Systematic evaluation of lecithin:cholesterol acyltransferase binding sites in apolipoproteins via peptide based nanodiscs: regulatory role of charged residues at positions 4 and 7.

Lecithin:cholesterol acyltransferase (LCAT) exhibits α-activity on high-density and ß-activity on low-density lipoproteins. However, the molecular determinants governing LCAT activation by different apolipoproteins remain elusive. Uncovering these determinants would offer the opportunity to design and explore advanced therapies against dyslipidemias. Here, we have conducted coarse-grained and all-atom molecular dynamics simulations of LCAT with nanodiscs made with α-helical amphiphilic peptides either derived from apolipoproteins A1 and E (apoA1 and apoE) or apoA1 mimetic peptide 22A that was optimized to activate LCAT. This study aims to explore what drives the binding of peptides to our previously identified interaction site in LCAT. We hypothesized that this approach could be used to screen for binding sites of LCAT in different apolipoproteins and would provide insights to differently localized LCAT activities. Our screening approach was able to discriminate apoA1 helixes 4, 6, and 7 as key contributors to the interaction with LCAT supporting the previous research data. The simulations provided detailed molecular determinants driving the interaction with LCAT: the formation of hydrogen bonds or salt bridges between peptides E4 or D4 and LCAT S236 or K238 residues. Additionally, salt bridging between R7 and D73 was observed, depending on the availability of R7. Expanding our investigation to diverse plasma proteins, we detected novel LCAT binding helixes in apoL1, apoB100, and serum amyloid A. Our findings suggest that the same binding determinants, involving E4 or D4 -S236 and R7-D73 interactions, influence LCAT ß-activity on low-density lipoproteins, where apoE and or apoB100 are hypothesized to interact with LCAT.

Effects of Recombinant Human Lecithin Cholesterol Acyltransferase on Lipoprotein Metabolism in Humans.

BACKGROUND: LCAT (lecithin cholesterol acyl transferase) catalyzes the conversion of unesterified, or free cholesterol, to cholesteryl ester, which moves from the surface of HDL (high-density lipoprotein) into the neutral lipid core. As this iterative process continues, nascent lipid-poor HDL is converted to a series of larger, spherical cholesteryl ester-enriched HDL particles that can be cleared by the liver in a process that has been termed reverse cholesterol transport. METHODS: We conducted a randomized, placebocontrolled, crossover study in 5 volunteers with atherosclerotic cardiovascular disease, to examine the effects of an acute increase of recombinant human (rh) LCAT via intravenous administration (300-mg loading dose followed by 150 mg at 48 hours) on the in vivo metabolism of HDL APO (apolipoprotein)A1 and APOA2, and the APOB100-lipoproteins, very low density, intermediate density, and low-density lipoproteins. RESULTS: As expected, recombinant human LCAT treatment significantly increased HDL-cholesterol (34.9 mg/dL; P≤0.001), and this was mostly due to the increase in cholesteryl ester content (33.0 mg/dL; P=0.014). This change did not affect the fractional clearance or production rates of HDL-APOA1 and HDL-APOA2. There were also no significant changes in the metabolism of APOB100-lipoproteins. CONCLUSIONS: Our results suggest that an acute increase in LCAT activity drives greater flux of cholesteryl ester through the reverse cholesterol transport pathway without significantly altering the clearance and production of the main HDL proteins and without affecting the metabolism of APOB100-lipoproteins. Long-term elevations of LCAT might, therefore, have beneficial effects on total body cholesterol balance and atherogenesis.

Impaired high-density lipoprotein function and endothelial barrier stability in severe anaphylaxis.

