Richard M. Epand

McMaster University, Hamilton, Ontario, Canada

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Publications (505)1971.74 Total impact

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    ABSTRACT: The epsilon isoform of mammalian diacylglycerol kinase (DGKϵ) is an enzyme that associates strongly with membranes and acts on a lipid substrate, diacylglycerol. The protein has one segment that is predicted to be a transmembrane helix, but appears to interconvert between a transmembrane helix and a re-entrant helix. Despite the hydrophobicity of this segment and the fact that the lipid substrate is also hydrophobic, removal of this hydrophobic segment by truncating the protein at the amino terminus has no effect on its enzymatic activity. The amino acid sequence of the catalytic segment of DGKϵ is highly homologous to that of a bacterial DGK, DgkB. This has allowed us to predict a conformation of DGKϵ based on the known crystal structure of DgkB. An important property of DGKϵ is that it is specific for diacylglycerol species containing an arachidonoyl group. The region of DGKϵ that interacts with this group is found within the accessory domain of the protein and not in the active site nor in the hydrophobic amino terminus. The nature of the acyl chain specificity of the enzyme indicates that DGKϵ is associated with the synthesis of phosphatidylinositol. Defects or deletion of the enzyme give rise to several disease states. Copyright © 2015. Published by Elsevier Ireland Ltd.
    Chemistry and Physics of Lipids 06/2015; DOI:10.1016/j.chemphyslip.2015.06.003 · 2.59 Impact Factor
  • Richard M. Epand, Diana Bach, Ellen Wachtel
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    ABSTRACT: In a recent article, Garg et al. used neutron scattering techniques to determine the limiting amount of cholesterol which vesicles of either POPS or POPC can accommodate. This amount was called “the cholesterol solubility limit”. In light of extensive literature on cholesterol phase separation in phospholipid bilayers, the way in which “solubility limit” is defined in this article and the conclusions derived are misleading and require some clarification.
    Soft Matter 06/2015; DOI:10.1039/C4SM02819H · 4.15 Impact Factor
  • Kenneth D'Souza, Richard M Epand
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    ABSTRACT: Phosphatidylinositol (PI) and its phosphorylated forms are required for many critical cellular processes. PI is highly enriched at its sn-1 and sn-2 positions, the major species being 1-stearoyl-2-arachidonoyl PI (18:0/20:4 PI). This enrichment is achieved in part through enzymatic acyl chain specificity in PI synthesis. Here we characterize the acyl chain specificity of phosphatidylinositol synthase (PIS), which is involved in the terminal step of PI synthesis. Unlike several other enzymes involved in PI synthesis, PIS appears to exhibit no acyl chain specificity towards its substrates. Thus, enrichment of newly formed PI occurs prior to the terminal synthesis step.
    Biochemistry 01/2015; 54(5). DOI:10.1021/bi5015634 · 3.01 Impact Factor
  • Richard M Epand
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    ABSTRACT: Biological membranes are composed largely of lipids and proteins. The most common arrangement of lipids in biological membranes is as a bilayer. This arrangement spontaneously forms a barrier for the passage of polar materials. The bilayer is thin but can have a large area in the dimension perpendicular to its thickness. The physical nature of the bilayer membrane will vary according to the conditions of the environment as well as the chemical structure of the lipid constituents of the bilayer. These physical properties determine the function of the membrane together with specific structural features of the lipids that allow them to have signaling properties.The lipids of the membrane are not uniformly distributed. There is an intrinsic asymmetry between the two monolayers that constitute the bilayer. In addition, some lipids tend to be enriched in particular regions of the membrane, termed domains. There is evidence that certain domains recruit specific proteins into that domain. This has been suggested to be important for allowing interaction among different proteins involved in certain signal transduction pathways.Membrane lipids have important roles in determining the physical properties of the membrane, in modulating the activity of membrane-bound proteins and in certain cases being specific secondary messengers that can interact with specific proteins. A large variety of lipids present in biological membranes result in them possessing many functions.
