Native Apo Research Articles

Conformational response to ligand binding of TMPRSS2, a protease involved in SARS-CoV-2 infection: Insights through computational modeling.

Thanks to the considerable research which has been undertaken in the last few years to improve our understanding of the biology and mechanism of action of SARS-CoV-2, we know how the virus uses its surface spike protein to infect host cells. The transmembrane prosthesis, serine 2 (TMPRSS2) protein, located on the surface of human cells, recognizes the cleavage site in the spike protein, leading to the release of the fusion peptide and entry of the virus into the host cells. Because of its role, TMPRSS2 has been proposed as a drug target to prevent infection by the virus. In this study, we aim to increase our understanding of TMPRSS2 using long scale microsecond atomistic molecular dynamics simulations, focusing on the conformational changes over time. The comparison between simulations conducted on the protein in the native (apo) and inhibited form (holo), has shown that in the holo form the inhibitor stabilizes the catalytic site and induces rearrangements in the extracellular domain of the protein. In turn, it leads to the formation of a new cavity in the vicinity of the ligand binding pocket that is stable in the microsecond time scale. Given the low specificity of known protease inhibitors, these findings suggest a new potential drug target site that can be used to improve TMPRSS2 specific recognition by newly designed inhibitors.

Abstract 677: Short Hydrocarbon-Stapled Apolipoprotein E Mimetic Peptides Enhance Lipoprotein Uptake

ApoE facilitates the clearance of apoB-containing lipoproteins by binding to heparan sulfate proteoglycans (HSPG), low density lipoprotein receptor (LDLR) and LDLR-related protein 1 (LRP1). ApoE has a receptor binding and a lipid binding domain, which are both needed for its biological function. Several apoE-based peptides have been developed that lower plasma lipid levels in animal models and in early-stage clinical trials. However, these peptides are long (28-61 residues), costly to synthesize and are potentially immunogenic as they often contain many amino acid substitutions that vary from the native apoE sequence. We developed novel short (10-12 residues) apoE mimetic peptides based on the apoE receptor binding region (residues 141-150) containing only 2 amino acid substitutions. We used hydrocarbon stapling to stabilize the native alpha helical structure of this region and to increase the lipid binding affinity of the peptides. In addition, octanoic or myristic acid was attached to the N-terminus of the peptides to further increase lipid binding affinity. Helical conformation and lipid binding of the peptides were confirmed by circular dichroism spectroscopy and phospholipid vesicle solubilization assay. Our peptides increased the uptake of fluorescent tagged VLDL and LDL in a concentration dependent manner (0.625-20 μM peptides) in both wild-type (WT) and LDLR knockout (KO) HepG2 cells as measured by flow cytometry. In addition, an all D-amino acid version of one of the peptides had a similar effect on LDL uptake as its all L-amino acid version. Altogether, this suggests that peptide-mediated uptake is LDLR independent. Treatment of WT HepG2 cells with heparinases diminished the low-dose effect (up to 2.5 μM) of the peptides on LDL uptake, indicating that HSPGs plays a role in peptide-mediated LDL uptake. Finally, the peptides (4.6 mg/kg, IV) lowered plasma total cholesterol (37% at 3h; 29% at 6h) and triglycerides (33% at 1h) in apoE-KO mice on a standard chow diet. In summary, the novel apoE peptides are shorter than previously reported peptides, are more similar to the native sequence of apoE, and are likely resistant to proteolysis, because of hydrocarbon stapling and, therefore, could potentially be developed into an oral therapy.

ApoE Mimetic Peptides to Improve the Vicious Cycle of Malnutrition and Enteric Infections by Targeting the Intestinal and Blood-Brain Barriers.

Apolipoprotein E (apoE) mimetic peptides are engineered fragments of the native apoE protein's LDL-receptor binding site that improve the outcomes following a brain injury and intestinal inflammation in a variety of models. The vicious cycle of enteric infections and malnutrition is closely related to environmental-driven enteric dysfunction early in life, and such chronic inflammatory conditions may blunt the developmental trajectories of children with worrisome and often irreversible physical and cognitive faltering. This window of time for microbiota maturation and brain plasticity is key to protecting cognitive domains, brain health, and achieving optimal/full developmental potential. This review summarizes the potential role of promising apoE mimetic peptides to improve the function of the gut-brain axis, including targeting the blood-brain barrier in children afflicted with malnutrition and enteric infections.