BACKGROUND: Growing evidence demonstrates the importance of high- and low-density lipoprotein cholesterol in certain immune and allergy-mediated diseases. OBJECTIVE: This study aimed to evaluate levels of high- and low-density lipoprotein cholesterol and apolipoproteins A1 and B in sera from a cohort of patients presenting with hypersensitivity reactions. We further assessed the function of high-density lipoprotein particles as well as their involvement in the molecular mechanisms of anaphylaxis. METHODS: Lipid profile determination was performed in paired (acute and baseline) serum samples from 153 patients. Thirty-eight experienced a non-anaphylactic reaction and 115 had an anaphylactic reaction (88 moderate and 27 severe). Lecithin cholesterol acyl transferase activity was assessed in patient sera, and we also evaluated macrophage cholesterol efflux in response to the serum samples. Last, the effect of anaphylactic-derived high-density lipoprotein (HDL) particles on the endothelial barrier was studied. Detailed methods are provided in the Methods section in this article's Online Repository available at www.jacionline.org. RESULTS: Serum samples from severe anaphylactic reactions show statistically significant low levels of HDL cholesterol, low-density lipoprotein cholesterol, and apolipoproteins A1 and B, which points to their possible role as biomarkers. Specifically, HDL particles play a protective role in cardiovascular diseases. Using functional human serum cell assays, we observed impaired capacity of apolipoprotein B-depleted serum to induce macrophage cholesterol efflux in severe anaphylactic reactions. In addition, purified HDL particles from human anaphylactic sera failed to stabilize and maintain the endothelial barrier. CONCLUSION: These results encourage further research on HDL functions in severe anaphylaxis, which may lead to new diagnostic and therapeutic strategies.

Low LCAT activity is linked to acute decompensated heart failure and mortality in patients with CKD.

Chronic kidney disease (CKD) is often associated with decreased activity of lecithin-cholesterol acyltransferase (LCAT), an enzyme essential for HDL maturation. This reduction in LCAT activity may potentially contribute to an increased risk of cardiovascular mortality in patients with CKD. The objective of this study was to investigate the association between LCAT activity in patients with CKD and the risk of adverse outcomes. We measured serum LCAT activity and characterized lipoprotein profiles using nuclear magnetic resonance spectroscopy in 453 non-dialysis CKD patients from the CARE FOR HOMe study. LCAT activity correlated directly with smaller HDL particle size, a type of HDL potentially linked to greater cardiovascular protection. Over a mean follow-up of 5.0 ± 2.2 years, baseline LCAT activity was inversely associated with risk of death (standardized HR 0.62, 95% CI 0.50-0.76; P < 0.001) and acute decompensated heart failure (ADHF) (standardized HR 0.67, 95% CI 0.52-0.85; P = 0.001). These associations remained significant even after adjusting for other risk factors. Interestingly, LCAT activity was not associated with the incidence of atherosclerotic cardiovascular events or kidney function decline during the follow-up. To conclude, our findings demonstrate that low LCAT activity is independently associated with all-cause mortality and ADHF in patients with CKD, and is directly linked to smaller, potentially more protective HDL subclasses.

Rescue of Familial Lecithin:Cholesterol Acyltranferase Deficiency Mutations with an Allosteric Activator.

Lecithin:cholesterol acyltransferase (LCAT) deficiencies represent severe disorders characterized by aberrant cholesterol esterification in plasma, leading to life-threatening conditions. This study investigates the efficacy of Compound 2, a piperidinyl pyrazolopyridine allosteric activator that binds the membrane-binding domain of LCAT, in rescuing the activity of LCAT variants associated with disease. The variants K218N, N228K, and G230R, all located in the cap and lid domains of LCAT, demonstrated notable activity restoration in response to Compound 2. Molecular dynamics simulations and structural modeling indicate that these mutations disrupt the lid and membrane binding domain, with Compound 2 potentially dampening these structural alterations. Conversely, variants such as M252K and F382V in the cap and α/ß-hydrolase domain, respectively, exhibited limited or no rescue by Compound 2. Future research should prioritize in vivo investigations that would validate the therapeutic potential of Compound 2 and related activators in familial LCAT deficiency patients with mutations in the cap and lid of the enzyme. SIGNIFICANCE STATEMENT: Lecithin:cholesterol acyltranferase (LCAT) catalyzes the first step of reverse cholesterol transport, namely the esterification of cholesterol in high density lipoprotein particles. Somatic mutations in LCAT lead to excess cholesterol in blood plasma and, in severe cases, kidney failure. In this study, we show that recently discovered small molecule activators can rescue function in LCAT-deficient variants when the mutations occur in the lid and cap domains of the enzyme.

