Structural studies of a serum amyloid A octamer that is primed to scaffold lipid nanodiscs.

Serum amyloid A (SAA) is a highly conserved acute-phase protein that plays roles in activating multiple pro-inflammatory pathways during the acute inflammatory response and is commonly used as a biomarker of inflammation. It has been linked to beneficial roles in tissue repair through improved clearance of lipids and cholesterol from sites of damage. In patients with chronic inflammatory diseases, elevated levels of SAA may contribute to increased severity of the underlying condition. The majority of circulating SAA is bound to lipoproteins, primarily high-density lipoprotein (HDL). Interaction with HDL not only stabilizes SAA but also alters its functional properties, likely through altered accessibility of protein-protein interaction sites on SAA. While high-resolution structures for lipid-free, or apo-, forms of SAA have been reported, their relationship with the HDL-bound form of the protein, and with other possible mechanisms of SAA binding to lipids, has not been established. Here, we have used multiple biophysical techniques, including SAXS, TEM, SEC-MALS, native gel electrophoresis, glutaraldehyde crosslinking, and trypsin digestion to characterize the lipid-free and lipid-bound forms of SAA. The SAXS and TEM data show the presence of soluble octamers of SAA with structural similarity to the ring-like structures reported for lipid-free ApoA-I. These SAA octamers represent a previously uncharacterized structure for lipid-free SAA and are capable of scaffolding lipid nanodiscs with similar morphology to those formed by ApoA-I. The SAA-lipid nanodiscs contain four SAA molecules and have similar exterior dimensions as the lipid-free SAA octamer, suggesting that relatively few conformational rearrangements may be required to allow SAA interactions with lipid-containing particles such as HDL. This study suggests a new model for SAA-lipid interactions and provides new insight into how SAA might stabilize protein-lipid nanodiscs or even replace ApoA-I as a scaffold for HDL particles during inflammation.

Interactions of variants of human apolipoprotein A-I with biopolymeric model matrices. Effect of collagen and heparin.

BACKGROUND: The extracellular matrix (ECM) is a complex tridimensional scaffold that actively participates in physiological and pathological events. The objective of this study was to test whether structural proteins of the ECM and glycosaminoglycans (GAGs) may favor the retention of human apolipoprotein A-I (apoA-I) variants associated with amyloidosis and atherosclerosis. METHODS: Biopolymeric matrices containing collagen type I (Col, a main macromolecular component of the ECM) with or without heparin (Hep, a model of GAGs) were constructed and characterized, and used to compare the binding of apoA-I having the native sequence (Wt) or Arg173Pro, a natural variant inducing cardiac amyloidosis. Protein binding was observed by fluorescence microscopy and unbound proteins quantified by a colorimetric assay. RESULTS: Both, Wt and Arg173Pro bound to the scaffolds containing Col, but the presence of Hep diminished the binding efficiency. Col-Hep matrices retained Arg173Pro more than the Wt. The retained protein was only partially removed from the matrices with saline solutions, indicating that electrostatic interactions may occur but are not the main driving force. Using in addition thermodynamic molecular simulations and size exclusion chromatography approaches, we suggest that the binding of apoA-I variants to the biopolymeric matrices is driven by many low affinity interactions. CONCLUSIONS: Under this scenario Col-Hep scaffolds contribute to the binding of Arg173Pro, as a cooperative platform which could modify the native protein conformation affecting protein folding. GENERAL SIGNIFICANCE: We show that the composition of the ECM is key to the protein retention, and well characterized biosynthetic matrices offer an invaluable in vitro model to mimic the hallmark of pathologies with interstitial infiltration such as cardiac amyloidosis.

Beneficial effects of recombinant CER-001 high-density lipoprotein infusion in sepsis: results from a bench to bedside translational research project.