    Methods in Molecular Biology 01/2015; 1232:1-6. DOI:10.1007/978-1-4939-1752-5_1 · 1.29 Impact Factor
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    ABSTRACT: CDP-diacylglycerol Synthases (CDS)1 are critical enzymes that catalyze the formation of CDP-diacylglycerol (CDP-DAG) from phosphatidic acid (PA). Here we show in vitro that the two isoforms of human CDS, CDS1 and CDS2, show different acyl chain specificities for its lipid substrate. CDS2 is selective for the acyl chains at the sn-1 and sn-2 positions, the most preferred species being 1-stearoyl-2-arachidonoyl-sn-phosphatidic acid. CDS1, conversely, shows no particular substrate specificity, displaying similar activities for almost all substrates tested. Additionally, we show that inhibition of CDS2 by phosphatidylinositol is also acyl chain dependent, with the greatest inhibition seen with the 1-stearoyl-2-arachidonoyl species. CDS1 shows no acyl chain dependent inhibition. Both CDS1 and CDS2 are inhibited by their anionic phospholipid end products, with phosphatidylinositol-(4,5)-bisphosphate showing the greatest inhibition. Our results indicate that CDS1 and CDS2 could create different CDP-DAG pools that may serve to enrich different phospholipid species with specific acyl chains.
    Biochemistry 11/2014; 53(47). DOI:10.1021/bi501250m · 3.01 Impact Factor
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    ABSTRACT: A novel paradigm for the function of the mitochondrial nucleoside diphosphate kinase NM23-H4/NDPK-D is proposed: acting as a bifunctional nanoswitch in bioenergetics and cardiolipin (CL) trafficking and signaling. Similar to some other mitochondrial proteins like cytochrome c or AIF, NM23-H4 seems to have dual functions in bioenergetics and apoptotic signaling. In its bioenergetic phosphotransfer mode, the kinase reversibly phosphorylates NDPs into NTPs, driven by mitochondrially generated ATP. Among others, this reaction can locally supply GTP to mitochondrial GTPases as shown for the dynamin-like GTPase OPA1, found in a complex together with NM23-H4. Further, NM23-H4 is functionally coupled to adenylate translocase (ANT) of the mitochondrial inner membrane (MIM), so generated ADP can stimulate respiration to rapidly regenerate ATP. The lipid transfer mode of NM23-H4 can support, dependent on the presence of CL, the transfer of anionic lipids between membranes in vitro and the sorting of CL from its mitochondrial sites of synthesis (MIM) to the mitochondrial outer membrane (MOM) in vivo. Such (partial) collapse of MIM/MOM CL asymmetry results in CL externalization on the mitochondrial surface, where CL can serve as pro-apoptotic or pro-mitophagic "eat me"-signal. The functional state of NM23-H4 depends on its degree of CL-membrane interaction. In vitro assays have shown that only NM23-H4 that fully cross-links two membranes is lipid transfer competent, but at the same time phosphotransfer (kinase) inactive. Thus, the two functions of NM23-H4 seem to be mutually exclusive. This novel mitochondrial regulatory circuit has potential for the development of interventions in various human pathologies.
    Archiv für Experimentelle Pathologie und Pharmakologie 09/2014; 388(2). DOI:10.1007/s00210-014-1047-4 · 2.36 Impact Factor
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    ABSTRACT: In addition to specific intermolecular interactions, biological processes at membranes are also modulated by the physical properties of the membrane. One of these properties is membrane curvature. NMR methods are useful for studying how membrane curvature affects the binding and insertion of proteins into membranes as well as how proteins can affect membrane curvature properties. In many cases these interactions result in a marked change in protein activity. We have reviewed examples from a range of systems having varied mechanisms by which membrane curvature is linked to protein activity. Among the examples discussed are antimicrobial peptides, proteins affecting membrane fusion, rhodopsin, protein kinase C, phospholipase C-delta1, phosphatidylinositol-3 kinase-related kinases and tafazzin. This article is part of a Special Issue entitled: NMR Spectroscopy for Atomistic Views of Biomembranes and Cell Surfaces.