ALKBH5 regulates chicken adipogenesis by mediating LCAT mRNA stability depending on m6A modification.

BACKGROUND: Previous studies have demonstrated the role of N6-methyladenosine (m6A) RNA methylation in various biological processes, our research is the first to elucidate its specific impact on LCAT mRNA stability and adipogenesis in poultry. RESULTS: The 6 100-day-old female chickens were categorized into high (n = 3) and low-fat chickens (n = 3) based on their abdominal fat ratios, and their abdominal fat tissues were processed for MeRIP-seq and RNA-seq. An integrated analysis of MeRIP-seq and RNA-seq omics data revealed 16 differentially expressed genes associated with to differential m6A modifications. Among them, ELOVL fatty acid elongase 2 (ELOVL2), pyruvate dehydrogenase kinase 4 (PDK4), fatty acid binding protein 9 (PMP2), fatty acid binding protein 1 (FABP1), lysosomal associated membrane protein 3 (LAMP3), lecithin-cholesterol acyltransferase (LCAT) and solute carrier family 2 member 1 (SLC2A1) have ever been reported to be associated with adipogenesis. Interestingly, LCAT was down-regulated and expressed along with decreased levels of mRNA methylation methylation in the low-fat group. Mechanistically, the highly expressed ALKBH5 gene regulates LCAT RNA demethylation and affects LCAT mRNA stability. In addition, LCAT inhibits preadipocyte proliferation and promotes preadipocyte differentiation, and plays a key role in adipogenesis. CONCLUSIONS: In conclusion, ALKBH5 mediates RNA stability of LCAT through demethylation and affects chicken adipogenesis. This study provides a theoretical basis for further understanding of RNA methylation regulation in chicken adipogenesis.

Effects of dietary interventions and intermittent fasting on HDL function in obese individuals with T2DM: a randomized controlled trial.

BACKGROUND: Cardiovascular disease represents a significant risk factor for mortality in individuals with type 2 diabetes mellitus (T2DM). High-density lipoprotein (HDL) is believed to play a crucial role in maintaining cardiovascular health through its multifaceted atheroprotective effects and its capacity to enhance glycemic control. The impact of dietary interventions and intermittent fasting (IF) on HDL functionality remains uncertain. The objective of this study was to assess the effects of dietary interventions and IF as a strategy to safely improve glycemic control and reduce body weight on functional parameters of HDL in individuals with T2DM. METHODS: Before the 12-week intervention, all participants (n = 41) of the INTERFAST-2 study were standardized to a uniform basal insulin regimen and randomized to an IF or non-IF group. Additionally, all participants were advised to adhere to dietary recommendations that promoted healthy eating patterns. The IF group (n = 19) followed an alternate-day fasting routine, reducing their calorie intake by 75% on fasting days. The participants' glucose levels were continuously monitored. Other parameters were measured following the intervention: Lipoprotein composition and subclass distribution were measured by nuclear magnetic resonance spectroscopy. HDL cholesterol efflux capacity, paraoxonase 1 (PON1) activity, lecithin cholesterol acyltransferase (LCAT) activity, and cholesterol ester transfer protein (CETP) activity were assessed using cell-based assays and commercially available kits. Apolipoprotein M (apoM) levels were determined by ELISA. RESULTS: Following the 12-week intervention, the IF regimen significantly elevated serum apoM levels (p = 0.0144), whereas no increase was observed in the non-IF group (p = 0.9801). ApoM levels correlated with weight loss and fasting glucose levels in the IF group. Both groups exhibited a robust enhancement in HDL cholesterol efflux capacity (p < 0.0001, p = 0.0006) after 12 weeks. Notably, only the non-IF group exhibited significantly elevated activity of PON1 (p = 0.0455) and LCAT (p = 0.0117) following the 12-week intervention. In contrast, the changes observed in the IF group did not reach statistical significance. CONCLUSIONS: A balanced diet combined with meticulous insulin management improves multiple metrics of HDL function. While additional IF increases apoM levels, it does not further enhance other aspects of HDL functionality. TRIAL REGISTRATION: The study was registered at the German Clinical Trial Register (DRKS) on 3 September 2019 under the number DRKS00018070.