Apolipoprotein E4 Effects a Distinct Transcriptomic Profile and Dendritic Arbor Characteristics in Hippocampal Neurons Cultured in vitro.

The APOE gene is diversified by three alleles ε2, ε3, and ε4 encoding corresponding apolipoprotein (apo) E isoforms. Possession of the ε4 allele is signified by increased risks of age-related cognitive decline, Alzheimer’s disease (AD), and the rate of AD dementia progression. ApoE is secreted by astrocytes as high-density lipoprotein-like particles and these are internalized by neurons upon binding to neuron-expressed apoE receptors. ApoE isoforms differentially engage neuronal plasticity through poorly understood mechanisms. We examined here the effects of native apoE lipoproteins produced by immortalized astrocytes homozygous for ε2, ε3, and ε4 alleles on the maturation and the transcriptomic profile of primary hippocampal neurons. Control neurons were grown in the presence of conditioned media from Apoe–/– astrocytes. ApoE2 and apoE3 significantly increase the dendritic arbor branching, the combined neurite length, and the total arbor surface of the hippocampal neurons, while apoE4 fails to produce similar effects and even significantly reduces the combined neurite length compared to the control. ApoE lipoproteins show no systemic effect on dendritic spine density, yet apoE2 and apoE3 increase the mature spines fraction, while apoE4 increases the immature spine fraction. This is associated with opposing effects of apoE2 or apoE3 and apoE4 on the expression of NR1 NMDA receptor subunit and PSD95. There are 1,062 genes differentially expressed across neurons cultured in the presence of apoE lipoproteins compared to the control. KEGG enrichment and gene ontology analyses show apoE2 and apoE3 commonly activate expression of genes involved in neurite branching, and synaptic signaling. In contrast, apoE4 cultured neurons show upregulation of genes related to the glycolipid metabolism, which are involved in dendritic spine turnover, and those which are usually silent in neurons and are related to cell cycle and DNA repair. In conclusion, our work reveals that lipoprotein particles comprised of various apoE isoforms differentially regulate various neuronal arbor characteristics through interaction with neuronal transcriptome. ApoE4 produces a functionally distinct transcriptomic profile, which is associated with attenuated neuronal development. Differential regulation of neuronal transcriptome by apoE isoforms is a newly identified biological mechanism, which has both implication in the development and aging of the CNS.

Are apolipoprotein E fragments a promising new therapeutic target for Alzheimer’s disease?

Human apolipoprotein E (ApoE) is a 299-amino acid secreted glycoprotein that binds cholesterol and phospholipids. ApoE exists as three common isoforms (ApoE2, ApoE3, and ApoE4) and heterozygous carriers of the ε4 allele of the gene encoding ApoE (APOE) have a fourfold greater risk of developing Alzheimer’s disease (AD). The enzymes thrombin, cathepsin D, α-chymotrypsin-like serine protease, and high-temperature requirement serine protease A1 are responsible for ApoE proteolytic processing resulting in bioactive C-terminal-truncated fragments that vary depending on ApoE isoforms, brain region, aging, and neural injury. The objectives of the present narrative review were to describe ApoE processing, discussing current hypotheses about the potential role of various ApoE fragments in AD pathophysiology, and reviewing the current development status of different anti-ApoE drugs. The exact mechanism by which APOE gene variants increase/decrease AD risk and the role of ApoE fragments in the deposition are not fully understood, but APOE is known to directly affect tau-mediated neurodegeneration. ApoE fragments co-localize with neurofibrillary tangles and amyloid β (Aβ) plaques, and may cause neurodegeneration. Among anti-ApoE approaches, a fascinating strategy may be to therapeutically overexpress ApoE2 in APOE ε4/ε4 carriers through vector administration or liposomal delivery systems. Another approach involves reducing ApoE4 expression by intracerebroventricular antisense oligonucleotides that significantly decreased Aβ pathology in transgenic mice. Differences in the proteolytic processing of distinct ApoE isoforms and the use of ApoE fragments as mimetic peptides in AD treatment are also under investigation. Treatment with peptides that mimic the structural and biological properties of native ApoE may reduce Aβ deposition, tau hyperphosphorylation, and glial activation in mouse models of Aβ pathology. Alternative strategies involve the use of ApoE4 structure correctors, passive immunization to target a certain form of ApoE, conversion of the ApoE4 aminoacid sequence into that of ApoE3 or ApoE2, and inhibition of the ApoE-Aβ interaction.

