RESUMO
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.
Assuntos
Amiloide , Humanos , Amiloide/metabolismo , Amiloide/química , Osso e Ossos/metabolismo , Osso e Ossos/diagnóstico por imagem , Amiloidose/metabolismo , Amiloidose/diagnóstico por imagem , Amiloidose/diagnóstico , Pré-Albumina/química , Pré-Albumina/metabolismo , Miocárdio/metabolismo , Cálcio/metabolismoRESUMO
Calcific aortic valve disease (CAVD) is a widely prevalent heart disorder in need of pharmacological interventions. Calcified areas in aortic valves often contain amyloid fibrils that promote calcification in vitro. This opinion paper suggests that amyloid contributes to CAVD development; amyloid-assisted nucleation can accelerate hydroxyapatite deposition onto collagen matrix. Notably, acidic arrays in amyloid match calcium-calcium spacing in the amorphous hydroxyapatite precursor, while oscillating hemodynamic perturbations promote amyloid deposition in the valve. Lipoprotein(a), a genetic risk factor for CAVD, augments calcification via several mechanisms, wherein hydrolysis of oxidized phospholipids (oxPLs) by Lp(a)-associated enzymes helps generate orthophosphate, and apolipoprotein(a) blocks plasmin-induced fibril degradation. Current studies of amyloid-calcium-collagen interactions in solution and in fibrillar complexes allow deeper insight into the role of amyloid in calcification.
RESUMO
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.
Assuntos
Cirurgia Bariátrica , Doenças Cardiovasculares , Humanos , Receptores de LDL/metabolismo , Obesidade/cirurgia , Inflamação , Matriz Extracelular/metabolismo , Redução de Peso , Lipoproteínas LDL/metabolismoRESUMO
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.
Assuntos
Fosfolipases A2 Secretórias , Proteína Amiloide A Sérica , Humanos , Camundongos , Animais , Proteína Amiloide A Sérica/metabolismo , Lipoproteínas , Fosfolipídeos , Fosfolipases A2 Secretórias/metabolismo , Heparitina Sulfato , Mamíferos/metabolismoRESUMO
Hydrolysis of VLDL triacylglycerol (TG) by lipoprotein lipase (LpL) is a major step in energy metabolism and VLDL-to-LDL maturation. Most functional LpL is anchored to the vascular endothelium, yet a small amount circulates on TG-rich lipoproteins. As circulating LpL has low catalytic activity, its role in VLDL remodeling is unclear. We use pre-heparin plasma and heparin-sepharose affinity chromatography to isolate VLDL fractions from normolipidemic, hypertriglyceridemic, or type-2 diabetic subjects. LpL is detected only in the heparin-bound fraction. Transient binding to heparin activates this VLDL-associated LpL, which hydrolyses TG, leading to gradual VLDL remodeling into IDL/LDL and HDL-size particles. The products and the timeframe of this remodeling closely resemble VLDL-to-LDL maturation in vivo. Importantly, the VLDL fraction that does not bind heparin is not remodeled. This relatively inert LpL-free VLDL is rich in TG and apoC-III, poor in apoE and apoC-II, shows impaired functionality as a substrate for the exogenous LpL or CETP, and likely has prolonged residence time in blood, which is expected to promote atherogenesis. This non-bound VLDL fraction increases in hypertriglyceridemia and in type-2 diabetes but decreases upon diabetes treatment that restores the glycemic control. In stark contrast, heparin binding by LDL increases in type-2 diabetes triggering pro-atherogenic LDL modifications. Therefore, the effects of heparin binding are associated negatively with atherogenesis for VLDL but positively for LDL. Collectively, the results reveal that binding to glycosaminoglycans initiates VLDL remodeling by circulating LpL, and suggest heparin binding as a marker of VLDL functionality and a readout for treatment of metabolic disorders.