Novel therapeutic opportunities for familial lecithin:cholesterol acyltransferase deficiency: promises and challenges.

PURPOSE OF REVIEW: Genetic lecithin:cholesterol acyltransferase (LCAT) deficiency is a rare, inherited, recessive disease, which manifests as two different syndromes: Familial LCAT deficiency (FLD) and Fish-eye disease (FED), characterized by low HDL-C and corneal opacity. FLD patients also develop anaemia and renal disease. There is currently no therapy for FLD, but novel therapeutics are at different stages of development. Here, we summarize the most recent advances and the opportunities for and barriers to the further development of such therapies. RECENT FINDINGS: Recent publications highlight the heterogeneous phenotype of FLD and the uncertainty over the natural history of disease and the factors contributing to disease progression. Therapies that restore LCAT function (protein and gene replacement therapies and LCAT activators) showed promising effects on markers of LCAT activity. Although they do not restore LCAT function, HDL mimetics may slow renal disease progression. SUMMARY: The further development of novel therapeutics requires the identification of efficacy endpoints, which include quantitative biomarkers of disease progression. Because of the heterogeneity of renal disease progression among FLD individuals, future treatments for FLD will have to be tailored based on the specific clinical characteristics of the patient. Extensive studies of the natural history and biomarkers of the disease will be required to achieve this goal.

Randomized, Placebo-Controlled Phase 2b Study to Evaluate the Safety and Efficacy of Recombinant Human Lecithin Cholesterol Acyltransferase in Acute ST-Segment-Elevation Myocardial Infarction: Results of REAL-TIMI 63B.

BACKGROUND: High-density lipoprotein plays a key role in reverse cholesterol transport. In addition, high-density lipoprotein particles may be cardioprotective and reduce infarct size in the setting of myocardial injury. Lecithin-cholesterol acyltransferase is a rate-limiting enzyme in reverse cholesterol transport. MEDI6012 is a recombinant human lecithin-cholesterol acyltransferase that increases high-density lipoprotein cholesterol. Administration of lecithin-cholesterol acyltransferase has the potential to reduce infarct size and regress coronary plaque in acute ST-segment-elevation myocardial infarction. METHODS: REAL-TIMI 63B (A Randomized, Placebo­controlled Phase 2b Study to Evaluate the Safety and Efficacy of MEDI6012 in Acute ST Elevation Myocardial Infarction) was a phase 2B multinational, placebo-controlled, randomized trial. Patients with ST-segment-elevation myocardial infarction within 6 hours of symptom onset and planned for percutaneous intervention were randomly assigned 2:1 to MEDI6012 (2- or 6-dose regimen) or placebo and followed for 12 weeks. The primary outcome was infarct size as a percentage of left ventricular mass by cardiac MRI at 10 to 12 weeks, with the primary analysis in patients with TIMI Flow Grade 0 to 1 before percutaneous intervention who received at least 2 doses of MEDI6012. The secondary outcome was change in noncalcified plaque volume on coronary computed tomographic angiography from baseline to 10 to 12 weeks with the primary analysis in patients who received all 6 doses of MEDI6012. RESULTS: A total of 593 patients were randomly assigned. Patients were a median of 62 years old, 77.9% male, and 95.8% statin naive. Median time from symptom onset to randomization was 146 (interquartile range [IQR], 103-221) minutes and from hospitalization to randomization was 12.7 (IQR, 6.6-24.0) minutes, and the first dose of drug was administered a median of 8 (IQR, 3-13) minutes before percutaneous intervention. The index myocardial infarction was anterior in 69.6% and TIMI Flow Grade 0 to 1 in 65.1% of patients. At 12 weeks, infarct size did not differ between treatment groups (MEDI6012: 9.71%, IQR 4.79-16.38; placebo: 10.48%, [IQR, 4.92-16.61], 1-sided P=0.79. There was also no difference in noncalcified plaque volume (geometric mean ratio, 0.96 [95% CI, NA-1.10], 1-sided P=0.30). There was no significant difference in treatment emergent serious adverse events. CONCLUSIONS: Administration of MEDI6012 in patients with acute ST-segment-elevation myocardial infarction did not result in a significant reduction in infarct size or noncalcified plaque volume at 12 weeks. MEDI6012 was well tolerated with no excess in overall serious adverse events. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier: NCT03578809.