BACKGROUND: Sepsis is characterized by a dysregulated immune response and metabolic alterations, including decreased high-density lipoprotein cholesterol (HDL-C) levels. HDL exhibits beneficial properties, such as lipopolysaccharides (LPS) scavenging, exerting anti-inflammatory effects and providing endothelial protection. We investigated the effects of CER-001, an engineered HDL-mimetic, in a swine model of LPS-induced acute kidney injury (AKI) and a Phase 2a clinical trial, aiming to better understand its molecular basis in systemic inflammation and renal function. METHODS: We carried out a translational approach to study the effects of HDL administration on sepsis. Sterile systemic inflammation was induced in pigs by LPS infusion. Animals were randomized into LPS (n = 6), CER20 (single dose of CER-001 20 mg/kg; n = 6), and CER20 × 2 (two doses of CER-001 20 mg/kg; n = 6) groups. Survival rate, endothelial dysfunction biomarkers, pro-inflammatory mediators, LPS, and apolipoprotein A-I (ApoA-I) levels were assessed. Renal and liver histology and biochemistry were analyzed. Subsequently, we performed an open-label, randomized, dose-ranging (Phase 2a) study included 20 patients with sepsis due to intra-abdominal infection or urosepsis, randomized into Group A (conventional treatment, n = 5), Group B (CER-001 5 mg/kg BID, n = 5), Group C (CER-001 10 mg/kg BID, n = 5), and Group D (CER-001 20 mg/kg BID, n = 5). Primary outcomes were safety and efficacy in preventing AKI onset and severity; secondary outcomes include changes in inflammatory and endothelial dysfunction markers. RESULTS: CER-001 increased median survival, reduced inflammatory mediators, complement activation, and endothelial dysfunction in endotoxemic pigs. It enhanced LPS elimination through the bile and preserved liver and renal parenchyma. In the clinical study, CER-001 was well-tolerated with no serious adverse events related to study treatment. Rapid ApoA-I normalization was associated with enhanced LPS removal and immunomodulation with improvement of clinical outcomes, independently of the type and gravity of the sepsis. CER-001-treated patients had reduced risk for the onset and progression to severe AKI (stage 2 or 3) and, in a subset of critically ill patients, a reduced need for organ support and shorter ICU length of stay. CONCLUSIONS: CER-001 shows promise as a therapeutic strategy for sepsis management, improving outcomes and mitigating inflammation and organ damage. TRIAL REGISTRATION: The study was approved by the Agenzia Italiana del Farmaco (AIFA) and by the Local Ethic Committee (N° EUDRACT 2020-004202-60, Protocol CER-001- SEP_AKI_01) and was added to the EU Clinical Trials Register on January 13, 2021.

Unravelling the influence of surface lipids on the structure, dynamics and interactome of high-density lipoproteins.

Surface lipids influence the biological activities of high-density lipoproteins (HDLs) but their species-specific effects on HDL structure, dynamics, and surface interactome has remained unclear. Building upon the five-lipid species HDL models developed and characterised in previous work, representative models of the major HDL subpopulations found in human plasma containing apolipoprotein A-I (apoA-I) have been studied using molecular dynamics simulation to describe their varying degrees of surface lipidome complexity. Specifically, two additional sets of representative HDL subpopulation particles were developed, one with sphingomyelin (SM) and the other with SM, phosphatidylethanolamine, phosphatidylinositol, and ceramide in quantities reflecting average levels characterised for HDL subpopulations derived from normolipidemic patients. These lipid species were assessed in terms of HDL size, morphology, dynamics, and overall interactome. The findings reveal that the presence of a representative SM fraction marginally enhanced HDL interfacial curvature and surface monolayer rigidity, manifesting in tighter phospholipid packing and slower surface lipid dynamics relative to SM-deficient HDL models. Furthermore, the presence of SM resulted in a reduction in the solvent exposure of core lipids and cholesterol molecules, whilst also enhancing apolipoprotein conformational flexibility and its overall twisting across the HDL surface. The hydrophobicity of apoA-I-bound lipid patches and the proportion of apoA-I hydrophobic surface area is enhanced by the overall lipidation of apoA-I irrespective of lipid composition. These findings offer new insights into how the surface lipid composition of different HDL subpopulations can significantly impact the overall interactome of HDL particles, potentially influencing subpopulation-specific biological functions like lipid scavenging and receptor interactions.

Delineating the impact of pathogenic mutations on the conformational dynamics of HDL's vital protein ApoA1: a combined computational and molecular dynamic simulation approach.

