33*01 and IGKVLD-39*01. Hydrogen-deuterium exchange mass spectrometry analysis of local conformational dynamics in full-length recombinant LCs and their fragments was integrated with studies of thermal stability, proteolytic susceptibility, amyloid formation, and amyloidogenic sequence propensities using spectroscopic, electron microscopic and bioinformatics tools. The results were mapped on the atomic structures of native and fibrillary proteins. Proteins from two κ1 subfamilies showed unexpected differences. Compared to their germline counterparts, amyloid LC related to IGKVLD-33*01 was less stable and formed amyloid faster, whereas amyloid LC related to IGKVLD-39*01 had similar stability and formed amyloid slower. These and other differences suggest different major factors influencing amyloid formation. In 33*01-related amyloid LC, these factors involved mutation-induced destabilization of the native structure and probable stabilization of amyloid. The atypical behaviour of 39*01-related amyloid LC tracked back to increased dynamics/exposure of amyloidogenic segments in ßC' V and ßE V that could initiate aggregation, combined with decreased dynamics/exposure near the Cys23-Cys88 disulfide whose rearrangement is rate-limiting to amyloidogenesis. The results suggest distinct amyloidogenic pathways for closely related LCs and point to the antigen-binding, complementarity-determining regions CDR1 and CDR3, which are linked via the conserved internal disulfide, as key factors in amyloid formation by various LCs.

Role of complementarity-determining regions 1 and 3 in pathologic amyloid formation by human immunoglobulin κ1 light chains.

BACKGROUND: Immunoglobulin light chain (LC) amyloidosis is a life-threatening disease complicated by vast numbers of patient-specific mutations. We explored 14 patient-derived and engineered proteins related to κ1-family germline genes IGKVLD-33*01 and IGKVLD-39*01. METHODS: Hydrogen-deuterium exchange mass spectrometry analysis of conformational dynamics in recombinant LCs and their fragments was integrated with studies of thermal stability, proteolytic susceptibility, amyloid formation and amyloidogenic sequence propensity. The results were mapped on the structures of native and fibrillary proteins. RESULTS: Proteins from two κ1 subfamilies showed unexpected differences. Compared to their germline counterparts, amyloid LC related to IGKVLD-33*01 was less stable and formed amyloid faster, whereas amyloid LC related to IGKVLD-39*01 had similar stability and formed amyloid slower, suggesting different major factors influencing amyloidogenesis. In 33*01-related amyloid LC, these factors involved destabilization of the native structure and probable stabilization of amyloid. The atypical behavior of 39*01-related amyloid LC stemmed from increased dynamics/exposure of amyloidogenic segments in ßC'V and ßEV that could initiate aggregation and decreased dynamics/exposure near the Cys23-Cys88 disulfide. CONCLUSIONS: The results suggest distinct amyloidogenic pathways for closely related LCs and point to the complementarity-defining regions CDR1 and CDR3, linked via the conserved internal disulfide, as key factors in amyloid formation.

Mapping cellular response to destabilized transthyretin reveals cell- and amyloidogenic protein-specific signatures.

BACKGROUND: In ATTR amyloidosis, transthyretin (TTR) protein is secreted from the liver and deposited as toxic aggregates at downstream target tissues. Despite recent advancements in treatments for ATTR amyloidosis, the mechanisms underlying misfolded TTR-mediated cellular damage remain elusive. METHODS: In an effort to define early events of TTR-associated stress, we exposed neuronal (SH-SY5Y) and cardiac (AC16) cells to wild-type and destabilized TTR variants (TTRV122I (p.V142I) and TTRL55P (p.L70P)) and performed transcriptional (RNAseq) and epigenetic (ATACseq) profiling. We subsequently compared TTR-responsive signatures to cells exposed to destabilized antibody light chain protein associated with AL amyloidosis as well as ER stressors (thapsigargin, heat shock). RESULTS: In doing so, we observed overlapping, yet distinct cell type- and amyloidogenic protein-specific signatures, suggesting unique responses to each amyloidogenic variant. Moreover, we identified chromatin level changes in AC16 cells exposed to mutant TTR that resolved upon pre-incubation with kinetic stabilizer tafamidis. CONCLUSIONS: Collectively, these data provide insight into the mechanisms underlying destabilized protein-mediated cellular damage and provide a robust resource representing cellular responses to aggregation-prone proteins and ER stress.

