Characterization of nanodisc-forming peptides for membrane protein studies.

Lipid-bilayer nanodiscs provide a stable, native-like membrane environment for the functional and structural studies of membrane proteins and other membrane-binding molecules. Peptide-based nanodiscs having unique properties are developed for membrane protein studies and other biological applications. While the self-assembly process rendering the formation of peptide-nanodiscs is attractive, it is important to understand the stability and suitability of these nanodisc systems for membrane protein studies. In this study, we investigated the nanodiscs formation by the anti-inflammatory and tumor-suppressing peptide AEM28. AEM28 is a chimeric peptide containing a cationic-rich heparan sulfate proteoglycan- (HSPG)-binding domain from human apolipoprotein E (hapoE) (141-150) followed by the 18A peptide's amino acid sequence. AEM28-based nanodiscs made with different types of lipids were characterized using various biophysical techniques and compared with the nanodiscs formed using 2F or 4F peptides. Variable temperature dynamic light-scattering and 31P NMR experiments indicated the fusion and size heterogeneity of nanodiscs at high temperatures. The suitability of AEM28 and Ac-18A-NH2- (2F-) based nanodiscs for studying membrane proteins is demonstrated by reconstituting and characterizing a drug-metabolizing enzyme, cytochrome-P450 (CYP450), or the redox complex CYP450-CYP450 reductase. AEM28 and 2F were also tested for their efficacies in solubilizing E. coli membranes to understand the possibility of using them for detergent-free membrane protein isolation. Our experimental results suggest that AEM28 nanodiscs are suitable for studying membrane proteins with a net positive charge, whereas 2F-based nanodiscs are compatible with any membrane proteins and their complexes irrespective of their charge. Furthermore, both peptides solubilized E. coli cell membranes, indicating their use in membrane protein isolation and other applications related to membrane solubilization.

Peptides as Therapeutic Agents for Atherosclerosis.

More than three decades ago, as a test for the amphipathic helix theory, an 18 amino acid residue peptide and its analogs were designed with no sequence homology to any of the exchangeable apolipoproteins. Based on the apolipoprotein A-I (the major protein component of high density lipoproteins, HDL) mimicking properties, they were termed as ApoA-I mimicking peptides. Several laboratories around the world started studying such de novo-designed peptides for their antiatherogenic properties. The present chapter describes the efforts in bringing these peptides as therapeutic agents for atherosclerosis and several lipid-mediated disorders.

Nanodisc reconstitution of flavin mononucleotide binding domain of cytochrome-P450-reductase enables high-resolution NMR probing.

Cytochrome-P450-reductase transfers electrons to cytochrome-P450 through its flavin mononucleotide binding domain (FBD). Despite the importance of membrane-anchoring for FBD function, studies have focused on its soluble domain lacking the transmembrane-domain. Here we demonstrate that the reconstitution of FBD in nanodiscs enables high-resolution NMR measurements and renders a stable conformation.

Apolipoprotein Mimetic Peptides: An Emerging Therapy against Diabetic Inflammation and Dyslipidemia.

Obesity has achieved epidemic status in the United States, resulting in an increase in type 2 diabetes mellitus, dyslipidemia, and cardiovascular disease. Numerous studies have shown that inflammation plays a key role in the development of insulin resistance and diabetic complications. HDL cholesterol levels are inversely associated with coronary heart disease in humans. The beneficial effect of HDL is due, in part, to apolipoproteins A-I and E, which possess anti-inflammatory properties. The functional quality of HDL, however, may be reduced in the context of diabetes. Thus, raising levels of functional HDL is an important target for reducing inflammation and diabetic complications. Apo A-I possesses eight alpha-helical sequences, most of which form class A amphipathic helical structures. Peptides belonging to this class inhibit atherogenesis in several mouse models. Additional peptides based on structural components of apoE have been shown to mediate a rapid clearance of atherogenic lipoproteins in dyslipidemic mice. In this review, we discuss the efficacy of apolipoprotein mimetic peptides in improving lipoprotein function, reducing inflammation, and reversing insulin resistance and cardiometabolic disease processes in diabetic animals.