Improving of HDL capacity for macrophages cholesterol efflux after plasma incubation with phospholipid nanoparticles

In connection with recent data about antiatherogenic importance of not only plasma HDL concentration, but of their cell cholesterol efflux capacity as well, the possibility of its correction by phospholipid (PL) nanoparticles was studied. Blood plasma was incubated with earlier elaborated PL nanoparticles emulsion with the particle diameter up to 30 nm, and HDL cholesterol efflux capacity of apo B-depleted plasma was studied. Using macrophages THP-1 preloaded 3H-cholesterol were used. The addition of incubated plasma supernatants with the elevated PL/apo A-1 ratio to cell media resulted in almost increase in two fold 3H-cholesterol efflux as compared with native HDL. The maximal efflux was observed at the PL/apo A-1 ratio of 1.06 as compared with native apo B-depleted plasma (the PL/apo A-1 ratio of 0.85). Results suggest possible usage of ultrasmall PL nanoparticles for regeneration of impaired antiatherogenic HDL functionality. This approach seems to be predominant compared with the usage of PL emulsions with detergent or apoprotein A1.

The SGLT2 Inhibitor Luseogliflozin Rapidly Normalizes Aortic mRNA Levels of Inflammation-Related but Not Lipid-Metabolism-Related Genes and Suppresses Atherosclerosis in Diabetic ApoE KO Mice.

Recent clinical studies have revealed the treatment of diabetic patients with sodium glucose co-transporter2 (SGLT2) inhibitors to reduce the incidence of cardiovascular events. Using nicotinamide and streptozotocin (NA/STZ) -treated ApoE KO mice, we investigated the effects of short-term (seven days) treatment with the SGLT2 inhibitor luseogliflozin on mRNA levels related to atherosclerosis in the aorta, as well as examining the long-term (six months) effects on atherosclerosis development. Eight-week-old ApoE KO mice were treated with NA/STZ to induce diabetes mellitus, and then divided into two groups, either untreated, or treated with luseogliflozin. Seven days after the initiation of luseogliflozin administration, atherosclerosis-related mRNA levels in the aorta were compared among four groups; i.e., wild type C57/BL6J, native ApoE KO, and NA/STZ-treated ApoE KO mice, with or without luseogliflozin. Short-term luseogliflozin treatment normalized the expression of inflammation-related genes such as F4/80, TNFα, IL-1β, IL-6, ICAM-1, PECAM-1, MMP2 and MMP9 in the NA/STZ-treated ApoE KO mice, which showed marked elevations as compared with untreated ApoE KO mice. In contrast, lipid metabolism-related genes were generally unaffected by luseogliflozin treatment. Furthermore, after six-month treatment with luseogliflozin, in contrast to the severe and widely distributed atherosclerotic changes in the aortas of NA/STZ-treated ApoE KO mice, luseogliflozin treatment markedly attenuated the progression of atherosclerosis, without affecting serum lipid parameters such as high density lipoprotein, low density lipoprotein and triglyceride levels. Given that luseogliflozin normalized the aortic mRNA levels of inflammation-related, but not lipid-related, genes soon after the initiation of treatment, it is not unreasonable to speculate that the anti-atherosclerotic effect of this SGLT2 inhibitor emerges rapidly, possibly via the prevention of inflammation rather than of hyperlipidemia.

Effects of native and oxidized apolipoprotein A-I on lipid bilayer microviscosity of erythrocyte plasma membrane

Using a pyrene as a fluorescent probe, we investigated the influence of native and oxidized apolipoprotein A-I (apo A-I) and their complexes with tetrahydrocortisol (THC) on the microviscosity of the erythrocyte plasma membrane. The addition of THC to isolated membranes led to a 17% increase in the membrane microviscosity. In contrast, native apo A-I reduced the microviscosity (i.e., increased the fluidity) of the membranes by 15%. A more pronounced increase (by 25%) in the membrane fluidity was found in the presence of the complex of apo A-I with THC. Unlike native apo A-I, oxidized apo A-I and its complex with THC did not change the membrane viscosity. In view of the fact that apo A-I plays an important role in the binding of membrane cholesterol we suggest that the observed increase in the membrane fluidity under the influence of the native apo A-I is associated with the cholesterol efflux from plasma membrane. Oxidative modification of apo A-I likely disturbs the mechanisms of the cholesterol efflux and prevents the decrease in the membrane microviscosity.