Apolipoprotein A1 (ApoA1), is the important component of high-density lipoproteins (HDL), that has key role in HDL biogenesis, cholesterol trafficking, and reverse cholesterol transport (RCT). Non-synonymous Single Nucleotide Polymorphisms (nsSNPs) in ApoA1 have been linked to cardiovascular diseases and amyloidosis as they alter the protein's native structure and function. Therefore in this study, we attempted to understand the molecular pathogenicity profile of nsSNPs of ApoA1 using various computational approaches. We used state-of-the-art computational methods to thoroughly investigate the 295 ApoA1 nsSNPs at sequence and structural levels. Seven nsSNPs (L13R, L84R, L84P, L99P, R173P, L187P, and L238P) out of 295 were classified as the most deleterious and destabilizing. In order to estimate the effect of such destabilizing mutations on the protein conformation, all-atom molecular dynamics simulations (MDS) of ApoA1 wild-type (WT), L99P and R173P for 100 ns, was carried out using GROMACS 5.0.1 package. The MD simulation investigation revealed significant structural alterations in L99P and R173P. In addition, they had changed principal component analysis and electrostatic surface potential, decreased structural compactness, and intramolecular hydrogen bonds, which supported the rationale underpinning ApoA1 dysfunction with such mutations. This work sheds light on ApoA1 dysfunction due to single amino acid alterations, and offers new insight into the molecular basis of ApoA1-related diseases progression.Communicated by Ramaswamy H. Sarma.

Differential impact of glycation on apolipoprotein A-I of high-density lipoprotein: a review.

Hyperglycemia is a poorly controlled diabetic condition, affects about 70% of people all round the world. In the year 2015, about 41.5 crore people were diabetic and is expected to reach around 64.3 crore by the year 2040. Cardiovascular diseases (CVDs) are considered as one of the major risk factors that cause more than half of the death of diabetic patients and promote related comorbidities. Atherosclerosis and amyloidosis are the prime factors linked with CVDs. Apolipoprotein A-I (ApoA-I) of HDL has protective action against CVDs, participates in reverse cholesterol transport mechanism and lipid metabolism, but gets easily glycated under prolonged hyperglycemic aura, i.e. glycation. ApoA-I has a potent role in maintenance of glucose level, providing a compelling link between diabetes and CVDs. Increased protein glycation in people with diabetes promotes atherosclerosis, which might play possible role in promotion of protein aggregation by altering the protein structure and its conformation. Here, we intend to investigate the mechanistic behavior of ApoA-I under the menace of glycation and its impact on ApoA-I structure and function that possibly link with aggregation or amyloidosis.

The role of C-terminal ionic residues in self-association of apolipoprotein A-I.

Apolipoprotein A-I (apoA-I) is the main protein of high-density lipoprotein and is comprised of a helical bundle domain and a C-terminal (CT) domain encompassing the last ~65 amino acid residues of the 243-residue protein. The CT domain contains three putative helices (helix 8, 9, and 10) and is critical for initiating lipid binding and harbors sites that mediate self-association of the lipid-free protein. Three lysine residues reside in helix-8 (K195, 206, 208), and three in helix-10 (K226, 238, 239). To determine the role of each CT lysine residue in apoA-I self-association, single, double and triple lysine to glutamine mutants were engineered via site-directed mutagenesis. Circular dichroism and chemical denaturation analysis revealed all mutants retained their structural integrity. Chemical crosslinking and size-exclusion chromatography showed a small effect on self-association when helix-8 lysine residues were changed into glutamine. In contrast, mutation of the three helix-10 lysine residues resulted in a predominantly monomeric protein and K226 was identified as a critical residue. When helix-10 glutamate residues 223, 234, or 235 were substituted with glutamine, reduced self-association was observed similar to that of the helix-10 lysine variants, suggesting ionic interactions between these residues. Thus, helix-10 is a critical part of apoA-I mediating self-association, and disruption of ionic interactions changes apoA-I from an oligomeric state into a monomer. Since the helix-10 triple mutant solubilized phospholipid vesicles at higher rates compared to wild-type apoA-I, this indicates monomeric apoA-I is more potent in lipid binding, presumably because helix-10 is fully accessible to interact with lipids.

Systematic evaluation of the effect of different apolipoprotein A-I mimetic peptides on the performance of synthetic high-density lipoproteins in vitro and in vivo.

