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.

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.

Bovine HDL and Dual Domain HDL-Mimetic Peptides Inhibit Tumor Development in Mice.

A growing body of literature supports the role of apolipoproteins present in HDL in the treatment of pro-inflammatory diseases including cancer. We examined whether bovine HDL (bHDL) and three dual-domain peptides, namely AEM-28 and its analog AEM-28-2, and HM-10/10, affect tumor growth and development in mouse models of ovarian and colon cancer. We demonstrate that bHDL inhibits mouse colorectal cancer cell line CT26-mediated lung tumor development, and mouse ovarian cancer cell line ID8-mediated tumor burden.

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.

Expression, purification, and functional reconstitution of F-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.

Structural Interaction of Apolipoprotein A-I Mimetic Peptide with Amyloid-β 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 β-amyloid (Aβ) 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 Aβ(M1-42) and characterize their toxicity.

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

Cytochrome P450 (cytP450) interacts with two redox partners, cytP450 reductase and cytochrome-b, 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.

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.

High-Resolution Structural Studies Elucidate Antiatherogenic and Anti-Inflammatory Properties of Peptides Designed to Mimic Amphipathic α-Helical Domains of Apolipoprotein A-I.

Peptides designed to mimic the antiatherogenic and anti-inflammatory properties of apolipoprotein A-I show that although lipid association is required, not all lipid-associating peptides exhibit these properties. Our studies of a series of peptides showed that peptides with aromatic residues at the center of the nonpolar face were able to interact with inflammatory lipids and inhibited inflammation, which resulted in the amelioration of several lipid-mediated disorders such as lesion development, tumor formation, and Alzheimer's plaque formation. The p values determined using C nuclear magnetic resonance (NMR) spectroscopy of K residues located at the polar-nonpolar interface provided the first clue to the relative orientations of the peptide helices with respect to each other and around the edge of the lipid discoidal complexes.

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).

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- NH) was designed to mimic the structure of tandem domains of class A amphipathic helices and has served as a lead peptide for biomedical applications.

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.

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.

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

Ac-hE18A-NH 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-NH reduces plasma cholesterol in animal models and exhibits anti-inflammatory properties independent of its cholesterol-reducing effect. Ac-hE18A-NH has already undergone phase I clinical trials as a lipid-lowering agent.

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.

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.

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.

A Minimal Functional Complex of Cytochrome P450 and FBD of Cytochrome P450 Reductase in Nanodiscs.

Structural interactions that enable electron transfer to cytochrome-P450 (CYP450) from its redox partner CYP450-reductase (CPR) are a vital prerequisite for its catalytic mechanism. The first structural model for the membrane-bound functional complex to reveal interactions between the full-length CYP450 and a minimal domain of CPR is now reported. The results suggest that anchorage of the proteins in a lipid bilayer is a minimal requirement for CYP450 catalytic function.

Cytochrome-P450-Induced Ordering of Microsomal Membranes Modulates Affinity for Drugs.

Although membrane environment is known to boost drug metabolism by mammalian cytochrome P450s, the factors that stabilize the structural folding and enhance protein function are unclear. In this study, we use peptide-based lipid nanodiscs to "trap" the lipid boundaries of microsomal cytochrome P450 2B4. We report the first evidence that CYP2B4 is able to induce the formation of raft domains in a biomimetic compound of the endoplasmic reticulum.

Membrane environment drives cytochrome P450's spin transition and its interaction with cytochrome b.

Heme's spin-multiplicity is key in determining the enzymatic function of cytochrome P450 (cytP450). The origin of the low-spin state in ferric P450 is still under debate. Here, we report the first experimental demonstration of P450's membrane interaction altering its spin equilibrium which is accompanied by a stronger affinity for cytochrome b.