Molecular interactions of small molecule inhibitors targeting cytoplasmic TIR domains.

Abstract Regulation of Toll-like receptor (TLR) signaling using small molecule agonists and antagonists have been widely sought after for controlling inflmation and disease. Most studies have focused on therapeutic targeting of TLR extracellular ligand binding domains. Recently, Computer-aided drug design (CADD) screens specifically targeting intracellular Toll-Interleukin-1 receptor resistance domains have identified and functionally characterized several small molecule and peptide inhibitors which protect against lethality in animal models of infection, inflammation and disease. We have sought to structurally characterize the molecular interactions of CADD derived small molecule inhibitors with respective TLR-TIR domains by determining the X-ray co-crystal structures of TLR2 –TIR domain in the presence of C29 and its substructure O-vanillin. Positive electron density is observed near the BB loop and residue Ile 685 of native TLR2-TIR in the presence of the small molecule inhibitor (C29) exhibit compared with the native apo form of this structure. However, the presence of (S-(DIMETHYLARSENIC) CYSTEINE) in this crystallization condition complicate analysis. To rule out the effects of DMSO and (S-(DIMETHYLARSENIC) CYSTEINE) we sought to examine small molecule inhibitor C29L substructure (o-Vanillin) in a second crystal form of TLR2 and which contains a more simplified crystallization condition grown in the presence of 1mM of C29L. This co-crystal exhibits positive electron density located in an around the BB loop compared with apo forms of this structure. Additionally, in this recent structure form we observe additional DD loop density previously not defined in the originally reported crystal form.

Novel Apo E-Derived ABCA1 Agonist Peptide (CS-6253) Promotes Reverse Cholesterol Transport and Induces Formation of preβ-1 HDL In Vitro.

Apolipoprotein (apo) mimetic peptides replicate some aspects of HDL function. We have previously reported the effects of compound ATI-5261 on its ability to replicate many functions of native apo A-I in the process of HDL biogenesis. ATI-5261 induced muscle toxicity in wild type C57Bl/6 mice, increased CPK, ALT and AST and increase in triglyceride (Tg) levels. Aromatic phenylalanine residues on the non-polar face of ATI-5261, together with positively charged arginine residues at the lipid-water interface were responsible for these effects. This information was used to create a novel analog (CS-6253) that was non-toxic. We evaluated this peptide designed from the carboxyl terminus of apo E, in its ability to mimic apo A-I functionality. Our data shows that the lipidated particles generated by incubating cells overexpressing ABCA1 with lipid free CS-6253 enhances the rate of ABCA1 lipid efflux with high affinity interactions with native ABCA1 oligomeric forms and plasma membrane micro-domains. Interaction between ABCA1 and lipid free CS-6253 resulted in formation of nascent HDL-CS-6253 particles that are actively remodeled in plasma. Mature HDL-CS-6253 particles deliver cholesterol to liver cells via SR-BI in-vitro. CS-6253 significantly increases cholesterol efflux in murine macrophages and in human THP-1 macrophage-derived foam cells expressing ABCA1. Addition of CS-6253 to plasma dose-dependently displaced apo A-I from α-HDL particles and led to de novo formation of preβ-1 HDL that stimulates ABCA1 dependent cholesterol efflux efficiently. When incubated with human plasma CS-6253 was also found to bind with HDL and LDL and promoted the transfer of cholesterol from HDL to LDL predominantly. Our data shows that CS-6253 mimics apo A-I in its ability to promote ABCA1-mediated formation of nascent HDL particles, and enhances formation of preβ-1 HDL with increase in the cycling of apo A-I between the preβ and α-HDL particles in-vitro. These mechanisms are potentially anti-atherogenic.