Assuntos
Diabetes Mellitus Tipo 2/genética , Hipertrigliceridemia/genética , Lipoproteínas LDL/metabolismo , Lipoproteínas VLDL/genética , Diabetes Mellitus Tipo 2/metabolismo , Endotélio Vascular/metabolismo , Metabolismo Energético/genética , Heparina/genética , Heparina/metabolismo , Humanos , Hipertrigliceridemia/metabolismo , Hipertrigliceridemia/patologia , Lipase Lipoproteica/genética , Lipase Lipoproteica/metabolismo , Lipoproteínas LDL/genética , Triglicerídeos/genéticaRESUMO
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?
Assuntos
AmiloideRESUMO
Low-density lipoprotein (LDL) binding to arterial proteoglycans initiates LDL retention and modification in the arterial wall, triggering atherosclerosis. The details of this binding, its effectors, and its ramifications are incompletely understood. We combined heparin affinity chromatography with biochemical, spectroscopic and electron microscopic techniques to show that brief binding to heparin initiates irreversible pro-atherogenic remodeling of human LDL. This involved decreased structural stability of LDL and increased susceptibility to hydrolysis, oxidation and fusion. Furthermore, phospholipid hydrolysis, mild oxidation and/or glycation of LDL in vitro increase the proteolytic susceptibility of apoB and its heparin binding affinity, perhaps by unmasking additional heparin-binding sites. For LDL from hyperglycemic type-2 diabetic patients, heparin binding was particularly destabilizing and caused apoB fragmentation and LDL fusion. However, for similar patients whose glycemic control was restored upon therapy, LDL-heparin binding affinity was rectified and LDL structural stability was partially restored. These results complement previous studies of LDL binding to arterial proteoglycans and suggest that such interactions may produce a particularly pro-atherogenic subclass of electronegative LDL. In summary, binding to heparin alters apoB conformation, perhaps by partially peeling it off the lipid, and triggers pro-atherogenic LDL modifications including hydrolysis, oxidation, and destabilization. Furthermore, phospholipid lipolysis, mild oxidation and glycation of LDL in vitro strengthen its binding to heparin, which helps explain stronger binding observed in hyperglycemic LDL. Combined effects of hyperglycemia and heparin binding are especially deleterious but are largely rectified upon diabetes therapy. These findings help establish a mechanistic link between diabetes and atherosclerosis.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Heparina/metabolismo , Hiperglicemia/metabolismo , Lipoproteínas LDL/metabolismo , Sítios de Ligação , Humanos , Hidrólise , Lipoproteínas LDL/sangue , Lipoproteínas LDL/química , Tamanho da Partícula , Agregados Proteicos , Conformação Proteica , Propriedades de SuperfícieRESUMO
Serum amyloid A (SAA) is an evolutionally conserved enigmatic biomarker of inflammation. In acute inflammation, SAA plasma levels increase ~1,000 fold, suggesting that this protein family has a vital beneficial role. SAA increases simultaneously with secretory phospholipase A2 (sPLA2), compelling us to determine how SAA influences sPLA2 hydrolysis of lipoproteins. SAA solubilized phospholipid bilayers to form lipoproteins that provided substrates for sPLA2. Moreover, SAA sequestered free fatty acids and lysophospholipids to form stable proteolysis-resistant complexes. Unlike albumin, SAA effectively removed free fatty acids under acidic conditions, which characterize inflammation sites. Therefore, SAA solubilized lipid bilayers to generate substrates for sPLA2 and removed its bioactive products. Consequently, SAA and sPLA2 can act synergistically to remove cellular membrane debris from injured sites, which is a prerequisite for tissue healing. We postulate that the removal of lipids and their degradation products constitutes a vital primordial role of SAA in innate immunity; this role remains to be tested in vivo.