The expanding role of lyso-phosphatidylcholine acyltransferase-3 (LPCAT3), a phospholipid remodeling enzyme, in health and disease.

PURPOSE OF REVIEW: The turnover of fatty acids (FAs) at the sn-2 position of phospholipids is mediated by the reciprocal actions of phospholipases A2 and lyso-PL acyltransferases (LPLAT). LPCAT3, a major LPLAT isoform, exhibits a strong specificity for polyunsaturated FAs s (PUFAs). Although the enzyme was originally studied in the context of cardiometabolism, recent investigations have shed light on the role of LPCAT3 in other tissues such as skeletal muscle and in unexpected biological processes such as cell death and oncogenesis. RECENT FINDINGS: The three-dimensional structure of LPCAT3 has been elucidated allowing further understanding of the mechanism of the acylation reaction as well as the substrate specificity of the enzyme. In skeletal muscle, LPCAT3-mediated phospholipid remodeling modulates membrane domain clustering and insulin signalingLPCAT3 plays an important role in the process of ferroptosis by modulating the PUFA content of phospholipids and possibly of plasmalogens.In tumor-associated macrophages, LPCAT3 can prevent ER stress induced by the tumor microenvironment and may equally modulate antitumor immunity. SUMMARY: LPCAT3 is an attractive therapeutic target in the cardiometabolic disorders. Nevertheless, the involvement of LPCAT3 in processes such as cell death and oncogenesis demands caution with respect to the potential deleterious effects of enzyme modulation.

Plasma FA composition in familial LCAT deficiency indicates SOAT2-derived cholesteryl ester formation in humans.

Mutations in the LCAT gene cause familial LCAT deficiency (Online Mendelian Inheritance in Man ID: #245900), a very rare metabolic disorder. LCAT is the only enzyme able to esterify cholesterol in plasma, whereas sterol O-acyltransferases 1 and 2 are the enzymes esterifying cellular cholesterol in cells. Despite the complete lack of LCAT activity, patients with familial LCAT deficiency exhibit circulating cholesteryl esters (CEs) in apoB-containing lipoproteins. To analyze the origin of these CEs, we investigated 24 carriers of LCAT deficiency in this observational study. We found that CE plasma levels were significantly reduced and highly variable among carriers of two mutant LCAT alleles (22.5 [4.0-37.8] mg/dl) and slightly reduced in heterozygotes (218 [153-234] mg/dl). FA distribution in CE (CEFA) was evaluated in whole plasma and VLDL in a subgroup of the enrolled subjects. We found enrichment of C16:0, C18:0, and C18:1 species and a depletion in C18:2 and C20:4 species in the plasma of carriers of two mutant LCAT alleles. No changes were observed in heterozygotes. Furthermore, plasma triglyceride-FA distribution was remarkably similar between carriers of LCAT deficiency and controls. CEFA distribution in VLDL essentially recapitulated that of plasma, being mainly enriched in C16:0 and C18:1, while depleted in C18:2 and C20:4. Finally, after fat loading, chylomicrons of carriers of two mutant LCAT alleles showed CEs containing mainly saturated FAs. This study of CEFA composition in a large cohort of carriers of LCAT deficiency shows that in the absence of LCAT-derived CEs, CEs present in apoB-containing lipoproteins are derived from hepatic and intestinal sterol O-acyltransferase 2.