Abstract 65: ABCA1 Ligand Peptides That Stimulate Cellular Cholesterol Efflux With High Potency Increase Insulin Sensitivity in Metabolic and Genetic Mouse Models of Obesity and Type 2 Diabetes

Anti-diabetic activities of HDL apolipoprotein(apo)s have been described, complementing ability to reduce atherosclerosis. These effects have been attributed, in part, to apos stimulating cholesterol efflux from cells via ABCA1. This suggests apo mimetic peptides that promote ABCA1 cholesterol efflux may be useful to combat diabetes and its cardiovascular complications. Two such peptides (CS6253 and T6991-2) with optimized safety features (NOAEL = 500 mg/kg) and little TG elevating effects have been designed by us through structure-activity studies of a prototypic ABCA1 ligand peptide, ATI-5261, derived from the C-terminal (CT) domain of apoE. Glucose lowering and insulin sensitizing effects of these new peptides were presently evaluated in mouse models of obesity and diabetes. Peptides CS6253 and T6991-2 stimulated cholesterol efflux from macrophages via ABCA1 with high potency similar to the native apoE CT domain (Km= 0.33±0.14, 0.24±02, 0.21±0.02 μM). Administration of CS6253 at a dose of 30 mg/kg (SC) on alternate days for 6 weeks reduced atherosclerosis by 32% in apoE deficient mice fed high-fat, Western diet for 14 weeks (15±2 vs. 22±4% plaque lesions, CS6253 vs. control, p<0.01). T6991-2 administered (SC) at a dose of 10 mg/kg for 6 weeks in chow-fed ob/ob mice showed little effect on steady-state plasma glucose levels vs. controls (126±22 vs. 135±11 mg/dl, respectively); however, the levels of glucose were greatly attenuated with peptide treatment vs. controls in response to GTT (1.8±0.5 vs. 2.8±0.4 fold increases in plasma glucose at 60 min, respectively, p<0.01). Treatment of C57BL/6J mice fed high-fat diet (HFD) with T6991-2 (6 weeks, 30 mg/kg) also enhanced insulin sensitivity by 2.2±0.7 fold vs. controls (55±17 vs. 25±5% reduction in basal glucose levels, respectively, at 15 minutes post insulin, 0.75 Units/kg, p<0.01). The favorable anti-diabetic effects of T6991-2 were not due to changes in total body weight or β-cell function (i.e. unchanged plasma C-peptide levels). Our data indicate that single amphipathic α-helix peptides derived from apoE CT domain are sufficient to confer potent cellular cholesterol efflux and anti-diabetic activities, with therapeutic potential for diabetes and cardiovascular disease.

Nature-inspired nanoformulations for contrast-enhanced in vivo MR imaging of macrophages.

Magnetic resonance imaging (MRI) of macrophages in atherosclerosis requires the use of contrast-enhancing agents. Reconstituted lipoprotein particles that mimic native high-density lipoproteins (HDL) are a versatile delivery platform for Gd-based contrast agents (GBCA) but require targeting moieties to direct the particles to macrophages. In this study, a naturally occurring methionine oxidation in the major HDL protein, apolipoprotein (apo) A-I, was exploited as a novel way to target HDL to macrophages. We also tested if fully functional GBCA-HDL can be generated using synthetic apo A-I peptides. The fluorescence and MRI studies reveal that specific oxidation of apo A-I or its peptides increases the in vitro macrophage uptake of GBCA-HDL by 2-3 times. The in vivo imaging studies using an apo E-deficient mouse model of atherosclerosis and a 3.0 T MRI system demonstrate that this modification significantly improves atherosclerotic plaque detection using GBCA-HDL. At 24 h post-injection of 0.05 mmol Gd kg(-1) GBCA-HDL containing oxidized apo A-I or its peptides, the atherosclerotic wall/muscle normalized enhancement ratios were 90 and 120%, respectively, while those of GBCA-HDL containing their unmodified counterparts were 35 and 45%, respectively. Confocal fluorescence microscopy confirms the accumulation of GBCA-HDL containing oxidized apo A-I or its peptides in intraplaque macrophages. Together, the results of this study confirm the hypothesis that specific oxidation of apo A-I targets GBCA-HDL to macrophages in vitro and in vivo. Furthermore, our observation that synthetic peptides can functionally replace the native apo A-I protein in HDL further encourages the development of these contrast agents for macrophage imaging.

