Molecular basis for non-invasive diagnostics of cardiac amyloids using bone tracers.

Amyloid diseases including Alzheimer's, Parkinson's and over 30 others are incurable life-threatening disorders caused by abnormal protein deposition as fibrils in various organs. Cardiac amyloidosis is particularly challenging to diagnose and treat. Identification of the fibril-forming protein, which in the heart is usually amyloid transthyretin (ATTR) or amyloid immunoglobulin light chain (AL), is paramount to treatment. A transformative non-invasive diagnostic modality is imaging using technetium-labeled pyrophosphate or diphosphonate bone tracers, 99mTc-PYP/DPD/HMDP. For unknown reasons, these tracers show preferential uptake by ATTR deposits. The tracer-binding moiety is unknown and potentially involves amyloid fibrils and/or amyloid-associated calcific deposits. We propose that, like in the bone, the tracers chelate to surface-bound Ca2+ in amyloid. In high-affinity protein sites, Ca2+ is coordinated by pairs of acidic residues. To identify such residues on amyloids, we harnessed atomic structures of patient-derived cardiac amyloids determined using cryogenic electron microscopy since 2019. These structures help explain why most but not all ATTR deposits uptake 99mTc-PYP/DPD/HMDP radiotracers, while in AL the opposite is true. Moreover, fibril structures help explain greater microcalcification observed in ATTR vs. AL deposits. These findings may aid the diagnostics and therapeutic targeting of cardiac amyloidosis and are relevant to other amyloids.

LDL binding to cell receptors and extracellular matrix is proatherogenic in obesity but improves after bariatric surgery.

Obesity is a major global public health issue involving dyslipidemia, oxidative stress, inflammation, and increased risk of CVD. Weight loss reduces this risk, but the biochemical underpinnings are unclear. We explored how obesity and weight loss after bariatric surgery influence LDL interactions that trigger proatherogenic versus antiatherogenic processes. LDL was isolated from plasma of six patients with severe obesity before (basal) and 6-12 months after bariatric surgery (basal BMI = 42.7 kg/m2; 6-months and 12-months postoperative BMI = 34.1 and 30 kg/m2). Control LDL were from six healthy subjects (BMI = 22.6 kg/m2). LDL binding was quantified by ELISA; LDL size and charge were assessed by chromatography; LDL biochemical composition was determined. Compared to controls, basal LDL showed decreased nonatherogenic binding to LDL receptor, which improved postoperatively. Conversely, basal LDL showed increased binding to scavenger receptors LOX1 and CD36 and to glycosaminoglycans, fibronectin and collagen, which is proatherogenic. One year postoperatively, this binding decreased but remained elevated, consistent with elevated lipid peroxidation. Serum amyloid A and nonesterified fatty acids were elevated in basal and postoperative LDL, indicating obesity-associated inflammation. Aggregated and electronegative LDL remained elevated, suggesting proatherogenic processes. These results suggest that obesity-induced inflammation contributes to harmful LDL alterations that probably increase the risk of CVD. We conclude that in obesity, LDL interactions with cell receptors and extracellular matrix shift in a proatherogenic manner but are partially reversed upon postoperative weight loss. These results help explain why the risk of CVD increases in obesity but decreases upon weight loss.

Lipid clearance and amyloid formation by serum amyloid A: exploring the links between beneficial and pathologic actions of an enigmatic protein.

Serum amyloid A (SAA) is named after a life-threatening disease, yet this small evolutionarily conserved protein must have played a vital role in host defense. Most circulating SAA binds plasma lipoproteins and modulates their metabolism. However, this hardly justifies the rapid and dramatic SAA upregulation in inflammation, which is concomitant with upregulation of secretory phospholipase A2 (sPLA2). We proposed that these proteins synergistically clear cell membrane debris from the sites of injury. The present study uses biochemical and biophysical approaches to further explore the beneficial function of SAA and its potential links to amyloid formation. We show that murine and human SAA1 are powerful detergents that solubilize diverse lipids, including mammalian biomembranes, converting them into lipoprotein-size nanoparticles. These nanoparticles provide ligands for cell receptors, such as scavenger receptor CD36 or heparin/heparan sulfate, act as substrates of sPLA2, and sequester toxic products of sPLA2. Together, these functions enable SAA to rapidly clear unprotected lipids. SAA can also adsorb, without remodeling, to lipoprotein-size nanoparticles such as exosomal liposomes, which are proxies for lipoproteins. SAA in complexes with zwitterionic phospholipids stabilizes α-helices, while SAA in complexes containing anionic lipids or micelle-forming sPLA2 products forms metastable ß-sheet-rich species that readily aggregate to form amyloid. Consequently, the synergy between SAA and sPLA2 extends from the beneficial lipid clearance to the pathologic amyloid formation. Furthermore, we show that lipid composition alters SAA conformation and thereby can influence the metabolic fate of SAA-lipid complexes, including their proamyloidogenic and proatherogenic binding to heparan sulfate.