Synthetic high-density lipoproteins nanomedicine (sHDL) composed of apolipoprotein A-I (ApoA-I) mimetic peptides and lipids have shown very promising results for the treatment of various cardiovascular diseases. Numerous efforts have also been made to design different ApoA-I mimetic peptides to improve the potency of sHDL, especially the efficiency of reverse cholesterol transport. However, the way in which ApoA-I mimetic peptides affect the properties of sHDL, including stability, cholesterol efflux, cholesterol esterification, elimination in vivo, and the relationship of these properties, is still poorly understood. Revealing the effect of these factors on the potency of sHDL is important for the design of better ApoA-I mimetic peptides. In this study, three widely used ApoA-I mimetic peptides with different sequences, lengths, LCAT activation and lipid binding affinities were used for the preparation of sHDL and were evaluated in terms of physical/chemical properties, cholesterol efflux, cholesterol esterification, remodeling, and pharmacokinetics/pharmacodynamics. Our results showed that ApoA-I mimetic peptides with the highest cholesterol efflux and cholesterol esterification in vitro did not exhibit the highest cholesterol mobilization in vivo. Further analysis indicated that other factors, such as pharmacokinetics and remodeling of sHDL, need to be considered in order to predict the efficiency of cholesterol mobilization in vivo. Thus, our study highlights the importance of using the overall performance, rather than in vitro results alone, as the blueprint for the design and optimization of ApoA-I mimetic peptides.

Apolipoprotein-mimetic Peptides: Current and Future Prospectives.

Apolipoprotein-mimetic peptides, mimicking the biological properties of apolipoproteins, have shown beneficial properties against various diseases (central and peripheral diseases) and have emerged as potential candidates for their treatments. Progress has been made from first-generation to second-generation apolipoprotein-mimetic peptides. Understanding these peptides from the first generation to the second generation is discussed in this review. First, we discussed the structural and therapeutic potentials of first-generation apolipoprotein-mimetic peptides. Further, we discussed the development of second-generation apolipoprotein-mimetic peptides, like dual-domain and bihelical peptides the emergence of second-generation apolipoprotein-mimetic peptides as potential candidates in different preclinical and clinical studies has also been emphasized.

Analysis of the orientation of cholesterol in high-density lipoprotein nanodiscs using solid-state NMR.

Cholesterol is an essential component of eukaryotic cellular membranes that regulates the order and phase behaviour of dynamic lipid bilayers. Although cholesterol performs many vital physiological roles, hypercholesterolaemia and the accumulation of cholesterol in atherosclerotic plaques can increase the risk of coronary heart disease morbidity. The risk is mitigated by the transportation of cholesterol from peripheral tissue to the liver by high-density lipoprotein (HDL), 6-20 nm-diameter particles of lipid bilayers constrained by an annular belt of the protein apolipoprotein A-I (apoA-I). Information on the dynamics and orientation of cholesterol in HDL is pertinent to the essential role of HDL in cholesterol cycling. This work investigates whether the molecular orientation of cholesterol in HDL differs from that in the unconstrained lipid bilayers of multilamellar vesicles (MLVs). Solid-state NMR (ssNMR) measurements of dynamically-averaged 13C-13C and 13C-1H dipolar couplings were used to determine the average orientation of triple 13C-labelled cholesterol in palmitoyloleoylphosphatidylcholine (POPC) lipid bilayers in reconstituted HDL (rHDL) nanodiscs and in MLVs. Individual 13C-13C dipolar couplings were measured from [2,3,4-13C3]cholesterol in a one-dimensional NMR experiment, by using a novel application of a method to excite double quantum coherence at rotational resonance. The measured dipolar couplings were compared with average values calculated from orientational distributions of cholesterol generated using a Gaussian probability density function. The data were consistent with small differences in the average orientation of cholesterol in rHDL and MLVs, which may reflect the effects of the constrained and unconstrained lipid bilayers in the two environments. The calculated distributions of cholesterol in rHDL and MLVs that were consistent with the NMR data also agreed well with orientational distributions extracted from previous molecular dynamics simulations of HDL nanodiscs and planar POPC bilayers.

Mechanistic Insights into the Activation of Lecithin-Cholesterol Acyltransferase in Therapeutic Nanodiscs Composed of Apolipoprotein A-I Mimetic Peptides and Phospholipids.