Assuntos
Fosfolipases A2 Secretórias/metabolismo , Proteína Amiloide A Sérica/metabolismo , Albuminas/metabolismo , Animais , Humanos , Hidrólise , Bicamadas Lipídicas/metabolismo , Lipólise , Lipoproteínas/sangue , Camundongos , Fosfolipídeos/metabolismoRESUMO
Very low-density lipoprotein (VLDL) is the main plasma carrier of triacylglycerol that is elevated in pathological conditions such as diabetes, metabolic syndrome, obesity and dyslipidemia. How variations in triacylglycerol levels influence structural stability and remodeling of VLDL and its metabolic product, low-density lipoproteins (LDL), is unknown. We applied a biochemical and biophysical approach using lipoprotein remodeling by lipoprotein lipase and cholesterol ester transfer protein, along with thermal denaturation that mimics key aspects of lipoprotein remodeling in vivo. The results revealed that increasing the triacylglycerol content in VLDL promotes changes in the lipoprotein size and release of the exchangeable apolipoproteins. Similarly, increased triacylglycerol content in LDL promotes lipoprotein remodeling and fusion. These effects were observed in single-donor lipoproteins from healthy subjects enriched in exogenous triolein, in single-donor lipoproteins from healthy subjects with naturally occurring differences in endogenous triacylglycerol, and in LDL and VLDL from pooled plasma of diabetic and normolipidemic patients. Consequently, triacylglycerol-induced destabilization is a general property of plasma lipoproteins. This destabilization reflects a direct effect of triacylglycerol on lipoproteins. Moreover, we show that TG can act indirectly by increasing lipoprotein susceptibility to oxidation and lipolysis and thereby promoting the generation of free fatty acids that augment fusion. These in vitro findings are relevant to lipoprotein remodeling and fusion in vivo. In fact, fusion of LDL and VLDL enhances their retention in the arterial wall and, according to the response-to-retention hypothesis, triggers atherosclerosis. Therefore, enhanced fusion of triacylglycerol-rich lipoproteins suggests a new causative link between elevated plasma triacylglycerol and atherosclerosis.
Assuntos
Lipoproteínas LDL/química , Lipoproteínas VLDL/química , Triglicerídeos/farmacologia , Aterosclerose/etiologia , Proteínas de Transferência de Ésteres de Colesterol/metabolismo , Humanos , Lipase Lipoproteica/metabolismo , Lipoproteínas/metabolismo , Lipoproteínas LDL/sangue , Lipoproteínas VLDL/sangue , Estrutura Molecular , Desnaturação ProteicaRESUMO
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.
Assuntos
Amiloidose Familiar/genética , Apolipoproteína A-I/química , Apolipoproteína A-I/genética , Aterosclerose/genética , Mutação Puntual , Amiloidose Familiar/metabolismo , Apolipoproteína A-I/metabolismo , Aterosclerose/metabolismo , Humanos , Metabolismo dos Lipídeos , Lipoproteínas HDL/química , Lipoproteínas HDL/metabolismo , Modelos Moleculares , Conformação Proteica , Estabilidade ProteicaRESUMO
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.
Assuntos
Ésteres/química , Fosfolipídeos/química , Proteólise , Proteína Amiloide A Sérica/química , HidróliseRESUMO
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 â¼35% 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.