    Biochimica et Biophysica Acta (BBA) - Biomembranes 05/2014; DOI:10.1016/j.bbamem.2014.05.004 · 3.43 Impact Factor
  • Biophysical Journal 01/2014; 106(2):184a. DOI:10.1016/j.bpj.2013.11.1042 · 3.97 Impact Factor
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    ABSTRACT: Host defense antimicrobial peptides are key components of human innate immunity that plays an indispensible role in human health. While there are multiple copies of cathelicidin genes in horses, cattle, pigs, and sheep, only one cathelicidin gene is found in humans. Interestingly, this single cathelicidin gene can be processed into different forms of antimicrobial peptides. LL-37, the most commonly studied form, is not only antimicrobial but also possesses other functional roles such as chemotaxis, apoptosis, wound healing, immune modulation, and cancer metastasis. This article reviews recent advances made in structural and biophysical studies of human LL-37 and its fragments, which serve as a basis to understand their antibacterial, anti-biofilm and antiviral activities. High-quality structures were made possible by using improved 2D NMR methods for peptide fragments and 3D NMR spectroscopy for intact LL-37. The two hydrophobic domains in the long amphipathic helix (residues 2-31) of LL-37 separated by a hydrophilic residue serine 9 explain its cooperative binding to bacterial lipopolysaccharides (LPS). Both aromatic rings (F5, F6, F17, and F27) and interfacial basic amino acids of LL-37 directly interact with anionic phosphatidylglycerols (PG). Although the peptide sequences reported in the literature vary slightly, there is a consensus that the central helix of LL-37 is essential for disrupting superbugs (e.g., MRSA), bacterial biofilms, and viruses such as human immunodeficiency virus 1 (HIV-1) and respiratory syncytial virus (RSV). In the central helix, the central arginine R23 is of particular importance in binding to bacterial membranes or DNA. Mapping the functional roles of the cationic amino acids of the major antimicrobial region of LL-37 provides a basis for designing antimicrobial peptides with desired properties. This article is part of a Special Issue entitled: Interfacially active peptides and proteins.
    Biochimica et Biophysica Acta 01/2014; DOI:10.1016/j.bbamem.2014.01.016 · 4.66 Impact Factor
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    ABSTRACT: Historically, cellular trafficking of lipids has received much less attention than protein trafficking, mostly because its biological importance was underestimated, involved sorting and translocation mechanisms were not known, and analytical tools were limiting. This has changed during the last decade, and we discuss here some progress made in respect to mitochondria and the trafficking of phospholipids, in particular cardiolipin. Different membrane contact site or junction complexes and putative lipid transfer proteins for intra- and intermembrane lipid translocation have been described, involving mitochondrial inner and outer membrane, and the adjacent membranes of the endoplasmic reticulum. An image emerges how cardiolipin precursors, remodeling intermediates, mature cardiolipin and its oxidation products could migrate between membranes, and how this trafficking is involved in cardiolipin biosynthesis and cell signaling events. Particular emphasis in this review is given to mitochondrial nucleoside diphosphate kinase D and mitochondrial creatine kinases, which emerge to have roles in both, membrane junction formation and lipid transfer.