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.

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.

The contribution of amyloid deposition in the aortic valve to calcification and aortic stenosis.

Calcific aortic valve disease (CAVD) and stenosis have a complex pathogenesis, and no therapies are available that can halt or slow their progression. Several studies have shown the presence of apolipoprotein-related amyloid deposits in close proximity to calcified areas in diseased aortic valves. In this Perspective, we explore a possible relationship between amyloid deposits, calcification and the development of aortic valve stenosis. These amyloid deposits might contribute to the amplification of the inflammatory cycle in the aortic valve, including extracellular matrix remodelling and myofibroblast and osteoblast-like cell proliferation. Further investigation in this area is needed to characterize the amyloid deposits associated with CAVD, which could allow the use of antisense oligonucleotides and/or isotype gene therapies for the prevention and/or treatment of CAVD.

Dynamic protein structures in normal function and pathologic misfolding in systemic amyloidosis.

Dynamic and disordered regions in native proteins are often critical for their function, particularly in ligand binding and signaling. In certain proteins, however, such regions can contribute to misfolding and pathologic deposition as amyloid fibrils in vivo. For example, dynamic and disordered regions can promote amyloid formation by destabilizing the native structure, by directly triggering the aggregation, by promoting protein condensation, or by acting as sites of early proteolytic cleavage that favor a release of aggregation-prone fragments or facilitate fibril maturation. At the same time, enhanced dynamics in the native protein state accelerates proteolytic degradation that counteracts amyloid accumulation in vivo. Therefore, the functional need for dynamic protein regions must be balanced against their inherently labile nature. How exactly this balance is achieved and how is it shifted upon amyloidogenic mutations or post-translational modifications? To illustrate possible scenarios, here we review the beneficial and pathologic roles of dynamic and disordered regions in the native states of three families of human plasma proteins that form amyloid precursors in systemic amyloidoses: immunoglobulin light chain, apolipoproteins, and serum amyloid A. Analysis of structure, stability and local dynamics of these diverse proteins and their amyloidogenic variants exemplifies how disordered/dynamic regions can provide a functional advantage as well as an Achilles heel in pathologic amyloid formation.

A Conservative Point Mutation in a Dynamic Antigen-binding Loop of Human Immunoglobulin λ6 Light Chain Promotes Pathologic Amyloid Formation.

Immunoglobulin light chain (LC) amyloidosis (AL) is a life-threatening human disease wherein free mono-clonal LCs deposit in vital organs. To determine what makes some LCs amyloidogenic, we explored patient-based amyloidogenic and non-amyloidogenic recombinant LCs from the λ6 subtype prevalent in AL. Hydrogen-deuterium exchange mass spectrometry, structural stability, proteolysis, and amyloid growth studies revealed that the antigen-binding CDR1 loop is the least protected part in the variable domain of λ6 LC, particularly in the AL variant. N32T substitution in CRD1 is identified as a driver of amyloid formation. Substitution N32T increased the amyloidogenic propensity of CDR1 loop, decreased its protection in the native structure, and accelerated amyloid growth in the context of other AL substitutions. The destabilizing effects of N32T propagated across the molecule increasing its dynamics in regions ∼30 Å away from the substitution site. Such striking long-range effects of a conservative point substitution in a dynamic surface loop may be relevant to Ig function. Comparison of patient-derived and engineered proteins showed that N32T interactions with other substitution sites must contribute to amyloidosis. The results suggest that CDR1 is critical in amyloid formation by other λ6 LCs.

Protein Amyloid Cofactors: Charged Side-Chain Arrays Meet Their Match?

Recent advances in high-resolution structural studies of protein amyloids have revealed parallel in-register cross-ß-sheets with periodic arrays of closely spaced identical residues. What do these structures tell us about the mechanisms of action of common amyloid-promoting factors, such as heparan sulfate (HS), nucleic acids, polyphosphates, anionic phospholipids, and acidic pH?