Assuntos
Amiloidose , Membranas Intracelulares , Lisossomos , Multimerização Proteica , Proteína Amiloide A Sérica , Amiloidose/metabolismo , Amiloidose/patologia , Animais , Concentração de Íons de Hidrogênio , Membranas Intracelulares/metabolismo , Membranas Intracelulares/patologia , Lisossomos/química , Lisossomos/metabolismo , Lisossomos/patologia , Camundongos , Estrutura Secundária de Proteína , Proteína Amiloide A Sérica/química , Proteína Amiloide A Sérica/metabolismoRESUMO
Serum amyloid A (SAA) is an acute-phase protein whose action in innate immunity and lipid homeostasis is unclear. Most circulating SAA binds plasma high-density lipoproteins (HDL) and reroutes lipid transport. In vivo SAA binds existing lipoproteins or generates them de novo upon lipid uptake from cells. We explored the products of SAA-lipid interactions and lipoprotein remodeling in vitro. SAA complexes with palmitoyl-oleoyl phosphocholine (POPC) were analyzed for structure and stability using circular dichroism and fluorescence spectroscopy, electron microscopy, gel electrophoresis and gel filtration. The results revealed the formation of 8-11nm lipoproteins that wereâ¼50% α-helical and stable at near-physiological conditions but were irreversibly remodeled at Tmâ¼52°C. Similar HDL-size nanoparticles formed spontaneously at ambient conditions or upon thermal remodeling of parent lipoproteins containing various amounts of proteins and lipids, including POPC and cholesterol. Therefore, such HDL-size particles formed stable kinetically accessible structures in a wide range of conditions. Based on their size and stoichiometry, each particle contained about 12 SAA and 72 POPC molecules, with a protein:lipid weight ratio circa 2.5:1, suggesting a structure distinct from HDL. High stability of these nanoparticles and their HDL-like size suggest that similar lipoproteins may form in vivo during inflammation or injury when SAA concentration is high and membranes from dead cells require rapid removal. We speculate that solubilization of membranes by SAA to generate lipoproteins in a spontaneous energy-independent process constitutes the primordial function of this ancient protein, providing the first line of defense in clearing cell debris from the injured sites.
Assuntos
Nanopartículas/química , Fosfatidilcolinas/química , Proteína Amiloide A Sérica/química , Animais , Colesterol/química , Cromatografia em Gel , Dicroísmo Circular , Eletroforese em Gel de Poliacrilamida , Lipoproteínas HDL/química , Camundongos , Microscopia Eletrônica , Tamanho da Partícula , Fosfolipídeos/química , Estabilidade Proteica , Proteína Amiloide A Sérica/imunologia , Espectrometria de FluorescênciaRESUMO
Lipids in the body are transported via lipoproteins that are nanoparticles comprised of lipids and amphipathic proteins termed apolipoproteins. This family of lipid surface-binding proteins is over-represented in human amyloid diseases. In particular, all major proteins of high-density lipoproteins (HDL), including apoA-I, apoA-II and serum amyloid A, can cause systemic amyloidoses in humans upon protein mutations, post-translational modifications or overproduction. Here, we begin to explore how the HDL lipid composition influences amyloid deposition by apoA-I and related proteins. First, we summarize the evidence that, in contrast to lipoproteins that are stabilized by kinetic barriers, free apolipoproteins are labile to misfolding and proteolysis. Next, we report original biochemical and biophysical studies showing that increase in triglyceride content in the core of plasma or reconstituted HDL destabilizes the lipoprotein assembly, making it more labile to various perturbations (oxidation, thermal and chemical denaturation and enzymatic hydrolysis), and promotes apoA-I release in a lipid-poor/free aggregation-prone form. Together, the results suggest that decreasing plasma levels of triglycerides will shift the dynamic equilibrium from the lipid-poor/free (labile) to the HDL-bound (protected) apolipoprotein state, thereby decreasing the generation of the protein precursor of amyloid. This prompts us to propose that triglyceride-lowering therapies may provide a promising strategy to alleviate amyloid diseases caused by the deposition of HDL proteins.
