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Structural Basis for Ligand Regulation of the Fatty Acid-binding Protein 5, Peroxisome Proliferator-activated Receptor β/δ (FABP5-PPARβ/δ) Signaling Pathway*

  • Eric H. Armstrong
    Footnotes
    Affiliations
    Department of Biochemistry

    Discovery and Developmental Therapeutics, Winship Cancer Institute, Emory University School of Medicine, Atlanta, Georgia 30322
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  • Devrishi Goswami
    Affiliations
    Department of Molecular Therapeutics, The Scripps Research Institute, Jupiter, Florida 33458
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  • Patrick R. Griffin
    Affiliations
    Department of Molecular Therapeutics, The Scripps Research Institute, Jupiter, Florida 33458
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  • Noa Noy
    Affiliations
    Departments of Pharmacology and Nutrition, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106
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  • Eric A. Ortlund
    Correspondence
    To whom correspondence should be addressed: Dept. of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322. Tel.: 404-727-5014; Fax: 404-727-2738
    Affiliations
    Department of Biochemistry

    Discovery and Developmental Therapeutics, Winship Cancer Institute, Emory University School of Medicine, Atlanta, Georgia 30322
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grant R01 DK060684 and Grant P30CA138292 from the Emory University Integrated Cellular Imaging Microscopy Core of the Winship Cancer Institute comprehensive cancer center. This work was also supported by start-up funds from Emory University (to E. A. O.).The atomic coordinates and structure factors (codes 4LKP and 4LKT) have been deposited in the Protein Data Bank (http://wwpdb.org/).
    1 Supported by National Institute of Health Graduate Training Grant 5T32GM008602 from Pharmacological Sciences, Emory University.
Open AccessPublished:April 01, 2014DOI:https://doi.org/10.1074/jbc.M113.514646
      Fatty acid-binding proteins (FABPs) are a widely expressed group of calycins that play a well established role in solubilizing cellular fatty acids. Recent studies, however, have recast FABPs as active participants in vital lipid-signaling pathways. FABP5, like its family members, displays a promiscuous ligand binding profile, capable of interacting with numerous long chain fatty acids of varying degrees of saturation. Certain “activating” fatty acids induce the protein's cytoplasmic to nuclear translocation, stimulating PPARβ/δ transactivation; however, the rules that govern this process remain unknown. Using a range of structural and biochemical techniques, we show that both linoleic and arachidonic acid elicit FABP5's translocation by permitting allosteric communication between the ligand-sensing β2 loop and a tertiary nuclear localization signal within the α-helical cap of the protein. Furthermore, we show that more saturated, nonactivating fatty acids inhibit nuclear localization signal formation by destabilizing this activation loop, thus implicating FABP5 specifically in cis-bonded, polyunsaturated fatty acid signaling.

      Introduction

      LCFAs,
      The abbreviations used are: LCFA
      long chain fatty acid
      FABP
      fatty acid-binding protein
      AA
      arachidonic acid
      NLS
      nuclear localization signal
      PPAR
      peroxisome proliferator-activated receptor
      LA
      linoleic acid
      HSD
      honestly significant difference
      ANOVA
      analysis of variance
      PoA
      palmitoleic acid
      iLBP
      intracellular lipid-binding protein
      DSm
      double-switch mutant
      SpA
      sapienic acid
      PA
      palmitic acid
      NES
      nuclear export signal
      PDB
      Protein Data Bank
      HDX
      hydrogen deuterium exchange
      EGFP
      enhanced green fluorescent protein
      h
      human
      1,8-ANS
      1-anilinonaphthalene-8-sulfonic acid
      CRBP-I
      cellular retinol-binding protein I
      CRABP-II
      cellular retinoic acid-binding protein 2.
      in addition to serving as a structural component and energy source of the cell, participate in cellular signaling by modulating the activity of a group of nuclear receptors known as the peroxisome proliferation-activated receptors (PPARs) (
      • Göttlicher M.
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      ). Because of the large number of target genes affected by this family of ligand-regulated transcription factors, LCFAs play a critical role in a variety of cellular processes and their related pathophysiologies, ranging from metabolic defects to cell differentiation and cancer progression (
      • Kersten S.