The Apolipoprotein A-I Mimetic L-4F Attenuates Monocyte Activation and Adverse Cardiac Remodeling after Myocardial Infarction.

Excessive inflammation after myocardial infarction (MI) can promote infarct expansion and adverse left ventricular (LV) remodeling. L-4F, a mimetic peptide of apolipoprotein A-I (apoA-I), exhibits anti-inflammatory and anti-atherogenic properties; however, whether L-4F imparts beneficial effects after myocardial infarction (MI) is unknown. Here we demonstrate that L-4F suppresses the expansion of blood, splenic, and myocardial pro-inflammatory monocytes and macrophages in a mouse model of reperfused MI. Changes in immune cell profiles were accompanied by alleviation of post-MI LV remodeling and dysfunction. In vitro, L-4F also inhibited pro-inflammatory and glycolytic gene expression in macrophages. In summary, L-4F treatment prevents prolonged and excessive inflammation after MI, in part through modulation of pro-inflammatory monocytes and macrophages, and improves post-MI LV remodeling. These data suggest that L-4F could be a used as a therapeutic adjunct in humans with MI to limit inflammation and alleviate the progression to heart failure.

Expression, purification, and functional reconstitution of 19F-labeled cytochrome b5 in peptide nanodiscs for NMR studies.

Microsomal cytochrome b5 (cytb5) is a membrane-bound protein capable of donating the second electron to cytochrome P450s (cytP450s) in the cytP450s monooxygenase reactions. Recent studies have demonstrated the importance of the transmembrane domain of cytb5 in the interaction with cytP450 by stabilizing its monomeric structure. While recent NMR studies have provided high-resolution insights into the structural interactions between the soluble domains of ~16-kDa cytb5 and ~57-kDa cytP450 in a membrane environment, there is need for studies to probe the residues in the transmembrane region as well as to obtain intermolecular distance constraints to better understand the very large size cytb5-cytP450 complex structure in a near native membrane environment. In this study, we report the expression, purification, functional reconstitution of 19F-labeled full-length rabbit cytb5 in peptide based nanodiscs for structural studies using NMR spectroscopy. Size exclusion chromatography, dynamic light scattering, transmission electron microscopy, and NMR experiments show a stable reconstitution of cytb5 in 4F peptide-based lipid-nanodiscs. The reported results demonstrate the use of 19F NMR experiments to study 19F-labeled (with 5-fluorotryptophan (5FW)) cytb5 reconstituted in peptide-nanodiscs and the detection of residues from the transmembrane domain by solution 19F NMR experiments. 19F NMR results revealing the interaction of the transmembrane domain of cytb5 with the full-length rabbit cytochrome P450 2B4 (CYP2B4) are also presented. We expect the results presented in this study to be useful to devise approaches to probe the structure, dynamics and functional roles of transmembrane domains of a membrane protein, and also to measure intermolecular 19F-19F distance constraints to determine the structural interactions between the transmembrane domains.

Structural Interaction of Apolipoprotein A-I Mimetic Peptide with Amyloid-ß Generates Toxic Hetero-oligomers.

Apolipoproteins are involved in pathological conditions of Alzheimer's disease (AD), and it has been reported that truncated apolipoprotein fragments and ß-amyloid (Aß) peptides coexist as neurotoxic heteromers within the plaques. Therefore, it is important to investigate these complexes at the molecular level to better understand their properties and roles in the pathology of AD. Here, we present a mechanistic insight into such heteromerization using a structurally homologue apolipoprotein fragment of apoA-I (4F) complexed with Aß(M1-42) and characterize their toxicity. The 4F peptide slows down the aggregation kinetics of Aß(M1-42) by constraining its structural plasticity. NMR and CD experiments identified 4F-Aß(M1-42) heteromers comprised of unstructured Aß(M1-42) and helical 4F. A uniform two-fold reduction in 15N/1H NMR signal intensities of Aß(M1-42) with no observable chemical shift perturbation indicated the formation of a large complex, which was further confirmed by diffusion NMR experiments. Microsecond-scale atomistic molecular dynamics simulations showed that 4F interaction with Aß(M1-42) is electrostatically driven and induces unfolding of Aß(M1-42). Neurotoxicity profiling of Aß(M1-42) complexed with 4F confirms a significant reduction in cell viability and neurite growth. Thus, the molecular architecture of heteromerization between 4F and Aß(M1-42) discovered in this study provides evidence toward our understanding of the role of apolipoproteins or their truncated fragments in exacerbating AD pathology.