The mechanistic details behind the activation of lecithin-cholesterol acyltransferase (LCAT) by apolipoprotein A-I (apoA-I) and its mimetic peptides are still enigmatic. Resolving the fundamental principles behind LCAT activation will facilitate the design of advanced HDL-mimetic therapeutic nanodiscs for LCAT deficiencies and coronary heart disease and for several targeted drug delivery applications. Here, we have combined coarse-grained molecular dynamics simulations with complementary experiments to gain mechanistic insight into how apoA-Imimetic peptide 22A and its variants tune LCAT activity in peptide-lipid nanodiscs. Our results highlight that peptide 22A forms transient antiparallel dimers in the rim of nanodiscs. The dimerization tendency considerably decreases with the removal of C-terminal lysine K22, which has also been shown to reduce the cholesterol esterification activity of LCAT. In addition, our simulations revealed that LCAT prefers to localize to the rim of nanodiscs in a manner that shields the membrane-binding domain (MBD), αA-αA', and the lid amino acids from the water phase, following previous experimental evidence. Meanwhile, the location and conformation of LCAT in the rim of nanodiscs are spatially more restricted when the active site covering the lid of LCAT is in the open form. The average location and spatial dimensions of LCAT in its open form were highly compatible with the electron microscopy images. All peptide 22A variants studied here had a specific interaction site in the open LCAT structure flanked by the lid and MBD domain. The bound peptides showed different tendencies to form antiparallel dimers and, interestingly, the temporal binding site occupancies of the peptide variants affected their in vitro ability to promote LCAT-mediated cholesterol esterification.

Human Serum Amyloid a Impaired Structural Stability of High-Density Lipoproteins (HDL) and Apolipoprotein (Apo) A-I and Exacerbated Glycation Susceptibility of ApoA-I and HDL.

Human serum amyloid A (SAA) is an exchangeable apolipoprotein (apo) in high-density lipoprotein (HDL) that influences HDL quality and functionality, particularly in the acute phase of inflammation. On the other hand, the structural and functional correlations of HDL containing SAA and apoA-I have not been reported. The current study was designed to compare the change in HDL quality with increasing SAA content in the lipid-free and lipid-bound states in reconstituted HDL (rHDL). The expressed recombinant human SAA1 (13 kDa) was purified to at least 98% and characterized in the lipid-free and lipid-bound states with apoA-I. The dimyristoyl phosphatidylcholine (DMPC) binding ability of apoA-I was impaired severely by the addition of SAA, while SAA alone could not bind with DMPC. The recombinant human SAA1 was incorporated into the rHDL (molar ratio 95:5:1, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC): cholesterol: apoA-I) with various apoA-I:SAA molar ratios from 1:0 to 1:0.5, 1:1 and 1:2. With increasing SAA1 content, the rHDL particle size was reduced from 98 Å to 93 Å, and the α-helicity of apoA-I:SAA was decreased from 73% to 40% for (1:0) and (1:2), respectively. The wavelength maximum fluorescence (WMF) of tryptophan in rHDL was red-shifted from 339 nm to 345 nm for (1:0) and (1:2) of apoA-I:SAA, respectively, indicating that the addition of SAA to rHDL destabilized the secondary structure of apoA-I. Upon denaturation by urea treatment from 0 M to 8 M, SAA showed only a 3 nm red-shift in WMF, while apoA-I showed a 16 nm red-shift in WMF, indicating that SAA is resistant to denaturation and apoA-I had higher conformational flexibility than SAA. The glycation reaction of apoA-I in the presence of fructose was accelerated up to 1.8-fold by adding SAA in a dose-dependent manner than that of apoA-I alone. In conclusion, the incorporation of SAA in rHDL impaired the structural stability of apoA-I and exacerbated glycation of HDL and apoA-I.

Molecular mechanisms for ABCA1-mediated cholesterol efflux.

The maintenance of cellular cholesterol homeostasis is essential for normal cell function and viability. Excessive cholesterol accumulation is detrimental to cells and serves as the molecular basis of many diseases, such as atherosclerosis, Alzheimer's disease, and diabetes mellitus. The peripheral cells do not have the ability to degrade cholesterol. Cholesterol efflux is therefore the only pathway to eliminate excessive cholesterol from these cells. This process is predominantly mediated by ATP-binding cassette transporter A1 (ABCA1), an integral membrane protein. ABCA1 is known to transfer intracellular free cholesterol and phospholipids to apolipoprotein A-I (apoA-I) for generating nascent high-density lipoprotein (nHDL) particles. nHDL can accept more free cholesterol from peripheral cells. Free cholesterol is then converted to cholesteryl ester by lecithin:cholesterol acyltransferase to form mature HDL. HDL-bound cholesterol enters the liver for biliary secretion and fecal excretion. Although how cholesterol is transported by ABCA1 to apoA-I remains incompletely understood, nine models have been proposed to explain this effect. In this review, we focus on the current view of the mechanisms underlying ABCA1-mediated cholesterol efflux to provide an important framework for future investigation and lipid-lowering therapy.