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      ,
      • Willson T.M.
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      The PPARs: from orphan receptors to drug discovery.
      ). However, the relative insolubility of these molecules makes them reliant upon a class of transport proteins, the FABPs, to exert their signaling effects (
      • Furuhashi M.
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      Fatty acid-binding proteins: role in metabolic diseases and potential as drug targets.
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      ).
      There are nine known FABP members in mammals, each ∼14–15 kDa in size with orthologs found throughout the animal kingdom (
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      The multigene family of fatty acid-binding proteins (FABPs): function, structure and polymorphism.
      ). Although they exhibit a wide range of sequence identity (∼20–70%), all form a twisted β-barrel, composed of 10 anti-parallel β-strands arranged into two orthogonal β-sheets, with a helix-turn-helix lid covering the ligand-binding site (
      • Smathers R.L.
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      The human fatty acid-binding protein family: evolutionary divergences and functions.
      ,
      • Storch J.
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      Structural and functional analysis of fatty acid-binding proteins.
      ,
      • Chmurzyńska A.
      The multigene family of fatty acid-binding proteins (FABPs): function, structure and polymorphism.
      ). As members of the intracellular lipid-binding protein (iLBP) family, they have traditionally been thought to be involved in the solubilization/protection of their various hydrophobic cargoes, facilitating ligand movement via passive diffusion between the various compartments of the cell (
      • Ong D.E.
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      The Retinoids: Biology, Chemistry, and Medicine.
      ,
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      The fatty acid transport function of fatty acid-binding proteins.
      ). Increasingly, however, FABPs are emerging as specific mediators of precise signaling pathways. For instance, FABP1 facilitates the polyunsaturated fatty acid and fibrate-induced transactivation of PPARα, via direct interaction with the nuclear receptor's ligand binding domain (
      • Petrescu A.D.
      • Huang H.
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      • McIntosh A.L.
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      Impact of L-FABP and glucose on polyunsaturated fatty acid induction of PPARα-regulated β-oxidative enzymes.
      ,
      • Petrescu A.D.
      • McIntosh A.L.
      • Storey S.M.
      • Huang H.
      • Martin G.G.
      • Landrock D.
      • Kier A.B.
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      High glucose potentiates L-FABP mediated fibrate induction of PPARα in mouse hepatocytes.
      ,
      • Hostetler H.A.
      • McIntosh A.L.
      • Atshaves B.P.
      • Storey S.M.
      • Payne H.R.
      • Kier A.B.
      • Schroeder F.
      L-FABP directly interacts with PPARα in cultured primary hepatocytes.
      ). A similar role has been observed with FABP4, whereby its ligand-mediated dimerization state governs nuclear import and subsequent ligand delivery to PPARγ (
      • Tan N.S.
      • Shaw N.S.
      • Vinckenbosch N.
      • Liu P.
      • Yasmin R.
      • Desvergne B.
      • Wahli W.
      • Noy N.
      Selective cooperation between fatty acid binding proteins and peroxisome proliferator-activated receptors in regulating transcription.
      ,
      • Ayers S.D.
      • Nedrow K.L.
      • Gillilan R.E.
      • Noy N.
      Continuous nucleocytoplasmic shuttling underlies transcriptional activation of PPARγ by FABP4.
      ). Recent findings have even revealed FABPs to be the once enigmatic N-acylethanolamine “transporter,” responsible for endocannabinoid cellular uptake, hydrolysis, and PPARα activation (
      • Kaczocha M.
      • Glaser S.T.
      • Deutsch D.G.
      Identification of intracellular carriers for the endocannabinoid anandamide.
      ,
      • Kaczocha M.
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      Fatty acid-binding proteins transport N-acylethanolamines to nuclear receptors and are targets of endocannabinoid transport inhibitors.
      ).
      FABP5 (E-FABP, KFABP, and mal1), first characterized almost 20 years ago in keratinocytes, is one of the most ubiquitously expressed proteins in its class, and it can be found across a broad spectrum of tissue/cell types such as the epidermis, adipose, macrophages, mammary glands, brain, kidney, liver, lung, heart, skeletal muscle, and testis (
      • Smathers R.L.