A systematic review of the natural history and biomarkers of primary lecithin:cholesterol acyltransferase deficiency.

Syndromes associated with LCAT deficiency, a rare autosomal recessive condition, include fish-eye disease (FED) and familial LCAT deficiency (FLD). FLD is more severe and characterized by early and progressive chronic kidney disease (CKD). No treatment is currently available for FLD, but novel therapeutics are under development. Furthermore, although biomarkers of LCAT deficiency have been identified, their suitability to monitor disease progression and therapeutic efficacy is unclear, as little data exist on the rate of progression of renal disease. Here, we systematically review observational studies of FLD, FED, and heterozygous subjects, which summarize available evidence on the natural history and biomarkers of LCAT deficiency, in order to guide the development of novel therapeutics. We identified 146 FLD and 53 FED patients from 219 publications, showing that both syndromes are characterized by early corneal opacity and markedly reduced HDL-C levels. Proteinuria/hematuria were the first signs of renal impairment in FLD, followed by rapid decline of renal function. Furthermore, LCAT activity toward endogenous substrates and the percentage of circulating esterified cholesterol (EC%) were the best discriminators between these two syndromes. In FLD, higher levels of total, non-HDL, and unesterified cholesterol were associated with severe CKD. We reveal a nonlinear association between LCAT activity and EC% levels, in which subnormal levels of LCAT activity were associated with normal EC%. This review provides the first step toward the identification of disease biomarkers to be used in clinical trials and suggests that restoring LCAT activity to subnormal levels may be sufficient to prevent renal disease progression.

Conformational flexibility of apolipoprotein A-I amino- and carboxy-termini is necessary for lipid binding but not cholesterol efflux.

Because of its critical role in HDL formation, significant efforts have been devoted to studying apolipoprotein A-I (APOA1) structural transitions in response to lipid binding. To assess the requirements for the conformational freedom of its termini during HDL particle formation, we generated three dimeric APOA1 molecules with their termini covalently joined in different combinations. The dimeric (d)-APOA1C-N mutant coupled the C-terminus of one APOA1 molecule to the N-terminus of a second with a short alanine linker, whereas the d-APOA1C-C and d-APOA1N-N mutants coupled the C-termini and the N-termini of two APOA1 molecules, respectively, using introduced cysteine residues to form disulfide linkages. We then tested the ability of these constructs to generate reconstituted HDL by detergent-assisted and spontaneous phospholipid microsolubilization methods. Using cholate dialysis, we demonstrate WT and all APOA1 mutants generated reconstituted HDL particles of similar sizes, morphologies, compositions, and abilities to activate lecithin:cholesterol acyltransferase. Unlike WT, however, the mutants were incapable of spontaneously solubilizing short chain phospholipids into discoidal particles. We found lipid-free d-APOA1C-N and d-APOA1N-N retained most of WT APOA1's ability to promote cholesterol efflux via the ATP binding cassette transporter A1, whereas d-APOA1C-C exhibited impaired cholesterol efflux. Our data support the double belt model for a lipid-bound APOA1 structure in nascent HDL particles and refute other postulated arrangements like the "double super helix." Furthermore, we conclude the conformational freedom of both the N- and C-termini of APOA1 is important in spontaneous microsolubilization of bulk phospholipid but is not critical for ABCA1-mediated cholesterol efflux.

Physaria fendleri and Ricinus communis lecithin:cholesterol acyltransferase-like phospholipases selectively cleave hydroxy acyl chains from phosphatidylcholine.