    Chemistry and Physics of Lipids 12/2013; 179. DOI:10.1016/j.chemphyslip.2013.12.008 · 2.59 Impact Factor
  • Valerian E Kagan, Richard M Epand
    Chemistry and Physics of Lipids 12/2013; DOI:10.1016/j.chemphyslip.2013.12.007 · 2.59 Impact Factor
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    ABSTRACT: Cationic antimicrobial peptides are recognized templates for developing a new generation of antimicrobials to combat superbugs. Human cathelicidin LL-37 is an essential host defense molecule in human innate immunity. Previously, we identified KR-12 as the smallest antibacterial peptide of LL-37. KR-12 has a narrow activity spectrum since it is active against Gram-negative Escherichia coli but not Gram-positive Staphylococcus aureus. The functional roles of the basic amino acids of KR-12, however, have not yet been elucidated. An alanine scan of cationic amino acids of KR-12 provided evidence for their distinct roles in the activities of the peptides. Bacterial killing and membrane permeation experiments indicate that the R23A and K25A mutants, as well as the lysine-to-arginine mutant, were more potent than KR-12. Another three cationic residues (K18, R19, and R29) of KR-12, which are located in the hydrophilic face of the amphiphathic helix, appeared to be more important in clustering anionic lipids or hemolysis than R23 and K25 in the interfacial region. While the loss of interfacial R23 or K25 reduced peptide helicity, underscoring its important role in membrane binding, the overall increase in peptide activity of KR-12 could be ascribed to the increased peptide hydrophobicity that outweighed the role of basic charge in this case. In contrast, the mutations of interfacial R23 or K25 reduced peptide bactericidal activity of GF-17, an overlapping, more hydrophobic and potent peptide also derived from LL-37. Thus, the hydrophobic context of the peptide determines whether an alanine substitution of an interfacial basic residue increases or decreases membrane permeation and peptide activity.
    RSC Advances 11/2013; 3(42). DOI:10.1039/C3RA42599A · 3.84 Impact Factor
  • Kenneth D'Souza, Richard M Epand
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    ABSTRACT: There are six major species of phospholipids in eukaryotes, each of which play unique structural and functional roles. One species, phosphatidylinositol (PI) only contributes about 2-10% of the total phospholipid pool. However, they are critical factors in the regulation of several fundamental processes such as in membrane dynamics and signal transduction pathways. Although numerous acyl species exist, PI species are enriched with one specific acyl chain composition at both the sn-1 and sn-2 positions. Recent work has identified several enzymes that act on lipids to lead to the formation or interconversion of PI species that exhibit acyl chain specificity. These enzymes contribute to this lipid's enrichment with specific acyl chains. The nature of the acyl chains on signaling lipids have been shown to contribute to their specificity. Here we review some of the critical functions of PI and the multiple pathways in which PI can be produced and metabolized. We also discuss a common motif that may confer arachidonoyl specificity to several of the enzymes involved.This article is part of a Special Issue entitled: Membrane structure and function: Relevance in the cell's physiology, pathology and therapy. This article is part of a Special Issue entitled: Membrane structure and function: Relevance in the cell's physiology, pathology and therapy.
    Biochimica et Biophysica Acta 10/2013; 1838(6). DOI:10.1016/j.bbamem.2013.10.003 · 4.66 Impact Factor
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    ABSTRACT: We have studied the relationship between diacylglycerol kinase delta (DGKδ) and lipogenesis. There is a marked increase in the expression of DGKδ during the differentiation of 3T3-L1 cells to adipocytes, as well as in the synthesis of neutral and polar lipids. When 3T3-L1 undifferentiated fibroblasts are transfected to express DGKδ there is increased triglyceride synthesis without differentiation to adipocytes. Hence, expression of DGKδ promotes lipogenesis. Lipid synthesis is decreased in DGKδ knockout mouse embryo fibroblasts, especially for lipids with shorter acyl chains and limited unsaturation. This reduction occurs for both neutral and polar lipids. These findings suggest reduced de novo lipid synthesis. This is confirmed by measuring the incorporation of glycerol into polar and neutral lipids that is higher in the wild type cells than in the DGKδ knockouts. In comparison, there was no change in lipid synthesis in DGKε knockout mouse embryo fibroblasts. We also demonstrate that the DGKδ knockout cells had a lower expression of acetyl-CoA carboxylase and fatty acid synthase as well as a lower degree of activation by phosphorylation of ATP citrate lyase. These three enzymes are involved in the synthesis of long chain fatty acids. Our results demonstrate that DGKδ markedly increases lipid synthesis, at least in part as a result of promoting the de novo synthesis of fatty acids.