Structural Basis for Vital Function and Malfunction of Serum Amyloid A: an Acute-Phase Protein that Wears Hydrophobicity on Its Sleeve.

PURPOSE OF REVIEW: This review addresses normal and pathologic functions of serum amyloid A (SAA), an enigmatic biomarker of inflammation and protein precursor of AA amyloidosis, a life-threatening complication of chronic inflammation. SAA is a small, highly evolutionarily conserved acute-phase protein whose plasma levels increase up to one thousand-fold in inflammation, infection, or after trauma. The advantage of this dramatic but transient increase is unclear, and the complex role of SAA in immune response is intensely investigated. This review summarizes recent advances in our understanding of the structure-function relationship of this intrinsically disordered protein, outlines its newly emerging beneficial roles in lipid transport and inflammation control, and discusses factors that critically influence its misfolding in AA amyloidosis. RECENT FINDINGS: High-resolution structures of lipid-free SAA in crystals and fibrils have been determined by x-ray crystallography and electron cryo-microscopy. Low-resolution structural studies of SAA-lipid complexes, together with biochemical, cell-based, animal model, genetic, and clinical studies, have provided surprising new insights into a wide range of SAA functions. An emerging vital role of SAA is lipid encapsulation to remove cell membrane debris from sites of injury. The structural basis for this role has been proposed. The lysosomal origin of AA amyloidosis has solidified, and its molecular and cellular mechanisms have emerged. Recent studies have revealed molecular underpinnings for understanding complex functions of this Cambrian protein in lipid transport, immune response, and amyloid formation. These findings help guide the search for much-needed targeted therapies to block the protein deposition in AA amyloidosis.

Novel clinical manifestations and treatment of hereditary apoA-I amyloidosis: when a good protein turns bad.

Amyloidoses are life-threatening diseases caused by the deposition of various proteins including apolipoprotein A-I, the major protein of plasma high-density lipoprotein. Timely diagnostics of amyloidoses are crucial for their treatment. Colombat et al. reported novel aspects of the hereditary apolipoprotein A-I amyloidosis, including its unexpected clinical presentation and genetic origins, as well as life- and vision-saving hepatorenal transplantation. This study improves the diagnostics of apolipoprotein A-I amyloidosis, optimizes its treatment, and expands our understanding of the molecular basis of this multipronged disease.

Molecular Insights into Human Hereditary Apolipoprotein A-I Amyloidosis Caused by the Glu34Lys Mutation.

Hereditary apolipoprotein A-I (apoA-I) amyloidosis is a life-threatening incurable genetic disorder whose molecular underpinnings are unclear. In this disease, variant apoA-I, the major structural and functional protein of high-density lipoprotein, is released in a free form, undergoes an α-helix to intermolecular cross-ß-sheet conversion along with a proteolytic cleavage, and is deposited as amyloid fibrils in various organs, which can cause organ damage and death. Glu34Lys is the only known charge inversion mutation in apoA-I that causes human amyloidosis. To elucidate the structural underpinnings of the amyloidogenic behavior of Glu34Lys apoA-I, we generated its recombinant globular N-terminal domain (residues 1-184) and compared the conformation and dynamics of its lipid-free form with those of two other naturally occurring apoA-I variants, Phe71Tyr (amyloidogenic) and Leu159Arg (non-amyloidogenic). All variants showed reduced structural stability and altered aromatic residue packing. The greatest decrease in stability was observed in the non-amyloidogenic variant, suggesting that amyloid formation is driven by local structural perturbations at sensitive sites. Molecular dynamics simulations revealed local helical unfolding and suggested that transient opening of the Trp72 side chain induced mutation-dependent structural perturbations in a sensitive region, including the major amyloid hot spot residues Leu14-Leu22. We posit that a shift from the "closed" to the "open" orientation of the Trp72 side chain modulates structural protection of amyloid hot spots, suggesting a previously unknown early step in the protein misfolding pathway.

Effects of Disease-Causing Mutations on the Conformation of Human Apolipoprotein A-I in Model Lipoproteins.