Assuntos
Amiloide/metabolismo , Apolipoproteína A-I/metabolismo , Lipoproteínas HDL/metabolismo , Triglicerídeos/metabolismo , Amiloide/sangue , Apolipoproteína A-I/sangue , Diabetes Mellitus/sangue , Diabetes Mellitus/metabolismo , Humanos , Lipólise , Lipoproteínas HDL/sangue , Modelos Moleculares , Oxirredução , Estabilidade Proteica , Proteólise , Triglicerídeos/sangueRESUMO
Oxidative stress and inflammation, which involve a dramatic increase in serum amyloid A (SAA) levels, are critical in the development of atherosclerosis. Most SAA circulates on plasma HDL particles, altering their cardioprotective properties. SAA-enriched HDL has diminished anti-oxidant effects on LDL, which may contribute to atherogenesis. We determined combined effects of SAA enrichment and oxidation on biochemical changes in HDL. Normal human HDLs were incubated with SAA, oxidized by various factors (Cu2+, myeloperoxidase, H2O2, OCl-), and analyzed for lipid and protein modifications and biophysical remodeling. Three novel findings are reported: addition of SAA reduces oxidation of HDL and LDL lipids; oxidation of SAA-containing HDL in the presence of OCl- generates a covalent heterodimer of SAA and apoA-I that resists the release from HDL; and mild oxidation promotes spontaneous release of proteins (SAA and apoA-I) from SAA-enriched HDL. We show that the anti-oxidant effects of SAA extend to various oxidants and are mediated mainly by the unbound protein. We propose that free SAA sequesters lipid hydroperoxides and delays lipoprotein oxidation, though much less efficiently than other anti-oxidant proteins, such as apoA-I, that SAA displaces from HDL. These findings prompt us to reconsider the role of SAA in lipid oxidation in vivo.
Assuntos
Antioxidantes/química , Lipoproteínas HDL/química , Lipoproteínas LDL/química , Proteína Amiloide A Sérica/química , Animais , Antioxidantes/fisiologia , Apolipoproteína A-I/química , Cobre/química , Humanos , Peroxidação de Lipídeos , Camundongos , Peroxidase/química , Proteína Amiloide A Sérica/fisiologiaRESUMO
Low-density lipoprotein (LDL) aggregation is central in triggering atherogenesis. A minor fraction of electronegative plasma LDL, termed LDL(-), plays a special role in atherogenesis. To better understand this role, we analyzed the kinetics of aggregation, fusion and disintegration of human LDL and its fractions, LDL(+) and LDL(-). Thermal denaturation of LDL was monitored by spectroscopy and electron microscopy. Initially, LDL(-) aggregated and fused faster than LDL(+), but later the order reversed. Most LDL(+) disintegrated and precipitated upon prolonged heating. In contrast, LDL(-) partially retained lipoprotein morphology and formed soluble aggregates. Biochemical analysis of all fractions showed no significant degradation of major lipids, mild phospholipid oxidation, and an increase in non-esterified fatty acid (NEFA) upon thermal denaturation. The main baseline difference between LDL subfractions was higher content of NEFA in LDL(-). Since NEFA promote lipoprotein fusion, increased NEFA content can explain rapid initial aggregation and fusion of LDL(-) but not its resistance to extensive disintegration. Partial hydrolysis of apoB upon heating was similar in LDL subfractions, suggesting that minor proteins importantly modulate LDL disintegration. Unlike LDL(+), LDL(-) contains small amounts of apoA-I and apoJ. Addition of exogenous apoA-I to LDL(+) hampered lipoprotein aggregation, fusion and precipitation, while depletion of endogenous apoJ had an opposite effect. Therefore, the initial rapid aggregation of LDL(-) is apparently counterbalanced by the stabilizing effects of minor proteins such as apoA-I and apoJ. These results help identify key determinants for LDL aggregation, fusion and coalescence into lipid droplets in vivo.