      • Petersen D.R.
      The human fatty acid-binding protein family: evolutionary divergences and functions.
      ,
      • Chmurzyńska A.
      The multigene family of fatty acid-binding proteins (FABPs): function, structure and polymorphism.
      ,
      • Siegenthaler G.
      • Hotz R.
      • Chatellard-Gruaz D.
      • Didierjean L.
      • Hellman U.
      • Saurat J.H.
      Purification and characterization of the human epidermal fatty acid-binding protein: localization during epidermal cell differentiation in vivo and in vitro.
      ). A member of the iLBP subfamily IV, FABP5 binds a wide array of ligands in a 1:1 ratio, including fatty acids and fatty acid metabolites spanning 10–22 carbons in length with various saturation states, as well as the vitamin A metabolite all-trans-retinoic acid and numerous synthetic drugs and probes (
      • Smathers R.L.
      • Petersen D.R.
      The human fatty acid-binding protein family: evolutionary divergences and functions.
      ,
      • Marcelino A.M.
      • Smock R.G.
      • Gierasch L.M.
      Evolutionary coupling of structural and functional sequence information in the intracellular lipid-binding protein family.
      ,
      • Simpson M.A.
      • LiCata V.J.
      • Ribarik Coe N.
      • Bernlohr D.A.
      Biochemical and biophysical analysis of the intracellular lipid binding proteins of adipocytes.
      ,
      • Schug T.T.
      • Berry D.C.
      • Shaw N.S.
      • Travis S.N.
      • Noy N.
      Opposing effects of retinoic acid on cell growth result from alternate activation of two different nuclear receptors.
      ). It has also been found to be involved in a range of pathologies, including the metabolic syndrome (
      • Maeda K.
      • Uysal K.T.
      • Makowski L.
      • Görgün C.Z.
      • Atsumi G.
      • Parker R.A.
      • Brüning J.
      • Hertzel A.V.
      • Bernlohr D.A.
      • Hotamisligil G.S.
      Role of the fatty acid binding protein mal1 in obesity and insulin resistance.
      ,
      • Hong J.
      • Gu W.
      • Zhang Y.
      • Yan Q.
      • Dai M.
      • Shi J.
      • Zhai Y.
      • Wang W.
      • Li X.
      • Ning G.
      Different association of circulating levels of adipocyte and epidermal fatty acid-binding proteins with metabolic syndrome and coronary atherosclerosis in Chinese adults.
      ), atherosclerosis (
      • Yeung D.C.
      • Wang Y.
      • Xu A.
      • Cheung S.C.
      • Wat N.M.
      • Fong D.Y.
      • Fong C.H.
      • Chau M.T.
      • Sham P.C.
      • Lam K.S.
      Epidermal fatty-acid-binding protein: a new circulating biomarker associated with cardio-metabolic risk factors and carotid atherosclerosis.
      ), cancer (
      • Schug T.T.
      • Berry D.C.
      • Toshkov I.A.
      • Cheng L.
      • Nikitin A.Y.
      • Noy N.
      Overcoming retinoic acid-resistance of mammary carcinomas by diverting retinoic acid from PPARβ/δ to RAR.
      ,
      • Morgan E.
      • Kannan-Thulasiraman P.
      • Noy N.
      Involvement of fatty acid binding protein 5 and PPARβ/δ in prostate cancer cell growth.
      ,
      • Jeong C.Y.
      • Hah Y.S.
      • Cho B.I.
      • Lee S.M.
      • Joo Y.T.
      • Jung E.J.
      • Jeong S.H.
      • Lee Y.J.
      • Choi S.K.
      • Ha W.S.
      • Park S.T.
      • Hong S.C.
      Fatty acid-binding protein 5 promotes cell proliferation and invasion in human intrahepatic cholangiocarcinoma.
      ,
      • Kannan-Thulasiraman P.
      • Seachrist D.D.
      • Mahabeleshwar G.H.
      • Jain M.K.
      • Noy N.
      Fatty acid-binding protein 5 and PPARβ/δ are critical mediators of epidermal growth factor receptor-induced carcinoma cell growth.
      ,
      • Levi L.
      • Lobo G.