Probing protein-protein and protein-substrate interactions in the dynamic membrane-associated ternary complex of cytochromes P450, b5, and reductase.

Cytochrome P450 (cytP450) interacts with two redox partners, cytP450 reductase and cytochrome-b5, to metabolize substrates. Using NMR, we reveal changes in the dynamic interplay when all three proteins are incorporated into lipid nanodiscs in the absence and presence of substrates.

Apolipoprotein A-I mimetics mitigate intestinal inflammation in COX2-dependent inflammatory bowel disease model.

Cyclooxygenase 2 (Cox2) total knockout and myeloid knockout (MKO) mice develop Crohn's-like intestinal inflammation when fed cholate-containing high fat diet (CCHF). We demonstrated that CCHF impaired intestinal barrier function and increased translocation of endotoxin, initiating TLR/MyD88-dependent inflammation in Cox2 KO but not WT mice. Cox2 MKO increased pro-inflammatory mediators in LPS-activated macrophages, and in the intestinal tissue and plasma upon CCHF challenge. Cox2 MKO also reduced inflammation resolving lipoxin A4 (LXA4) in intestinal tissue, while administration of an LXA4 analog rescued disease in Cox2 MKO mice fed CCHF. The apolipoprotein A-I (APOA1) mimetic 4F mitigated disease in both the Cox2 MKO/CCHF and piroxicam-accelerated Il10-/- models of inflammatory bowel disease (IBD) and reduced elevated levels of pro-inflammatory mediators in tissue and plasma. APOA1 mimetic Tg6F therapy was also effective in reducing intestinal inflammation in the Cox2 MKO/CCHF model. We further demonstrated that APOA1 mimetic peptides: i) inhibited LPS and oxidized 1-palmitoyl-2-arachidonoyl-sn-phosphatidylcholine (oxPAPC) dependent pro-inflammatory responses in human macrophages and intestinal epithelium; and ii) directly cleared pro-inflammatory lipids from mouse intestinal tissue and plasma. Our results support a causal role for pro-inflammatory and inflammation resolving lipids in IBD pathology and a translational potential for APOA1 mimetic peptides for the treatment of IBD.

Probing membrane enhanced protein-protein interactions in a minimal redox complex of cytochrome-P450 and P450-reductase.

Investigating the interplay in a minimal redox complex of cytochrome-P450 and its reductase is crucial for understanding cytochrome-P450's enzymatic activity. Probing the hotspots of dynamic structural interactions using NMR revealed the engagement of loop residues from P450-reductase to be responsible for the enhanced affinity of CYP450 towards its obligate redox partner.

Lipoprotein modulation of proteinuric renal injury.

High-density lipoprotein (HDL) and its main protein, apolipoprotein AI (apoAI), have established benefits in various cells, but whether these cytoprotective effects of HDL pertain to renal cells is unclear. We investigated the in vitro consequences of exposing damaged podocytes to normal apoAI, HDL, and apoAI mimetic (L-4F), and the in vivo effects of L-4F on kidney and atherosclerotic injury in a podocyte-specific injury model of proteinuria. In vitro, primary mouse podocytes were injured by puromycin aminonucleoside (PAN). Cellular viability, migration, production of reactive oxygen species (ROS), apoptosis, and the underlying signaling pathway were assessed. In vivo, we used a proteinuric model, Nphs1-hCD25 transgenic (NEP25+) mice, which express human CD25 on podocytes. Podocyte injury was induced by using immunotoxin (LMB2) and generated a proteinuric atherosclerosis model, NEP25+:apoE-/- mice, was generated by mating apoE-deficient (apoE-/-) mice with NEP25+ mice. Animals received L-4F or control vehicle. Renal function, podocyte injury, and atherosclerosis were assessed. PAN reduced podocyte viability, migration, and increased ROS production, all significantly lessened by apoAI, HDL, and L-4F. L-4F attenuated podocyte apoptosis and diminished PAN-induced inactivation of Janus family protein kinase-2/signal transducers and activators of transcription 3. In NEP25+ mice, L-4F significantly lessened overall proteinuria, and preserved podocyte expression of synaptopodin and cell density. Proteinuric NEP25+:apoE-/- mice had more atherosclerosis than non-proteinuric apoE-/- mice, and these lesions were significantly decreased by L-4F. Normal human apoAI, HDL, and apoAI mimetic protect against podocyte damage. ApoAI mimetic provides in vivo beneficial effects on podocytes that culminate in reduced albuminuria and atherosclerosis. The results suggest supplemental apoAI/apoAI mimetic may be a novel candidate to lessen podocyte damage and its complications.