Apolipoprotein A-I carboxy-terminal domain residues 187-243 are required for adiponectin-induced cholesterol efflux.

Adiponectin exerts its atheroprotection by stimulating adenosine triphosphate binding cassette transporter A1 (ABCA1)-mediated cholesterol efflux to apolipoprotein A-I (apoA-I). However, involvement of the apoA-I residues in this process have not been studied. In Tamm-Horsfall 1 (THP-1) macrophages and baby hamster kidney (BHK) cells we assessed adiponectin's potential to restore cholesterol efflux in the presence of apoA-I and ABCA1 mutants, respectively. Adiponectin was unable to restore efflux from THP-1 macrophages in the presence of apoA-I carboxy-terminal domain (CTD) successive mutants from residues 187-243 versus apoA-I mutants alone. Furthermore, adiponectin did not significantly influence cholesterol efflux to apoA-I from BHK-ABCA1 mutant cells. Adiponectin appears to require functional apoA-I CTD residues 187-243 and wild-type ABCA1 to mediate efficient cholesterol efflux from THP-1 macrophages and BHK cells, respectively. Therefore, adiponectin cannot rescue defective cholesterol efflux in apoA-I- or ABCA1-mutant conditions, but rather increases cholesterol efflux in wild-type apoA-I conditions compared to apoA-I exposure alone.

Evolutionary and structural constraints influencing apolipoprotein A-I amyloid behavior.

Apolipoprotein A-I (apoA-I) has a key function in the reverse cholesterol transport. However, aggregation of apoA-I single point mutants can lead to hereditary amyloid pathology. Although several studies have tackled the biophysical and structural consequences introduced by these mutations, there is little information addressing the relationship between the evolutionary and structural features that contribute to the amyloid behavior of apoA-I. We combined evolutionary studies, in silico mutagenesis and molecular dynamics (MD) simulations to provide a comprehensive analysis of the conservation and pathogenic role of the aggregation-prone regions (APRs) present in apoA-I. Sequence analysis demonstrated that among the four amyloidogenic regions described for human apoA-I, only two (APR1 and APR4) are evolutionary conserved across different species of Sarcopterygii. Moreover, stability analysis carried out with the FoldX engine showed that APR1 contributes to the marginal stability of apoA-I. Structural properties of full-length apoA-I models suggest that aggregation is avoided by placing APRs into highly packed and rigid portions of its native fold. Compared to silent variants extracted from the gnomAD database, the thermodynamic and pathogenic impact of amyloid mutations showed evidence of a higher destabilizing effect. MD simulations of the amyloid variant G26R evidenced the partial unfolding of the alpha-helix bundle with the concomitant exposure of APR1 to the solvent, suggesting an insight into the early steps involved in its aggregation. Our findings highlight APR1 as a relevant component for apoA-I structural integrity and emphasize a destabilizing effect of amyloid variants that leads to the exposure of this region.

Structural and Functional Changes of Reconstituted High-Density Lipoprotein (HDL) by Incorporation of α-synuclein: A Potent Antioxidant and Anti-Glycation Activity of α-synuclein and apoA-I in HDL at High Molar Ratio of α-synuclein.