      • Doud M.K.
      • von Lintig J.
      • Seachrist D.
      • Tochtrop G.P.
      • Noy N.
      Genetic ablation of the fatty acid-binding protein FABP5 suppresses HER2-induced mammary tumorigenesis.
      ), and potentially certain neurodegenerative diseases (
      • Cologna S.M.
      • Jiang X.S.
      • Backlund P.S.
      • Cluzeau C.V.
      • Dail M.K.
      • Yanjanin N.M.
      • Siebel S.
      • Toth C.L.
      • Jun H.S.
      • Wassif C.A.
      • Yergey A.L.
      • Porter F.D.
      Quantitative proteomic analysis of Niemann-Pick disease, type C1 cerebellum identifies protein biomarkers and provides pathological insight.
      ).
      Work conducted by Tan et al. demonstrated the ability of FABP5 to specifically enhance the transactivation of PPARβ/δ, whose known gene targets are involved in cellular glucose and lipid homeostasis (
      • Barak Y.
      • Liao D.
      • He W.
      • Ong E.S.
      • Nelson M.C.
      • Olefsky J.M.
      • Boland R.
      • Evans R.M.
      Effects of peroxisome proliferator-activated receptor δ on placentation, adiposity, and colorectal cancer.
      ,
      • Akiyama T.E.
      • Lambert G.
      • Nicol C.J.
      • Matsusue K.
      • Peters J.M.
      • Brewer Jr., H.B.
      • Gonzalez F.J.
      Peroxisome proliferator-activated receptor β/δ regulates very low density lipoprotein production and catabolism in mice on a Western diet.
      ,
      • Lee C.H.
      • Olson P.
      • Hevener A.
      • Mehl I.
      • Chong L.W.
      • Olefsky J.M.
      • Gonzalez F.J.
      • Ham J.
      • Kang H.
      • Peters J.M.
      • Evans R.M.
      PPARδ regulates glucose metabolism and insulin sensitivity.
      ), differentiation (
      • Nadra K.
      • Anghel S.I.
      • Joye E.
      • Tan N.S.
      • Basu-Modak S.
      • Trono D.
      • Wahli W.
      • Desvergne B.
      Differentiation of trophoblast giant cells and their metabolic functions are dependent on peroxisome proliferator-activated receptor β/δ.
      ,
      • Tan N.S.
      • Michalik L.
      • Noy N.
      • Yasmin R.
      • Pacot C.
      • Heim M.
      • Flühmann B.
      • Desvergne B.
      • Wahli W.
      Critical roles of PPARβ/δ in keratinocyte response to inflammation.
      ), and resistance to apoptosis (
      • Tan N.S.
      • Michalik L.
      • Noy N.
      • Yasmin R.
      • Pacot C.
      • Heim M.
      • Flühmann B.
      • Desvergne B.
      • Wahli W.
      Critical roles of PPARβ/δ in keratinocyte response to inflammation.
      ,
      • Di-Poï N.
      • Tan N.S.
      • Michalik L.
      • Wahli W.
      • Desvergne B.
      Antiapoptotic role of PPARβ in keratinocytes via transcriptional control of the Akt1 signaling pathway.
      ). Despite FABP5's promiscuous binding profile, only a subset of fatty acids and other ligands have been shown to result in the protein's nuclear translocation, where it is thought to engage PPARβ/δ, allowing for the channeling of ligand into the nuclear receptor's binding pocket (
      • Tan N.S.
      • Shaw N.S.
      • Vinckenbosch N.
      • Liu P.
      • Yasmin R.
      • Desvergne B.
      • Wahli W.
      • Noy N.
      Selective cooperation between fatty acid binding proteins and peroxisome proliferator-activated receptors in regulating transcription.
      ). Although previous structural studies have shed considerable light on the role of FABP4 in PPARγ signaling (
      • Ayers S.D.
      • Nedrow K.L.
      • Gillilan R.E.
      • Noy N.
      Continuous nucleocytoplasmic shuttling underlies transcriptional activation of PPARγ by FABP4.
      ,
      • Gillilan R.E.
      • Ayers S.D.
      • Noy N.
      Structural basis for activation of fatty acid-binding protein 4.