The apoA-I mimetic peptide 4F protects apolipoprotein A-I from oxidative damage.

High density lipoprotein (HDL) is prone to modification by the oxidizing and chlorinating agent hypochlorite anion (OCl-). Oxidation of apolipoprotein (apo) A-I, the major protein in HDL, reduces ABCA-1 mediated cholesterol efflux and other protective responses to HDL. The apoA-I mimetic peptide 4F has been shown to undergo oxidation; however, the ability of the peptide to mediate cholesterol efflux remains intact. Here, we show that 4F protects apoA-I from hypochlorite-mediated oxidation. Mass spectral analysis of apoA-I shows that tyrosine residues that are prone to hypochlorite-mediated chlorination are protected in the presence of 4F. Furthermore, 4F enhances the cholesterol efflux ability of apoA-I to a greater extent than either 4F or apoA-I alone, even after hypochlorite oxidation. These observations suggest that apoA-I in lipid complexes may be protected by the presence of 4F, resulting in the preservation of its anti-inflammatory and anti-atherogenic properties. These studies also form the basis for the future studies of nanoparticles possessing both apoA-I and 4F.

Solid-state NMR structural investigations of peptide-based nanodiscs and of transmembrane helices in bicellar arrangements.

The membrane topology of the peptide 18A, a derivative of apolipoprotein A-I, is investigated in structural detail. Apolipoprotein A-I is the dominant protein component of high density lipoproteins with important functions in cholesterol metabolism. 18A (Ac-DWLKA FYDKV AEKLK EAF- NH2) was designed to mimic the structure of tandem domains of class A amphipathic helices and has served as a lead peptide for biomedical applications. At low peptide-to-lipid ratios 18A partitions into phosphatidylcholine membranes with helix topologies parallel to the membrane surface, an alignment that is maintained when disc-like bicelles form at higher peptide-to-lipid ratios. Notably, the bicelles interact cooperatively with the magnetic field of the NMR spectrometer, thus the bilayer normal is oriented perpendicular to the magnetic field direction. A set of peptides that totals four 15N or 2H labelled positions of 18A allowed the accurate analysis of tilt and azimuthal angles relative to the membrane surface under different conditions. The topology agrees with a double belt arrangement forming a rim that covers the hydrophobic fatty acyl chains of the bicelles. In another set of experiments, it was shown that POPC nanodiscs prepared in the presence of diisobutylene/maleic acid (DIBMA) polymers can also be made to align in the magnetic field. Finally, the transmembrane domains of the DQ alpha-1 and DQ beta-1 subunits of the major histocomptability complex (MHC) class II have been prepared and reconstituted into magnetically oriented bicelles for NMR structural analysis.

Apolipoprotein A1 Forms 5/5 and 5/4 Antiparallel Dimers in Human High-density Lipoprotein.