Cognitive Deficits and Disruption of Neurogenesis in a Mouse Model of Apolipoprotein E4 Domain Interaction

Apolipoprotein E4 (apoE4) allele is the major genetic risk factor for sporadic Alzheimer disease (AD) due to the higher prevalence and earlier onset of AD in apoE4 carriers. Accumulating data suggest that the interaction between the N- and the C-terminal domains in the protein may be the main pathologic feature of apoE4. To test this hypothesis, we used Arg-61 mice, a model of apoE4 domain interaction, by introducing the domain interaction feature of human apoE4 into native mouse apoE. We carried out hippocampus-dependent learning and memory tests and related cellular and molecular assays on 12- and 3-month-old Arg-61 and age-matched background C57BL/6J mice. Learning and memory task performance were impaired in Arg-61 mice at both old and young ages compared with C57BL/6J mice. Surprisingly, young Arg-61 mice had more mitotic doublecortin-positive cells in the subgranular zone; mRNA levels of brain-derived neurotrophic factor (BDNF) and TrkB were also higher in 3-month-old Arg-61 hippocampus compared with C57BL/6J mice. These early-age neurotrophic and neurogenic (proliferative) effects in the Arg-61 mouse may be an inadequate compensatory but eventually detrimental attempt by the system to "repair" itself. This is supported by the higher cleaved caspase-3 levels in the young animals that not only persisted, but increased in old age, and the lower levels of doublecortin at old age in the hippocampus of Arg-61 mice. These results are consistent with human apoE4-dependent cognitive and neuro-pathologic changes, supporting the principal role of domain interaction in the pathologic effect of apoE4. Domain interaction is, therefore, a viable therapeutic/prophylactic target for cognitive impairment and AD in apoE4 subjects.

The complex structures of isocitrate dehydrogenase from Clostridium thermocellum and Desulfotalea psychrophila suggest a new active site locking mechanism

Isocitrate dehydrogenase (IDH) catalyzes the oxidative NAD(P)+-dependent decarboxylation of isocitrate into α-ketoglutarate and CO2 and is present in organisms spanning the biological range of temperature. We have solved two crystal structures of the thermophilic Clostridium thermocellum IDH (CtIDH), a native open apo CtIDH to 2.35Å and a quaternary complex of CtIDH with NADP+, isocitrate and Mg2+ to 2.5Å. To compare to these a quaternary complex structure of the psychrophilic Desulfotalea psychrophila IDH (DpIDH) was also resolved to 1.93Å. CtIDH and DpIDH showed similar global thermal stabilities with melting temperatures of 67.9 and 66.9°C, respectively. CtIDH represents a typical thermophilic enzyme, with a large number of ionic interactions and hydrogen bonds per residue combined with stabilization of the N and C termini. CtIDH had a higher activity temperature optimum, and showed greater affinity for the substrates with an active site that was less thermolabile compared to DpIDH. The uncompensated negative surface charge and the enlarged methionine cluster in the hinge region both of which are important for cold activity in DpIDH, were absent in CtIDH. These structural comparisons revealed that prokaryotic IDHs in subfamily II have a unique locking mechanism involving Arg310, Asp251′ and Arg255 (CtIDH). These interactions lock the large domain to the small domain and direct NADP+ into the correct orientation, which together are important for NADP+ selectivity.

Expression of the human apoE2 isoform in adipocytes: altered cellular processing and impaired adipocyte lipogenesis

Expression of apoE in adipocytes has been shown to have an important role in modulating adipocyte triglyceride (TG) metabolism and gene expression that is independent of circulating and extracellular apoE. The impact of adipocyte expression of common human apoE isoforms was evaluated using adipocytes harvested from human apoE2, -3, and -4 knock-in mice. Expression of the apoE2 isoform was associated with an increase in adipocyte apoE gene expression and apoE synthesis. Newly synthesized apoE2 was unstable in adipocytes and demonstrated increased degradation and decreased secretion. ApoE2-expressing mice were hyperlipidemic, and had increased size of gonadal fat pads and of adipocytes, compared with apoE3 mice. In isolated cells, however, expression of the apoE2 isoform produced defective lipogenesis and increased TG hydrolysis. Incubation of adipose tissue with apoE3-containing TG-rich lipoproteins resulted in a significant increase in TG in adipose tissue from apoE3 and -E4 mice, but not apoE2 mice. Reduced capacity to internalize FFA as lipogenic substrate contributed to defective lipogenesis. Newly synthesized apoE2 is unstable in adipocytes and results in decreased adipocyte TG synthesis and defective FA uptake. These changes recapitulate those observed in apoE knockout adipocytes and have implications for understanding metabolic disturbances in humans expressing the E2 isoform.