      ), the mechanism underlying select lipid activation (e.g. nuclear translocation) of FABP5 remains unknown. Using a combination of x-ray crystallography, hydrogen deuterium exchange (HDX)-mass spectroscopy, and biochemical and cellular approaches, we have established the presence of a ligand-sensitive tertiary nuclear localization signal (NLS) located on the α1 and α2 helices of FABP5. Furthermore, we show that interaction of a bound ligand with FABP5's β2 loop relays conformational information to the NLS, thereby serving as the driving force for fatty acid-specific nuclear translocation.

      DISCUSSION

      Since the discovery of the first FABP's by Ockner et al. (
      • Ockner R.K.
      • Manning J.A.
      • Poppenhausen R.B.
      • Ho W.K.
      A binding protein for fatty acids in cytosol of intestinal mucosa, liver, myocardium, and other tissues.
      ) over 40 years ago, a wealth of data have steadily accumulated regarding the structure and function of this class of proteins (
      • Furuhashi M.
      • Hotamisligil G.S.
      Fatty acid-binding proteins: role in metabolic diseases and potential as drug targets.
      ). Although the vast majority of structural studies have focused on the determinants of stability and ligand binding, almost no attention outside of FABP1 and -4 has been given to the physical mechanisms driving signal propagation and protein-protein interaction (
      • Ayers S.D.
      • Nedrow K.L.
      • Gillilan R.E.
      • Noy N.
      Continuous nucleocytoplasmic shuttling underlies transcriptional activation of PPARγ by FABP4.
      ,
      • Gillilan R.E.
      • Ayers S.D.
      • Noy N.
      Structural basis for activation of fatty acid-binding protein 4.
      ,
      • Hertzel A.V.
      • Hellberg K.
      • Reynolds J.M.
      • Kruse A.C.
      • Juhlmann B.E.
      • Smith A.J.
      • Sanders M.A.
      • Ohlendorf D.H.
      • Suttles J.
      • Bernlohr D.A.
      Identification and characterization of a small molecule inhibitor of fatty acid binding proteins.
      ,
      • Hostetler H.A.
      • Balanarasimha M.
      • Huang H.
      • Kelzer M.S.
      • Kaliappan A.
      • Kier A.B.
      • Schroeder F.
      Glucose regulates fatty acid binding protein interaction with lipids and peroxisome proliferator-activated receptor α.
      ,
      • Velkov T.
      Interactions between human liver fatty acid binding protein and peroxisome proliferator activated receptor selective drugs.
      ). We have expanded understanding of FABP signaling by identifying the molecular switch that dictates fatty acid-specific activation, whereby the conformation of bound LCFA relays information from what we now term the “activation loop” (βC-D) of the portal region to the protein's tertiary NLS, consisting of Lys-24, Arg-33, and Lys-34.
      In this way, FABP5 shares key mechanistic elements from both FABP4 and CRABP-II, yet ultimately undergoes a method of activation different from either. Like FABP4, only certain ligands cause nuclear localization of the protein (
      • Tan N.S.
      • Shaw N.S.
      • Vinckenbosch N.
      • Liu P.
      • Yasmin R.
      • Desvergne B.
      • Wahli W.
      • Noy N.
      Selective cooperation between fatty acid binding proteins and peroxisome proliferator-activated receptors in regulating transcription.
      ,
      • Gillilan R.E.
      • Ayers S.D.
      • Noy N.
      Structural basis for activation of fatty acid-binding protein 4.
      ). However, instead of dimer rearrangement driving the cytosolic exposure of the NLS, FABP5, like CRABP-II, remains monomeric, with binding of activating ligand resulting in stabilization of the NLS that is necessary for nuclear import (
      • Gillilan R.E.
      • Ayers S.D.
      • Noy N.
      Structural basis for activation of fatty acid-binding protein 4.
      ,
      • Sessler R.J.
      • Noy N.
      A ligand-activated nuclear localization signal in cellular retinoic acid binding protein-II.
      ). Interestingly, although this process can occur in as little as 30–60 min for all three proteins, the ensuing enhancement of nuclear receptor-driven gene transcription is most frequently tested 24 h after ligand introduction (
      • Ayers S.D.