α-synuclein (α-syn) is a major culprit of Parkinson's disease (PD), although lipoprotein metabolism is very important in the pathogenesis of PD. α-syn was expressed and purified using the pET30a expression vector from an E. coli expression system to elucidate the physiological effects of α-syn on lipoprotein metabolism. The human α-syn protein (140 amino acids) with His-tag (8 amino acids) was expressed and purified to at least 95% purity. Isoelectric focusing gel electrophoresis showed that the isoelectric point (pI) of α-syn and apoA-I were pI = 4.5 and pI = 6.4, respectively. The lipid-free α-syn showed almost no phospholipid-binding ability, while apoA-I showed rapid binding ability with a half-time (T1/2) = 8 ± 0.7 min. The α-syn and apoA-I could be incorporated into the reconstituted HDL (rHDL, molar ratio 95:5:1:1, palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC):cholesterol:apoA-I:α-syn with the production of larger particles (92 Å) than apoA-I-rHDL (86 and 78 Å) and α-syn-rHDL (65 Å). An rHDL containing both apoA-I and α-syn showed lower α-helicity around 45% with a red shift of the Trp wavelength maximum fluorescence (WMF) from 339 nm, while apoA-I-HDL showed 76% α-helicity and 337 nm of WMF. The denaturation by urea addition showed that the incorporation of α-syn in rHDL caused a larger increase in the WMF than apoA-I-rHDL, suggesting that the destabilization of the secondary structure of apoA-I by the addition of α-syn. On the other hand, the addition of α-syn induced two-times higher resistance to rHDL glycation at apoA-I:α-syn molar ratios of 1:1 and 1:2. Interestingly, low α-syn in rHDL concentrations, molar ratio of 1:0.5 (apoA-I:α-syn), did not prevent glycation with more multimerization of apoA-I. In the lipid-free and lipid-bound state, α-syn showed more potent antioxidant activity than apoA-I against cupric ion-mediated LDL oxidation. On the other hand, microinjection of α-syn (final 2 µM) resulted in 10% less survival of zebrafish embryos than apoA-I. A subcutaneous injection of α-syn (final 34 µM) resulted in less tail fin regeneration than apoA-I. Interestingly, incorporation of α-syn at a low molar ratio (apoA-I:α-syn, 1:0.5) in rHDL resulted destabilization of the secondary structure and impairment of apoA-I functionality via more oxidation and glycation. However, at a higher molar ratio of α-syn in rHDL (apoA-I:α-syn = 1:1 or 1:2) exhibited potent antioxidant and anti-glycation activity without aggregation. In conclusion, there might be a critical concentration of α-syn and apoA-I in HDL-like complex to prevent the aggregation of apoA-I via structural and functional enhancement.

New insights into the emerging effects of inflammatory response on HDL particles structure and function.

According to the increasing results, it has been well-demonstrated that the chronic inflammatory response, including systemic lupus erythematosus, rheumatoid arthritis, and inflammatory bowel disease are associated with an increased risk of atherosclerotic cardiovascular disease. The mechanism whereby inflammatory response up-regulates the risk of cardio-metabolic disorder disease is multifactorial; furthermore, the alterations in high density lipoprotein (HDL) structure and function which occur under the inflammatory response could play an important modulatory function. On the other hand, the serum concentrations of HDL cholesterol (HDL-C) have been shown to be reduced significantly under inflammatory status with remarked alterations in HDL particles. Nevertheless, the potential mechanism whereby the inflammatory response reduces serum HDL-C levels is not simply defined but reduces apolipoprotein A1 production. The alterations in HDL structure mediated by the inflammatory response has been also confirmed to decrease the ability of HDL particle to play an important role in reverse cholesterol transport and protect the LDL particles from oxidation. Recently, it has been shown that under the inflammatory condition, diverse alterations in HDL structure could be observed which lead to changes in HDL function. In the current review, the emerging effects of inflammatory response on HDL particles structure and function are well-summarized to elucidate the potential mechanism whereby different inflammatory status modulates the pathogenic development of dyslipidemia.

Structural and Functional Impairments of Reconstituted High-Density Lipoprotein by Incorporation of Recombinant ß-Amyloid42.