    Biochemistry 10/2013; 52(44). DOI:10.1021/bi401178y · 3.01 Impact Factor
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    ABSTRACT: Sticholysins (Sts) I and II (StI/II) are pore-forming toxins (PFTs) produced by the Caribbean Sea anemone Stichodactyla helianthus belonging to the actinoporin family, a unique class of eukaryotic PFTs exclusively found in sea anemones. The role of lipid phase co-existence in the mechanism of action of membranolytic proteins and peptides is not clearly understood. As for actinoporins, it has been proposed that phase separation promotes pore forming activity. However little is known about the effect of sticholysins on the phase separation of lipids in membranes. To gain insight into the mechanism of action of sticholysins, we evaluated the effect of these proteins on lipid segregation using differential scanning calorimetry (DSC) and atomic force microscopy (AFM). New evidence was obtained reflecting that these proteins reduce line tension in the membrane by promoting lipid mixing. In terms of the relevance for the mechanism of action of actinoporins, we hypothesize that expanding lipid disordered phases into lipid ordered phases decreases the lipid packing at the borders of the lipid raft, turning it into a more suitable environment for N-terminal insertion and pore formation.
    Biochimica et Biophysica Acta 08/2013; 1828(11). DOI:10.1016/j.bbamem.2013.08.001 · 4.66 Impact Factor
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    ABSTRACT: Aggregatibacter actinomycetemcomitans produces a repeats-in-toxin (RTX) leukotoxin (LtxA) that selectively kills human immune cells. Binding of LtxA to its β2 integrin receptor (lymphocyte function-associated antigen-1, LFA-1) results in the clustering of the toxin/receptor complex in lipid rafts. Clustering occurs only in the presence of LFA-1 and cholesterol, and LtxA is unable to kill cells lacking either LFA-1 or cholesterol. Here, the interaction of LtxA with cholesterol was measured using surface plasmon resonance and differential scanning calorimetry. The binding of LtxA to phospholipid bilayers increased by four orders of magnitude in the presence of 40% cholesterol relative to the absence of cholesterol. The affinity was specific to cholesterol and required an intact secondary structure. LtxA contains two cholesterol recognition/amino acid consensus (CRAC) sites; CRAC336 (333LEEYSKR339) is highly conserved among RTX toxins, while CRAC503 (501VDYLK505) is unique to LtxA. A peptide corresponding to CRAC336 inhibited the ability of LtxA to kill Jurkat (Jn.9) cells. Although peptides corresponding to both CRAC336 and CRAC503 bind cholesterol, only CRAC336 competitively inhibited LtxA binding to this sterol. A panel of full-length LtxA CRAC mutants demonstrated that an intact CRAC336 site was essential for LtxA cytotoxicity. The conservation of CRAC336 among RTX toxins suggests that this mechanism may be conserved among RTX toxins.
    Journal of Biological Chemistry 06/2013; DOI:10.1074/jbc.M113.486654 · 4.57 Impact Factor
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    ABSTRACT: Toward generating new tools for fighting multidrug-resistant (MDR) bacteria, we assessed the ability of a membrane-active peptide to sensitize gram-negative bacteria to various antibiotics. The mechanism for affecting inner and/or outer membrane functions was assessed by complementary biophysical methods (SPR, DSC, ITC). The implication of efflux pumps was examined using Acr-AB mutants, as tested with representative antibiotics, host defense peptides, and synthetic mimics. The ability to affect disease course systemically was compared for a single therapy and combination therapy, using the mouse thigh-infection model. The data show that potent antibiotic action can be provoked in vitro and in vivo, by a treatment combining two antibacterial compounds whose individual inefficiency against gram-negative bacteria stems from their efflux. Thus, at subminimal inhibitory concentrations, the lipopeptide-like sequence, N(α)(ω7)dodecenoyl-lysyl-[lysyl-aminododecanoyl-lysyl]-amide (designated C12(ω7)K-β12), has, nonetheless, rapidly achieved a transient membrane depolarization, which deprived bacteria of the proton-motive force required for active efflux. Consequently, bacteria became significantly sensitive to intracellular targeting antibiotics. Collectively, these findings suggest a potentially useful approach for expanding the antibiotics sensitivity spectrum of MDR gram-negative bacteria to include efflux substrates.-Goldberg, K., Sarig, H., Zaknoon, F., Epand, R. F., Epand, R. M., Mor, A. Sensitization of gram-negative bacteria by targeting the membrane potential.