Assuntos
Apolipoproteína A-I/metabolismo , Aterosclerose/genética , Clusterina/metabolismo , Lipoproteínas LDL/metabolismo , Aterosclerose/metabolismo , Aterosclerose/patologia , Humanos , Cinética , Gotículas Lipídicas/metabolismo , Gotículas Lipídicas/patologia , Lipoproteínas LDL/química , Oxirredução , Agregação Patológica de Proteínas/genética , Estabilidade Proteica , TemperaturaRESUMO
Serum amyloid A (SAA) is an acute-phase protein that circulates mainly on plasma HDL. SAA interactions with its functional ligands and its pathogenic deposition in reactive amyloidosis depend, in part, on the structural disorder of this protein and its propensity to oligomerize. In vivo, SAA can displace a substantial fraction of the major HDL protein, apoA-I, and thereby influence the structural remodeling and functions of acute-phase HDL in ways that are incompletely understood. We use murine SAA1.1 to report the first structural stability study of human plasma HDL that has been enriched with SAA. Calorimetric and spectroscopic analyses of these and other SAA-lipid systems reveal two surprising findings. First, progressive displacement of the exchangeable fraction of apoA-I by SAA has little effect on the structural stability of HDL and its fusion and release of core lipids. Consequently, the major determinant for HDL stability is the nonexchangeable apoA-I. A structural model explaining this observation is proposed, which is consistent with functional studies in acute-phase HDL. Second, we report an α-helix folding/unfolding transition in SAA in the presence of lipid at near-physiological temperatures. This new transition may have potentially important implications for normal functions of SAA and its pathogenic misfolding.
Assuntos
Reação de Fase Aguda/metabolismo , Lipoproteínas HDL/química , Lipoproteínas HDL/metabolismo , Proteína Amiloide A Sérica/farmacologia , Temperatura , Reação de Fase Aguda/sangue , Animais , Dimiristoilfosfatidilcolina/metabolismo , Humanos , Camundongos , Fosfatidilcolinas/metabolismo , Desnaturação Proteica/efeitos dos fármacos , Dobramento de Proteína/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Proteína Amiloide A Sérica/química , Proteína Amiloide A Sérica/metabolismo , SoluçõesRESUMO
High plasma levels of apolipoprotein A-I (apoA-I) correlate with cardiovascular health, whereas dysfunctional apoA-I is a cause of atherosclerosis. In the atherosclerotic plaques, amyloid deposition increases with aging. Notably, apoA-I is the main component of these amyloids. Recent studies identified high levels of oxidized lipid-free apoA-I in atherosclerotic plaques. Likely, myeloperoxidase (MPO) secreted by activated macrophages in atherosclerotic lesions is the promoter of such apoA-I oxidation. We hypothesized that apoA-I oxidation by MPO levels similar to those present in the artery walls in atherosclerosis can promote apoA-I structural changes and amyloid fibril formation. ApoA-I was exposed to exhaustive chemical (H2O2) oxidation or physiological levels of enzymatic (MPO) oxidation and incubated at 37 °C and pH 6.0 to induce fibril formation. Both chemically and enzymatically oxidized apoA-I produced fibrillar amyloids after a few hours of incubation. The amyloid fibrils were composed of full-length apoA-I with differential oxidation of the three methionines. Met to Leu apoA-I variants were used to establish the predominant role of oxidation of Met-86 and Met-148 in the fibril formation process. Importantly, a small amount of preformed apoA-I fibrils was able to seed amyloid formation in oxidized apoA-I at pH 7.0. In contrast to hereditary amyloidosis, wherein specific mutations of apoA-I cause protein destabilization and amyloid deposition, oxidative conditions similar to those promoted by local inflammation in atherosclerosis are sufficient to transform full-length wild-type apoA-I into an amyloidogenic protein. Thus, MPO-mediated oxidation may be implicated in the mechanism that leads to amyloid deposition in the atherosclerotic plaques in vivo.