Beta (ß)-amyloid (Aß) is a causative protein of Alzheimer's disease (AD). In the pathogenesis of AD, the apolipoprotein (apo) A-I and high-density lipoprotein (HDL) metabolism is essential for the clearance of Aß. In this study, recombinant Aß42 was expressed and purified via the pET-30a expression vector and E.coli production system to elucidate the physiological effects of Aß on HDL metabolism. The recombinant human Aß protein (51 aa) was purified to at least 95% purity and characterized in either the lipid-free and lipid-bound states with apoA-I. Aß was incorporated into the reconstituted HDL (rHDL) (molar ratio 95:5:1, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC):cholesterol:apoA-I) with various apoA-I:Aß ratios from 1:0 to 1:0.5, 1:1 and 1:2. With an increasing molar ratio of Aß, the α-helicity of apoA-I was decreased from 62% to 36% with a red shift of the Trp wavelength maximum fluorescence from 337 to 340 nm in apoA-I. The glycation reaction of apoA-I was accelerated further by the addition of Aß. The treatment of fructose and Aß caused more multimerization of apoA-I in the lipid-free state and in HDL. The phospholipid-binding ability of apoA-I was impaired severely by the addition of Aß in a dose-dependent manner. The phagocytosis of LDL into macrophages was accelerated more by the presence of Aß with the production of more oxidized species. Aß severely impaired tissue regeneration, and a microinjection of Aß enhanced embryotoxicity. In conclusion, the beneficial functions of apoA-I and HDL were severely impaired by the addition of Aß via its detrimental effect on secondary structure. The impairment of HDL functionality occurred more synergistically by means of the co-addition of fructose and Aß.

Purification and HDL-like particle formation of apolipoprotein A-I after co-expression with the EDDIE mutant of Npro autoprotease.

Apolipoprotein A-I (ApoA-I) is the major protein constituent of high-density lipoprotein particles, and as such is involved in cholesterol transport and activation of LCAT (the lecithin:cholesterol acyltransferase). It may also form amyloidal deposits in the body, showing the multifaceted interactions of ApoA-I. In order to facilitate the study of ApoA-I in various systems, we have developed a protocol based on recombinant expression in E. coli. ApoA-I is protected from degradation by driving its expression to inclusion bodies using a tag: the EDDIE mutant of Npro autoprotease from classical swine fever virus. Upon refolding, EDDIE will cleave itself off from the target protein. The result is a tag-free ApoA-I, with its N-terminus intact. ApoA-I was then purified using a five-step procedure composed of anion exchange chromatography, immobilized metal ion affinity chromatography, hydrophobic interaction chromatography, boiling and size exclusion chromatography. This led to protein of high purity as confirmed with SDS-PAGE and mass spectrometry. The purified ApoA-I formed discoidal objects in the presence of zwitterionic phospholipid DMPC, showing its retained function of interacting with lipids. The protocol was also tested by expression and purification of two ApoA-I mutants, both of which could be purified in the same manner as the wildtype, showing the robustness of the protocol.

SARS-CoV-2 infection as a trigger of humoral response against apolipoprotein A-1.

BACKGROUND: Unravelling autoimmune targets triggered by SARS-CoV-2 infection may provide crucial insights into the physiopathology of the disease and foster the development of potential therapeutic candidate targets and prognostic tools. We aimed at determining (a) the association between anti-SARS-CoV-2 and anti-apoA-1 humoral response and (b) the degree of linear homology between SARS-CoV-2, apoA-1 and Toll-like receptor 2 (TLR2) epitopes. DESIGN: Bioinformatics modelling coupled with mimic peptides engineering and competition experiments were used to assess epitopes sequence homologies. Anti-SARS-CoV-2 and anti-apoA-1 IgG as well as cytokines were assessed by immunoassays on a case-control (n = 101), an intensive care unit (ICU; n = 126) and a general population cohort (n = 663) with available samples in the pre and post-pandemic period. RESULTS: Using bioinformatics modelling, linear sequence homologies between apoA-1, TLR2 and Spike epitopes were identified but without experimental evidence of cross-reactivity. Overall, anti-apoA-1 IgG levels were higher in COVID-19 patients or anti-SARS-CoV-2 seropositive individuals than in healthy donors or anti-SARS-CoV-2 seronegative individuals (P < .0001). Significant and similar associations were noted between anti-apoA-1, anti-SARS-CoV-2 IgG, cytokines and lipid profile. In ICU patients, anti-SARS-CoV-2 and anti-apoA-1 seroconversion rates displayed similar 7-day kinetics, reaching 82% for anti-apoA-1 seropositivity. In the general population, SARS-CoV-2-exposed individuals displayed higher anti-apoA-1 IgG seropositivity rates than nonexposed ones (34% vs 16.8%; P = .004). CONCLUSION: COVID-19 induces a marked humoral response against the major protein of high-density lipoproteins. As a correlate of poorer prognosis in other clinical settings, such autoimmunity signatures may relate to long-term COVID-19 prognosis assessment and warrant further scrutiny in the current COVID-19 pandemic.