Production of hydroxy fatty acids (HFAs) in transgenic crops represents a promising strategy to meet our demands for specialized plant oils with industrial applications. The expression of Ricinus communis (castor) OLEATE 12-HYDROXYLASE (RcFAH12) in Arabidopsis has resulted in only limited accumulation of HFAs in seeds, which probably results from inefficient transfer of HFAs from their site of synthesis (phosphatidylcholine; PC) to triacylglycerol (TAG), especially at the sn-1/3 positions of TAG. Phospholipase As (PLAs) may be directly involved in the liberation of HFAs from PC, but the functions of their over-expression in HFA accumulation and distribution at TAG in transgenic plants have not been well studied. In this work, the functions of lecithin:cholesterol acyltransferase-like PLAs (LCAT-PLAs) in HFA biosynthesis were characterized. The LCAT-PLAs were shown to exhibit homology to LCAT and mammalian lysosomal PLA2 , and to contain a conserved and functional Ser/His/Asp catalytic triad. In vitro assays revealed that LCAT-PLAs from the HFA-accumulating plant species Physaria fendleri (PfLCAT-PLA) and castor (RcLCAT-PLA) could cleave acyl chains at both the sn-1 and sn-2 positions of PC, and displayed substrate selectivity towards sn-2-ricinoleoyl-PC over sn-2-oleoyl-PC. Furthermore, co-expression of RcFAH12 with PfLCAT-PLA or RcLCAT-PLA, but not Arabidopsis AtLCAT-PLA, resulted in increased occupation of HFA at the sn-1/3 positions of TAG as well as small but insignificant increases in HFA levels in Arabidopsis seeds compared with RcFAH12 expression alone. Therefore, PfLCAT-PLA and RcLCAT-PLA may contribute to HFA turnover on PC, and represent potential candidates for engineering the production of unusual fatty acids in crops.

The HDL mimetic CER-001 remodels plasma lipoproteins and reduces kidney lipid deposits in inherited lecithin:cholesterol acyltransferase deficiency.

BACKGROUND: Kidney failure is the major cause of morbidity and mortality in familial lecithin:cholesterol acyltransferase deficiency (FLD), a rare inherited lipid disorder with no cure. Lipoprotein X (LpX), an abnormal lipoprotein, is primarily accountable for nephrotoxicity. METHODS: CER-001 was tested in an FLD patient with dramatic kidney disease for 12 weeks. RESULTS: Infusions of CER-001 normalized the lipoprotein profile, with a disappearance of the abnormal LpX in favour of normal-sized LDL. The worsening of kidney function was slowed by the treatment, and kidney biopsy showed a slight reduction of lipid deposits and a stabilization of the disease. In vitro experiments demonstrate that CER-001 progressively reverts lipid accumulation in podocytes by a dual effect: remodelling plasma lipoproteins and removing LpX-induced lipid deposit. CONCLUSION: This study demonstrates that CER-001 may represent a therapeutic option in FLD patients. It also has the potential to be beneficial in other renal diseases characterized by kidney lipid deposits.

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.

Positive allosteric modulators of lecithin: Cholesterol acyltransferase adjust the orientation of the membrane-binding domain and alter its spatial free energy profile.

Lecithin:cholesterol acyltransferase protein (LCAT) promotes the esterification reaction between cholesterol and phospholipid-derived acyl chains. Positive allosteric modulators have been developed to treat LCAT deficiencies and, plausibly, also cardiovascular diseases in the future. The mechanism of action of these compounds is poorly understood. Here computational docking and atomistic molecular dynamics simulations were utilized to study the interactions between LCAT and the activating compounds. Results indicate that all drugs bind to the allosteric binding pocket in the membrane-binding domain in a similar fashion. The presence of the compounds in the allosteric site results in a distinct spatial orientation and sampling of the membrane-binding domain (MBD). The MBD's different spatial arrangement plausibly affects the lid's movement from closed to open state and vice versa, as suggested by steered molecular dynamics simulations.