      • Nedrow K.L.
      • Gillilan R.E.
      • Noy N.
      Continuous nucleocytoplasmic shuttling underlies transcriptional activation of PPARγ by FABP4.
      ,
      • Budhu A.S.
      • Noy N.
      Direct channeling of retinoic acid between cellular retinoic acid-binding protein II and retinoic acid receptor sensitizes mammary carcinoma cells to retinoic acid-induced growth arrest.
      ,
      • Majumdar A.
      • Petrescu A.D.
      • Xiong Y.
      • Noy N.
      Nuclear translocation of cellular retinoic acid-binding protein II is regulated by retinoic acid-controlled SUMOylation.
      ). This time difference could explain, at least in part, why 10 μm AA and LA are sufficient for our localization assays, but not for FABP5-enhanced transactivation of PPARβ/δ, as ligand degradation and metabolism become more relevant over time.
      Sequence alignment in Clustal Omega (
      • Sievers F.
      • Wilm A.
      • Dineen D.
      • Gibson T.J.
      • Karplus K.
      • Li W.
      • Lopez R.
      • McWilliam H.
      • Remmert M.
      • Söding J.
      • Thompson J.D.
      • Higgins D.G.
      Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega.
      ) of all nine human FABP members with other iLBPs known to participate in ligand-mediated signaling reveals that FABP8, a major protein constituent of the peripheral nervous system myelin (
      • Greenfield S.
      • Brostoff S.
      • Eylar E.H.
      • Morell P.
      Protein composition of myelin of the peripheral nervous system.
      ), contains residues homologous to the cryptic NLS present in FABP4 and -5 and CRABP-II, as well as to the pair of bulky/hydrophobic amino acids that constitute the ligand-dependent activation switch (Fig. 10). This raises the possibility that myelin FABP could also undergo directed nuclear localization; however, the same NLS homology is also found within the α helices of the iLBP cellular retinol-binding protein I (CRBP-I), where it governs the protein's retinol-dependent interaction with the transmembrane receptor stimulated by retinoic acid 6 (STRA6) (
      • Berry D.C.
      • O'Byrne S.M.
      • Vreeland A.C.
      • Blaner W.S.
      • Noy N.
      Cross talk between signaling and vitamin A transport by the retinol-binding protein receptor STRA6.
      ). Therefore, the potential of FABP8 to engage in a ligand-driven signaling pathway other than nuclear translocation cannot be discounted. Based on its predicted amino acid sequence (data not shown), the same directed inquiries can also be made for the newly discovered FABP12, although its presence within cells has not been documented beyond the mRNA level (
      • Liu R.Z.
      • Li X.
      • Godbout R.
      A novel fatty acid-binding protein (FABP) gene resulting from tandem gene duplication in mammals: transcription in rat retina and testis.
      ).
      Figure thumbnail gr10
      FIGURE 10Alignment of FABP1–9 with CRABP-II and CRBP-I. Sequence alignment of the region in FABP5 shown to be most affected by ligand- induced activation with that of the other human FABPs as well as CRABP-II and CRBP-I. FABP8 is the only protein in its class that harbors all three homologous NLS residues (bold, blue) and appropriately bulky/hydrophobic “switch” residues (bold, green), yet is currently untested for ligand-driven nuclear translocation.
      Additionally, we have shown that a single fatty acid can adopt at least two unique conformations within the binding pocket of FABP5. Although FABP-bound fatty acid tail mobility has been noted previously via x-ray crystallography (
      • Angelucci F.
      • Johnson K.A.
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      • Miele A.E.
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      • Valle C.
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      • Liberti P.
      • Cioli D.
      • Klinkert M.Q.
      • Bellelli A.
      Schistosoma mansoni fatty acid binding protein: specificity and functional control as revealed by crystallographic structure.
      ), our delineation of an active U-conformation versus inactive L-conformer opens up exciting new possibilities for structure-based drug design. The overexpression of FABP5 has been linked to insulin resistance (
      • Maeda K.
      • Uysal K.T.
      • Makowski L.
      • Görgün C.Z.
      • Atsumi G.
      • Parker R.A.
      • Brüning J.