Apolipoprotein A1 (APOA1), the major protein of high-density lipoprotein (HDL), contains 10 helical repeats that play key roles in protein-protein and protein-lipid interactions. The current structural model for HDL proposes that APOA1 forms an antiparallel dimer in which helix 5 in monomer 1 associates with helix 5 in monomer 2 along a left-left (LL5/5) interface, forming a protein complex with a 2-fold axis of symmetry centered on helix 5. However, computational studies suggest that other orientations are possible. To test this idea, we used a zero-length chemical cross-linking reagent that forms covalent bonds between closely apposed basic and acidic residues. Using proteolytic digestion and tandem mass spectrometry, we identified amino acids in the central region of the antiparallel APOA1 dimer of HDL that were in close contact. As predicted by the current model, we found six intermolecular cross-links that were consistent with the antiparallel LL5/5 registry. However, we also identified three intermolecular cross-links that were consistent with the antiparallel LL5/4 registry. The LL5/5 is the major structural conformation of the two complexes in both reconstituted discoidal HDL particles and in spherical HDL from human plasma. Molecular dynamic simulations suggest that that LL5/5 and LL5/4 APOA1 dimers possess similar free energies of dimerization, with LL5/5 having the lowest free energy. Our observations indicate that phospholipidated APOA1 in HDL forms different antiparallel dimers that could play distinct roles in enzyme regulation, assembly of specific protein complexes, and the functional properties of HDL in humans.

Nanodisc-Forming Scaffold Protein Promoted Retardation of Amyloid-Beta Aggregation.

Peptidic nanodiscs are useful membrane mimetic tools for structural and functional studies of membrane proteins, and membrane interacting peptides including amyloids. Here, we demonstrate anti-amyloidogenic activities of a nanodisc-forming 18-residue peptide (denoted as 4F), both in lipid-bound and lipid-free states by using Alzheimer's amyloid-beta (Aß40) peptide as an example. Fluorescence-based amyloid fibrillation kinetic assays showed a significant delay in Aß40 amyloid aggregation by the 4F peptide. In addition, 4F-encased lipid nanodiscs, at an optimal concentration of 4F (>20 µM) and nanodisc size (<10 nm), significantly affect amyloid fibrillation. A comparison of experimental results obtained from nanodiscs with that obtained from liposomes revealed a substantial inhibitory efficacy of 4F-lipid nanodiscs against Aß40 aggregation and were also found to be suitable to trap Aß40 intermediates. A combination of atomistic molecular dynamics simulations with NMR and circular dichroism experimental results exhibited a substantial change in Aß40 conformation upon 4F binding through electrostatic and π-π interactions. Specifically, the 4F peptide was found to interfere with the central ß-sheet-forming residues of Aß40 through substantial hydrogen, π-π, and π-alkyl interactions. Fluorescence experiments and coarse-grained molecular dynamics simulations showed the formation of a ternary complex, where Aß40 binds to the proximity of peptidic belt and membrane surface that deaccelerate amyloid fibrillation. Electron microscopy images revealed short and thick amyloid fibers of Aß40 formed in the presence of 4F or 4F-lipid nanodsics. These findings could aid in the development of amyloid inhibitors as well as in stabilizing Aß40 intermediates for high-resolution structural and neurobiological studies.

Novel fatty acyl apoE mimetic peptides have increased potency to reduce plasma cholesterol in mice and macaques.

Ac-hE18A-NH2 is a dual-domain apoE mimetic peptide that possesses the putative receptor binding domain from apoE (LRKLRKRLLR, denoted hE; residues 141-150) covalently attached to lipid-associating peptide 18A. Like apoE, Ac-hE18A-NH2 reduces plasma cholesterol in animal models and exhibits anti-inflammatory properties independent of its cholesterol-reducing effect. Ac-hE18A-NH2 has already undergone phase I clinical trials as a lipid-lowering agent. To explore the therapeutic potential more, we designed and synthesized new analogues by linking ɑ-aminohexanoic acid, octanoic acid, or myristic acid to LRRLRRRLLR-18A-NH2 ([R]hE18A-NH2) and examined the cholesterol-lowering potency in animals. The modified peptides effectively reduced plasma cholesterol in apoE-null mice fed standard chow or a Western diet; the myristyl analogue was the most effective. A single administration of the myristyl analogue reduced plasma total and LDL cholesterol in a dose-dependent manner in hypercholesterolemic cynomolgus macaques for up to 1 week despite the continuation of a cholesterol-supplemented diet. The myristyl peptide (7.4 mg/kg) reduced total and LDL cholesterol at 24 h by 64% and 74%, respectively; plasma HDL levels were modestly reduced and returned to baseline by day 7. These new analogues should exhibit enhanced potency at lower doses than Ac-hE18A-NH2, which may make them attractive therapeutic candidates for clinical trials.