Plasma high-density lipoproteins (HDLs) are protein-lipid nanoparticles that transport lipids and protect against atherosclerosis. Human apolipoprotein A-I (apoA-I) is the principal HDL protein whose mutations can cause either aberrant lipid metabolism or amyloid disease. Hydrogen-deuterium exchange (HDX) mass spectrometry (MS) was used to study the apoA-I conformation in model discoidal lipoproteins similar in size to large plasma HDL. We examined how point mutations associated with hereditary amyloidosis (F71Y and L170P) or atherosclerosis (L159R) influence the local apoA-I conformation in model lipoproteins. Unlike other apoA-I forms, the large particles showed minimal conformational heterogeneity, suggesting a fully extended protein conformation. Mutation-induced structural perturbations in lipid-bound protein were attenuated compared to the free protein and indicated close coupling between the two belt-forming apoA-I molecules. These perturbations propagated to distant lipoprotein sites, either increasing or decreasing their protection. This HDX MS study of large model HDL, compared with previous studies of smaller particles, ascertained that apoA-I's central region helps accommodate the protein conformation to lipoproteins of various sizes. This study also reveals that the effects of mutations on lipoprotein conformational dynamics are much weaker than those in a lipid-free protein. Interestingly, the mutation-induced perturbations propagate to distant sites nearly 10 nm away and alter their protection in ways that cannot be predicted from the lipoprotein structure and stability. We propose that long-range mutational effects are mediated by both protein and lipid and can influence lipoprotein functionality.

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.

Serum amyloid A sequesters diverse phospholipids and their hydrolytic products, hampering fibril formation and proteolysis in a lipid-dependent manner.

Serum amyloid A action in immune response and deposition in inflammation-linked amyloidosis involve SAA-lipid interactions. We show that SAA sequesters neutral and anionic phospholipids and their hydrolytic products to form nanoparticles, suggesting a synergy with phospholipase A2. The lipid charge and shape affect SAA protection from proteolysis, aggregation and fibrillogenesis.

Serum amyloid A forms stable oligomers that disrupt vesicles at lysosomal pH and contribute to the pathogenesis of reactive amyloidosis.

Serum amyloid A (SAA) is an acute-phase plasma protein that functions in innate immunity and lipid homeostasis. SAA is a protein precursor of reactive AA amyloidosis, the major complication of chronic inflammation and one of the most common human systemic amyloid diseases worldwide. Most circulating SAA is protected from proteolysis and misfolding by binding to plasma high-density lipoproteins. However, unbound soluble SAA is intrinsically disordered and is either rapidly degraded or forms amyloid in a lysosome-initiated process. Although acidic pH promotes amyloid fibril formation by this and many other proteins, the molecular underpinnings are unclear. We used an array of spectroscopic, biochemical, and structural methods to uncover that at pH 3.5-4.5, murine SAA1 forms stable soluble oligomers that are maximally folded at pH 4.3 with ∼35% α-helix and are unusually resistant to proteolysis. In solution, these oligomers neither readily convert into mature fibrils nor bind lipid surfaces via their amphipathic α-helices in a manner typical of apolipoproteins. Rather, these oligomers undergo an α-helix to ß-sheet conversion catalyzed by lipid vesicles and disrupt these vesicles, suggesting a membranolytic potential. Our results provide an explanation for the lysosomal origin of AA amyloidosis. They suggest that high structural stability and resistance to proteolysis of SAA oligomers at pH 3.5-4.5 help them escape lysosomal degradation, promote SAA accumulation in lysosomes, and ultimately damage cellular membranes and liberate intracellular amyloid. We posit that these soluble prefibrillar oligomers provide a missing link in our understanding of the development of AA amyloidosis.

In vitro co-expression of human amyloidogenic immunoglobulin light and heavy chain proteins: a relevant cell-based model of AL amyloidosis.

Immunoglobulin (Ig) light chain (LC) amyloidosis (AL) is characterized by the overproduction and tissue deposition of monoclonal LC in various organs and tissues. The plasma circulating monoclonal LC is believed to be the precursor of the deposited protein and in vitro studies aimed at understanding AL pathobiology have mainly focused on LC and its variable domain. While 33% of patients have free circulating monoclonal LC, ∼40% feature LC complexed to heavy chain (HC) forming a monoclonal intact Ig; the significance of free vs. bound LC in the amyloid forming pathway is unknown. To address this issue, we developed a cell-based model using stable mouse plasmacytoma Sp2/0 cells that co-express patient-derived amyloidogenic LC and HC proteins. The system was designed using amyloidogenic kappa and lambda LC, and gamma HC sequences; stable production and secretion of either free LC and/or intact Ig were accomplished by varying the LC to HC ratios. This novel cell-based system provides a relevant tool to systematically investigate LC and HC interactions, and the molecular events leading to the development of AL amyloidosis.