Assuntos
Amiloide/metabolismo , Apolipoproteína A-I/metabolismo , Aterosclerose/metabolismo , Peroxidase/metabolismo , Placa Aterosclerótica/metabolismo , Amiloide/genética , Apolipoproteína A-I/genética , Aterosclerose/genética , Aterosclerose/patologia , Humanos , Peróxido de Hidrogênio/farmacologia , Concentração de Íons de Hidrogênio , Metionina/genética , Metionina/metabolismo , Oxidantes/farmacologia , Oxirredução/efeitos dos fármacos , Peroxidase/genética , Placa Aterosclerótica/genética , Placa Aterosclerótica/patologiaRESUMO
High-density lipoproteins and their major protein, apolipoprotein A-I (apoA-I), remove excess cellular cholesterol and protect against atherosclerosis. However, in acquired amyloidosis, nonvariant full-length apoA-I deposits as fibrils in atherosclerotic plaques; in familial amyloidosis, N-terminal fragments of variant apoA-I deposit in vital organs, damaging them. Recently, we used the crystal structure of Δ(185-243)apoA-I to show that amyloidogenic mutations destabilize apoA-I and increase solvent exposure of the extended strand 44-55 that initiates ß-aggregation. In the present study, we test this hypothesis by exploring naturally occurring human amyloidogenic mutations, W50R and G26R, within or close to this strand. The mutations caused small changes in the protein's α-helical content, stability, proteolytic pattern and protein-lipid interactions. These changes alone were unlikely to account for amyloidosis, suggesting the importance of other factors. Sequence analysis predicted several amyloid-prone segments that can initiate apoA-I misfolding. Aggregation studies using N-terminal fragments verified this prediction experimentally. Three predicted N-terminal amyloid-prone segments, mapped on the crystal structure, formed an α-helical cluster. Structural analysis indicates that amyloidogenic mutations or Met86 oxidation perturb native packing in this cluster. Taken together, the results suggest that structural perturbations in the amyloid-prone segments trigger α-helix to ß-sheet conversion in the N-terminal ~ 75 residues forming the amyloid core. Polypeptide outside this core can be proteolysed to form 9-11 kDa N-terminal fragments found in familial amyloidosis. Our results imply that apoA-I misfolding in familial and acquired amyloidosis follows a similar mechanism that does not require significant structural destabilization or proteolysis. This novel mechanism suggests potential therapeutic interventions for apoA-I amyloidosis.
Assuntos
Amiloidose Familiar/metabolismo , Apolipoproteína A-I/genética , Aterosclerose/metabolismo , Amiloidose Familiar/genética , Apolipoproteína A-I/metabolismo , Aterosclerose/genética , Humanos , Metionina/metabolismo , Mutação/genética , Dobramento de Proteína , Relação Estrutura-AtividadeRESUMO
In the acute phase of the inflammatory response, secretory phospholipase A2 (sPLA2) reaches its maximum levels in plasma, where it is mostly associated with high density lipoproteins (HDL). Overexpression of human sPLA2 in transgenic mice reduces both HDL cholesterol and apolipoprotein A-I (apoA-I) plasma levels through increased HDL catabolism by an unknown mechanism. To identify unknown PLA2-mediated activities on the molecular components of HDL, we characterized the protein and lipid products of the PLA2 reaction with HDL. Consistent with previous studies, hydrolysis of HDL phospholipids by PLA2 reduced the particle size without changing its protein composition. However, when HDL was destabilized in the presence of PLA2 by the action of cholesteryl ester transfer protein or by guanidine hydrochloride treatment, a fraction of apoA-I, but no other proteins, dissociated from the particle and was rapidly cleaved. Incubation of PLA2 with lipid-free apoA-I produced similar protein fragments in the range of 6-15 kDa, suggesting specific and direct reaction of PLA2 with apoA-I. Mass spectrometry analysis of isolated proteolytic fragments indicated at least two major cleavage sites at the C-terminal and the central domain of apoA-I. ApoA-I proteolysis by PLA2 was Ca(2+)-independent, implicating a different mechanism from the Ca(2+)-dependent PLA2-mediated phospholipid hydrolysis. Inhibition of proteolysis by benzamidine suggests that the proteolytic and lipolytic activities of PLA2 proceed through different mechanisms. Our study identifies a previously unknown proteolytic activity of PLA2 that is specific to apoA-I and may contribute to the enhanced catabolism of apoA-I in inflammation and atherosclerosis.