An additive destabilising effect of compound T60I and V122I substitutions in ATTRv amyloidosis.

BACKGROUND: The amyloidogenic transthyretin (TTR) variant, V122I, occurs in 4% of the African American population and frequently presents as a restricted cardiomyopathy. While heterozygosity for TTR V122I predominates, several compound heterozygous cases have been previously described. Herein, we detail features of ATTRv amyloidosis associated with novel compound heterozygous TTR mutation, T60I/V122I and provide evidence supporting the amyloidogenecity of T60I. METHODS: A 63-year-old African American female presented with atrial fibrillation, congestive heart failure, autonomic and peripheral neuropathy. In vitro studies of TTR T60I and V122I were undertaken to compare the biophysical properties of the proteins. RESULTS: Congophilic deposits in a rectal biopsy were immunohistochemically positive for TTR. Serum screening by isoelectric focussing revealed two TTR variants in the absence of wild-type protein. DNA sequencing identified compound heterozygous TTR gene mutations, c.239C > T and c.424G > A. Adipose amyloid deposits were composed of both T60I and V122I. While kinetic stabilities of T60I and V122I variants were similar, distinct thermodynamic stabilities and amyloid growth kinetics were observed. CONCLUSIONS: This report provides clinical and experimental results supporting the amyloidogenic nature of a novel TTR T60I variant. In vitro data indicate that the destabilising effect of individual T60I and V122I variants appears to be additive rather than synergistic.

Clinical Penetrance of the Transthyretin V122I Variant in Older Black Patients With Heart Failure: The SCAN-MP (Screening for Cardiac Amyloidosis With Nuclear Imaging in Minority Populations) Study.

Background Transthyretin amyloid cardiomyopathy (ATTR-CM) is an underdiagnosed cause of heart failure (HF) among patients ≥60 years of age. Although the V122I (valine to isoleucine substitution at position 122 of the transthyretin protein) variant associated with hereditary ATTR-CM is present in 3.4% of self-identified Black individuals in the United States (or 1.5 million people), the phenotypic penetrance is not known. Methods and Results The SCAN-MP (Screening for Cardiac Amyloidosis With Nuclear Imaging in Minority Populations) study is a currently accruing prospective multisite study designed to determine the prevalence of ATTR-CM using technetium-99m-pyrophosphate imaging in older (≥60 years of age) self-identified Black and Hispanic individuals with HF. Calculations of the penetrance and prevalence of the V122I allele, along with analyses of functional, biochemical, and echocardiographic parameters, were performed for the first 278 Black participants in SCAN-MP. The prevalence of ATTR-CM was 6.8% (95% CI, 4.2-10.5; n=19 cases), of whom 63% were ATTR wild-type. The prevalence of V122I was 6.5% (n=18 carriers), of whom 7 had ATTR-CM, yielding a phenotypic penetrance of 39% (95% CI, 17-64). V122I carriers with ATTR-CM evidenced more advanced HF than carriers without ATTR-CM. Prealbumin concentration was lowest among V122I carriers with ATTR-CM (12.9 mg/dL) versus carriers without ATTR-CM (21.0 mg/dL) and HF controls (25.0 mg/dL, P<0.0001). Conclusions Among older Black individuals with HF and increased left ventricular wall thickness, of those with ATTR-CM, 63% had wild-type, and of those with V122I, the phenotypic penetrance of ATTR-CM was 39% (95% CI, 17-64), suggesting that genotype alone is insufficient for diagnosis. Prealbumin concentration may be useful to identify V122I carriers with ATTR-CM. Registration URL: https://www.clinicaltrials.gov; Unique identifier: NCT03812172.