Regulation of microsomal triglyceride transfer protein by apolipoprotein A-IV in newborn swine intestinal epithelial cells

Apolipoprotein (apo) A-IV overexpression enhances chylomicron (CM) assembly and secretion in newborn swine intestinal epithelial cells by producing larger particles (Lu S, Yao Y, Cheng X, Mitchell S, Leng S, Meng S, Gallagher JW, Shelness GS, Morris GS, Mahan J, Frase S, Mansbach CM, Weinberg RB, Black DD. J Biol Chem 281: 3473-3483, 2006). To determine the impact of apo A-IV on microsomal triglyceride transfer protein (MTTP), IPEC-1 cell lines containing a tetracycline-regulatable expression system were used to overexpress native swine apo A-IV and "piglike" human apo A-IV, a mutant human apo A-IV with deletion of the EQQQ-rich COOH-terminus, previously shown to upregulate basolateral triglyceride (TG) secretion 5-fold and 25-fold, respectively. Cells were incubated 24 h with and without doxycycline and oleic acid (OA, 0.8 mM). Overexpression of the native swine apo A-IV and piglike human apo A-IV increased MTTP lipid transfer activity by 39.7% (P = 0.006) and 53.6% (P = 0.0001), respectively, compared with controls. Changes in mRNA and protein levels generally paralleled changes in activity. Interestingly, native swine apo A-IV overexpression also increased MTTP large subunit mRNA, protein levels, and lipid transfer activity in the absence of OA, suggesting a mechanism not mediated by lipid absorption. Overexpression of piglike human apo A-IV significantly increased partitioning of radiolabeled OA from endoplasmic reticulum (ER) membrane to lumen, suggesting increased net transfer of membrane TG to luminal particles. These results suggest that the increased packaging of TG into nascent CMs in the ER lumen, induced by apo A-IV, is associated with upregulation of MTTP activity at the pretranslational level. Thus MTTP is regulated by apo A-IV in a manner to promote increased packaging of TG into the CM core, which may be important in neonatal fat absorption.

Peroxisome Proliferator-activated Receptor γ Stimulation of Adipocyte ApoE Gene Transcription Mediated by the Liver Receptor X Pathway

Peroxisome proliferator-activated receptor (PPARgamma) agonists increase insulin sensitivity in humans and are useful for treating human diabetes. Treatment with these agonists leads to increased apoE expression and triglyceride accumulation in adipocytes. The importance of apoE for adipocyte triglyceride accumulation is demonstrated by observations that triglyceride accumulation is impaired in apoE knockout adipocytes treated with PPARgamma agonists. The current studies investigate the molecular mechanism for PPARgamma stimulation of the adipocyte apoE gene and demonstrate that the liver receptor X (LXR) response element within an apoE gene downstream enhancer is required for the apoE response to PPARgamma agonists. The response of the apoE gene to treatment with PPARgamma agonists was delayed beyond 12 h suggesting the involvement of an intermediary pathway. The combined addition of PPARgamma and LXR agonists did not increase apoE response beyond that observed with addition of either alone. Deletion or mutation of the LXR response element completely eliminated the adipocyte apoE gene response to a PPARgamma agonist. Chromatin immunoprecipitation analyses performed using isolated adipocytes, or adipose tissue from mice treated with PPARgamma agonists, showed increased LXR binding to the apoE gene after PPARgamma agonist treatment. Knockdown of LXR expression completely eliminated the increase in apoE message, protein, and triglyceride in response to PPARgamma stimulation. The LXR response element has been previously shown to mediate sterol responsiveness of the apoE gene, and apoE expression plays an important role in adipocyte triglyceride balance. The current observations suggest that the PPARgamma-LXR-apoE regulatory cascade could be an important molecular link for cross-talk between adipocyte triglyceride and cholesterol homeostasis.