    The FASEB Journal 06/2013; 27(9). DOI:10.1096/fj.13-227942 · 5.48 Impact Factor
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Publication Stats

15k Citations
1,971.74 Total Impact Points


  • 1975–2015
    • McMaster University
      • • Department of Biochemistry and Biomedical Sciences
      • • Health Sciences Centre
      • • Department of Chemistry and Chemical Biology
      Hamilton, Ontario, Canada
  • 2013
    • Technion - Israel Institute of Technology
      • Faculty of Biotechnology and Food Engineering
      H̱efa, Haifa, Israel
  • 2011
    • National Institute of Allergy and Infectious Diseases
      Maryland, United States
    • Vanderbilt University
      • Division of Clinical Pharmacology
      Nashville, MI, United States
  • 2010
    • University of Toronto
      • Institute of Biomaterials and Biomedical Engineering
      Toronto, Ontario, Canada
  • 2009–2010
    • Brigham Young University - Provo Main Campus
      • Department of Chemistry and Biochemistry
      Provo, Utah, United States
  • 2008
    • Emory University
      Atlanta, Georgia, United States
    • University of California, San Diego
      • Department of Chemistry and Biochemistry
      San Diego, CA, United States
  • 2001–2007
    • University of Utah
      Salt Lake City, Utah, United States
    • University of Alabama at Birmingham
      • Department of Medicine
      Birmingham, AL, United States
  • 2006
    • Weizmann Institute of Science
      • Department of Biological Chemistry
  • 2004
    • Université de Fribourg
      • Département de médecine
      Fribourg, FR, Switzerland
    • The Children's Hospital of Philadelphia
      Filadelfia, Pennsylvania, United States
    • University of Wisconsin–Madison
      • Department of Chemistry
      Madison, Wisconsin, United States
    • Indiana University-Purdue University Indianapolis
      • Department of Biochemistry and Molecular Biology
      Indianapolis, Indiana, United States
  • 1997–2003
    • Hamilton Health Sciences
      Hamilton, Ontario, Canada
  • 2002
    • The University of Calgary
      • Department of Biological Sciences
      Calgary, Alberta, Canada
  • 2000
    • University of California, Berkeley
      • Department of Chemistry
      Berkeley, MO, United States
  • 1995–1999
    • Vrije Universiteit Brussel
      Bruxelles, Brussels Capital, Belgium
    • Saint Vincent's Institute
      Fitzroy, Victoria, Australia
  • 1998
    • VU University Amsterdam
      Amsterdamo, North Holland, Netherlands
    • The University of Edinburgh
      • Royal (Dick) School of Veterinary Studies
      Edinburgh, SCT, United Kingdom
    • University of Illinois, Urbana-Champaign
      • Department of Biochemistry
      Urbana, Illinois, United States
  • 1996
    • Hebrew University of Jerusalem
      • Department of Soil and Water Sciences (Seagram Center)
      Yerushalayim, Jerusalem, Israel
    • Brock University
      • Department of Biological Sciences
      St. Catharines, Ontario, Canada
  • 1993
    • Centro Universitario de Ciencias de la Salud
      Guadalajara, Jalisco, Mexico
  • 1990–1991
    • Setsunan University
      • Faculty of Pharmaceutical Sciences
      Ōsaka-shi, Osaka-fu, Japan
  • 1982
    • Yale University
      • Department of Chemistry
      New Haven, Connecticut, United States
  • 1972–1975
    • University of Guelph
      • Department of Chemistry
      Guelph, Ontario, Canada