      • Hertzel A.V.
      • Bernlohr D.A.
      • Hotamisligil G.S.
      Role of the fatty acid binding protein mal1 in obesity and insulin resistance.
      ), and its signaling is related to cancer cell survival (
      • Schug T.T.
      • Berry D.C.
      • Shaw N.S.
      • Travis S.N.
      • Noy N.
      Opposing effects of retinoic acid on cell growth result from alternate activation of two different nuclear receptors.
      ), proliferation (
      • Schug T.T.
      • Berry D.C.
      • Toshkov I.A.
      • Cheng L.
      • Nikitin A.Y.
      • Noy N.
      Overcoming retinoic acid-resistance of mammary carcinomas by diverting retinoic acid from PPARβ/δ to RAR.
      ,
      • Kannan-Thulasiraman P.
      • Seachrist D.D.
      • Mahabeleshwar G.H.
      • Jain M.K.
      • Noy N.
      Fatty acid-binding protein 5 and PPARβ/δ are critical mediators of epidermal growth factor receptor-induced carcinoma cell growth.
      ), and metastasis (
      • Jeong C.Y.
      • Hah Y.S.
      • Cho B.I.
      • Lee S.M.
      • Joo Y.T.
      • Jung E.J.
      • Jeong S.H.
      • Lee Y.J.
      • Choi S.K.
      • Ha W.S.
      • Park S.T.
      • Hong S.C.
      Fatty acid-binding protein 5 promotes cell proliferation and invasion in human intrahepatic cholangiocarcinoma.
      ,
      • Levi L.
      • Lobo G.
      • Doud M.K.
      • von Lintig J.
      • Seachrist D.
      • Tochtrop G.P.
      • Noy N.
      Genetic ablation of the fatty acid-binding protein FABP5 suppresses HER2-induced mammary tumorigenesis.
      ), making the protein an ideal candidate for antagonist development. Theoretically, such compounds could exert their influence via one of several mechanisms of action. The first would be to bind and disrupt the portal region, forcing the activation loop into its inactive state. The second would be to bind completely within the binding pocket, allowing closure of the activation loop and subsequent nuclear translocation of the protein, although the compound itself would be unable to bind PPARβ/δ. The third, and likely most potent, would be to improve the nuclear accumulation of current PPARβ/δ antagonists by optimizing their ability to bind and activate FABP5.
      Conversely, our fatty acid binding model can be used for the prediction of additional FABP5 activators. We have demonstrated that the state of unsaturation is one of the major determinants of a fatty acid's activation potential, presumably due to its affect on U-conformation preference within the binding pocket. Judging from the configurations seen within our structures as well as that published by Hohoff et al. (
      • Hohoff C.
      • Börchers T.
      • Rüstow B.
      • Spener F.
      • van Tilbeurgh H.
      Expression, purification, and crystal structure determination of recombinant human epidermal-type fatty acid binding protein.
      ), the first 11–13 carbons share a remarkably close alignment regardless of fatty acid type, thereby placing a greater degree of importance for activator differentiation on the cis-double bonds located more distal to the carboxylate headgroup. As both LA and AA were found to be activators, yet PoA (an ω-7 FA) was not, this suggests an intriguing role for FABP5 as a specific mediator for ω-6 and possibly ω-3 fatty acid signaling. Because all unsaturated fatty acids tested, including oleic acid (data not shown), were able to significantly activate PPARβ/δ in the absence of FABP5, the presence of such a secondary control measure likely serves to ensure preferential activation of the nuclear receptor by this or a similar subset of fatty acids.

      Acknowledgments

      We gratefully acknowledge Alexa Mattheyses, Jason Fritz, and Debby Martinson (Integrated Cellular Imaging Core, Emory University) and Shuiliang Yu (Case Western Reserve University) for training in microscopy methods, and Katie Doud (Case Western Reserve University) for generous assistance with the PPARβ/δ activation assays. Data for 4LKP were collected at Southeast Regional Collaborative Access Team (SER-CAT) 22-BM beamline at the Advanced Photon Source, Argonne National Laboratory. Use of the Advanced Photon Source was supported by the United States Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract W-31-109-Eng-38.

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