Treatment with apo B peptide vaccines inhibits atherosclerosis in human apo B‐100 transgenic mice without inducing an increase in peptide‐specific antibodies

Autoantibodies to apolipoprotein (apo) B-100 peptides are present in human plasma and have been shown to be associated with decreased cardiovascular risk. The present study aimed to determine if apo B-100 peptide vaccines are atheroprotective in mice expressing human apo B-100 and if the effectiveness of the vaccines is influenced by the level of pre-existing peptide-specific autoantibodies. LDL receptor(-/-)/human apo B-100 transgenic mice were immunized with native human apo B-100 peptides p45 or p210 at 6, 9 and 11 weeks and the extent of atherosclerosis determined by en face Oil Red O staining of the aorta at 25 weeks. Autoantibody levels were determined by enzyme-linked immunosorbent assay, and RNA expression in the spleen was assessed by real time PCR. Control mice had high levels of autoantibodies against p210 but only low levels against p45. Immunization with native p45 and p210 reduced atherosclerosis by 66% (P < 0.02) and 59% (P = 0.06), respectively. The atheroprotective effect of apo B peptide immunization occurred in the absence of an increase in peptide-specific IgG, but was associated with an increase in IgM recognizing native and copper-oxidized LDL. Immunization with apo B peptide-based vaccines inhibits atherosclerosis in mice expressing human apo B-100 suggesting that they can interact with their target as expressed in humans. The protective effect is independent of the pre-existing level of apo B peptide autoantibodies and can occur without activating an increase in peptide-specific antibodies suggesting that atheroprotection can be mediated by cellular immune responses.

Systemic and Specific Delivery of Small Interfering RNAs to the Liver Mediated by Apolipoprotein A-I

Tissue-targeted delivery of small interfering RNA (siRNA) must be achieved before RNA interference (RNAi) technology can be used in practical therapeutic approaches. In this study, the potential of apolipoprotein A-I (apo A-I) for the systemic delivery of nucleic acids to the liver is demonstrated using real-time in vivo imaging. As a proof of concept, synthetic siRNAs against hepatitis B virus (HBV) were formulated into complexes of apo A-I and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/cholesterol (DTC-Apo) and injected intravenously (i.v.) into a mouse model carrying replicating HBV. We show that administration of these nanoparticles can significantly reduce viral protein expression by receptor-mediated endocytosis. The advantages of the apo A-I-mediated siRNA delivery method are its liver specificity, its effectiveness at low doses (< or = 2 mg/kg) in only a single treatment, and its persistent antiviral effect up to 8 days. The liver-targeted gene silencing was also shown by in vivo images, in which bioluminescent signals emitted from the liver were efficiently reduced after i.v. administration of luciferase-specific siRNA and DTC-Apo lipoplex. Thus, our unique approach to siRNA delivery creates a foundation for the development of a new class of promising therapeutics against hepatitis viruses or hepatocyte genes related to tumor growth.

Recombinant Neural Protein PrP Can Bind with Both Recombinant and Native Apolipoprotein E &amp;lt;italic&amp;gt;In Vitro&amp;lt;/italic&amp;gt;

The most essential and crucial step during the pathogenesis of transmissible spongiform encephalopathy is the conformational change of cellular prion protein (PrP(C)) to pathologic isoform (PrP(Sc)). A lot of data revealed that caveolae-like domains (CLDs) in the cell surface were the probable place where the conversion of PrP proteins happened. Apolipoprotein E (ApoE) is an apolipoprotein which is considered to play an important role in the development of Alzheimer's disease and other neurodegenerative diseases by forming protein complex through binding to the receptor located in the clathrin-coated pits of the cell surface. In this study, a 914-bp cDNA sequence encoding human ApoE3 was amplified from neuroblastoma cell line SH-SY5Y. Three human ApoE isomers were expressed and purified from Escherichia coli. ApoE-specific antiserum was prepared by immunizing rabbits with the purified ApoE3. GST/His pull-down assay, immunoprecipitation and ELISA revealed that three full-length ApoE isomers interact with the recombinant full-length PrP protein in vitro. The regions corresponding to protein binding were mapped in the N-terminal segment of ApoE (amino acid 1-194) and the N-terminal of PrP (amino acid 23-90). Moreover, the recombinant PrP showed the ability to form a complex with the native ApoE from liver tissues. Our data provided direct evidence of molecular interaction between ApoE and PrP. It also supplied scientific clues for assessing the significance of CLDs on the surface of cellular membrane in the process of conformational conversion from PrP(C) to PrP(Sc) and probing into the pathogenesis of transmissible spongiform encephalopathy.