Supramolecular Organization of Apolipoprotein-A-I-Derived Peptides within Disc-like Arrangements.

Apolipoprotein A-I is the major protein component of high-density lipoproteins and fulfils important functions in lipid metabolism. Its structure consists of a chain of tandem domains of amphipathic helices. Using this protein as a template membrane scaffolding protein, class A amphipathic helical peptides were designed to support the amphipathic helix theory and later as therapeutic tools in biomedicine. Here, we investigated the lipid interactions of two apolipoprotein-A-I-derived class A amphipathic peptides, 14A (Ac-DYLKA FYDKL KEAF-NH2) and 18A (Ac-DWLKA FYDKV AEKLK EAF- NH2), including the disc-like supramolecular structures they form with phospholipids. Thus, the topologies of 14A and 18A in phospholipid bilayers have been determined by oriented solid-state NMR spectroscopy. Whereas at a peptide-to-lipid ratio of 2 mol% the peptides align parallel to the bilayer surface, at 7.5 mol% disc-like structures are formed that spontaneously orient in the magnetic field of the NMR spectrometer. From a comprehensive data set of four 15N- or 2H-labeled positions of 14A, a tilt angle, which deviates from perfectly in-planar by 14°, and a model for the peptidic rim structure have been obtained. The tilt and helical pitch angles are well suited to cover the hydrophobic chain region of the bilayer when two peptide helices form a head-to-tail dimer. Thus, the detailed topology found in this work agrees with the peptides forming the rim of nanodiscs in a double belt arrangement.

Lipid-exchange in nanodiscs discloses membrane boundaries of cytochrome-P450 reductase.

Lipids are critical for the function of membrane proteins. NADPH-cytochrome-P450-reductase, the sole electron transferase for microsomal oxygenases, possesses a conformational dynamics entwined with its topology. Here, we use peptide-nanodiscs to unveil cytochrome-P450-reductase's lipid boundaries, demonstrating a protein-driven enrichment of ethanolamine lipids (by 25%) which ameliorates by 3-fold CPR's electron-transfer ability.

The Apolipoprotein E Mimetic Peptide AEM-2 Attenuates Mitochondrial Injury And Apoptosis In Human THP-1 Macrophages.

Cardiovascular disease, specifically atherosclerosis, is exacerbated by hypercholesterolemia. Current therapies that target lipid lowering, however, are not effective in all patients. Apolipoprotein E (apoE) plays an important role in mediating the clearance of plasma cholesterol and also exerts numerous cytoprotective responses. Our laboratory has synthesized novel therapeutics that mimic the ability of apoE to decrease plasma cholesterol. The apoE mimetic peptide AEM-2 is a dual domain peptide composed of an amphipathic helical region that binds phospholipids and a positively charged region that mediates the hepatic clearance of lipoproteins. Administration of AEM-2 to apoE null mice reduced plasma cholesterol concentration by 80% one hour post-administration. Since apoE is also known to exert anti-inflammatory effects that are independent of its ability to lower cholesterol, we tested effects of AEM-2 on lipopolysaccharide-induced responses in human THP-1 macrophages. Pre-treatment of THP-1 cells with AEM-2 significantly reduced the LPS-induced secretion of IL-6 and TNFα. Since LPS administration is associated with an increase in mitochondrial injury, we monitored effects of AEM-2 on mitochondrial function. AEM-2 significantly reduced mitochondrial superoxide formation, prevented the LPS-induced decrease in mitochondrial membrane potential and attenuated the release of cytochrome c. AEM-2 also inhibited the activities of initiator caspases 8 and 9 and effector caspase 3. The attenuation of apoptosis in AEM-2 treated cells was associated with an increase in cellular autophagy. These data suggest that AEM-2 attenuates cellular injury in LPS-treated THP-1 macrophages and facilitates the removal of cellular debris and damaged organelles via induction of autophagy.