Novel LCAT (Lecithin:Cholesterol Acyltransferase) Activator DS-8190a Prevents the Progression of Plaque Accumulation in Atherosclerosis Models.

OBJECTIVE: Enhancement of LCAT (lecithin:cholesterol acyltransferase) activity has possibility to be beneficial for atherosclerosis. To evaluate this concept, we characterized our novel, orally administered, small-molecule LCAT activator DS-8190a, which was created from high-throughput screening and subsequent derivatization. We also focused on its mechanism of LCAT activation and the therapeutic activity with improvement of HDL (high-density lipoprotein) functionality. Approach and Results: DS-8190a activated human and cynomolgus monkey but not mouse LCAT enzymes in vitro. DS-8190a was orally administered to cynomolgus monkeys and dose dependently increased LCAT activity (2.0-fold in 3 mg/kg group on day 7), resulting in HDL cholesterol elevation without drastic changes of non-HDL cholesterol. Atheroprotective effects were then evaluated using Ldl-r KO×hLcat Tg mice fed a Western diet for 8 weeks. DS-8190a treatment achieved significant reduction of atherosclerotic lesion area (48.3% reduction in 10 mg/kg treatment group). Furthermore, we conducted reverse cholesterol transport study using Ldl-r KO×hLcat Tg mice intraperitoneally injected with J774A.1 cells loaded with [3H]-cholesterol and confirmed significant increases of [3H] count in plasma (1.4-fold) and feces (1.4-fold on day 2 and 1.5-fold on day3) in the DS-8190a-treated group. With regard to the molecular mechanism involved, direct binding of DS-8190a to human LCAT protein was confirmed by 2 different approaches: affinity purification by DS-8190a-immobilized beads and thermal shift assay. In addition, the candidate binding site of DS-8190a in human LCAT protein was identified by photoaffinity labeling. CONCLUSIONS: This study demonstrates the potential of DS-8190a as a novel therapeutic for atherosclerosis. In addition, this compound proves that a small-molecule direct LCAT activator can achieve HDL-C elevation in monkey and reduction of atherosclerotic lesion area with enhanced HDL function in rodent.

Correction of Familial LCAT Deficiency by AAV-hLCAT Prevents Renal Injury and Atherosclerosis in Hamsters-Brief Report.

[Figure: see text].

Lipidomic Approaches to Study HDL Metabolism in Patients with Central Obesity Diagnosed with Metabolic Syndrome.

The metabolic syndrome (MetS) is a cluster of cardiovascular risk factors characterised by central obesity, atherogenic dyslipidaemia, and changes in the circulating lipidome; the underlying mechanisms that lead to this lipid remodelling have only been partially elucidated. This study used an integrated "omics" approach (untargeted whole serum lipidomics, targeted proteomics, and lipoprotein lipidomics) to study lipoprotein remodelling and HDL composition in subjects with central obesity diagnosed with MetS (vs. controls). Compared with healthy subjects, MetS patients showed higher free fatty acids, diglycerides, phosphatidylcholines, and triglycerides, particularly those enriched in products of de novo lipogenesis. On the other hand, the "lysophosphatidylcholines to phosphatidylcholines" and "cholesteryl ester to free cholesterol" ratios were reduced, pointing to a lower activity of lecithin cholesterol acyltransferase (LCAT) in MetS; LCAT activity (directly measured and predicted by lipidomic ratios) was positively correlated with high-density lipoprotein cholesterol (HDL-C) and negatively correlated with body mass index (BMI) and insulin resistance. Moreover, many phosphatidylcholines and sphingomyelins were significantly lower in the HDL of MetS patients and strongly correlated with BMI and clinical metabolic parameters. These results suggest that MetS is associated with an impairment of phospholipid metabolism in HDL, partially led by LCAT, and associated with obesity and underlying insulin resistance. This study proposes a candidate strategy to use integrated "omics" approaches to gain mechanistic insights into lipoprotein remodelling, thus deepening the knowledge regarding the molecular basis of the association between MetS and atherosclerosis.