Tillman, Matthew (Summer 2020)

Permanent URL: https://etd.library.emory.edu/concern/etds/mk61rh71d?locale=en


Lipids signal to control cellular homeostasis, metabolism, inflammation, and aging. Since lipids are hydrophobic, they are primarily sequestered within membranes, which limits their ability to signal through diffusion. Lipid transfer proteins (LTPs) solubilize lipids and mediate their signaling effects. LTPs are not simply passive carriers of lipids but are active participants in signaling that sense specific lipids that in turn regulate LTP function. In this study, we biochemically and structurally characterize two LTPs that control aging and lipid metabolism respectively. We determine the first structure of Lipid Binding Protein 8 (LBP-8), a fatty acid binding protein in Caenorhabditis elegans that extends lifespan through carrying lysosomal lipid signals into the nucleus to initiate expression of life prolonging genes. We identify a structurally conserved nuclear localization signal and describe a range of fatty acids LBP-8 is capable of binding, including life extending ligands such as oleic acid and oleoylethanolamide. Secondly, we characterize the functional role of the lipid binding StAR-related lipid transfer domain (StarD) of Thioesterase Superfamily Member 1 (Them1) to regulate lipid metabolism and thermogenesis in brown adipocytes. We show the StarD of Them1 acts as a lipid sensor, binding fatty acid and lysophosphatidylcholine species, which allosterically control the enzymatic activity of Them1. Furthermore, we also show how ADP and ATP allosterically control Them1 activity through a distinct mechanism. Together, lipids and ADP/ATP engage molecular switches that fine tune Them1 activity to regulate the thermogenic capacity of brown adipose tissue. Collectively, this work shows how lipids interact with LTPs to control their activity and vital biological processes.

Table of Contents

ABBREVIATIONS                                                                                                                       1

CHAPTER 1: INTRODUCTION                                                                                                6

    Lipids are signaling molecules                                                                                                   7

    Cytosolic lipid transfer proteins (LTPs)                                                                                     8

    Structure and binding preference of calycins                                                                             9

         Figure 1. Schematic of structural features of calycin family members.                              10

         Figure 2. Structure of FABP5 bound to linoleic acid.                                                        12

    Structure and binding preference of StarDs                                                                             14 

         Figure 3. Structure of StarD2 bound to palmitoyl-linoleoyl phosphatidylcholine.            15

    Functions of LTPs                                                                                                                    16

    Lipid Transporters                                                                                                                    16

         Figure 4. Schematic of functional roles of LTPs.                                                                17

    Lipid Chaperones                                                                                                                     19

    Lipid Sensors                                                                                                                            20

    Questions and Hypotheses Addressed in this Work                                                                 21

         Figure 5. Schematic of LTPS studied in this work.                                                             23    

References                                                                                                                                     24

CHAPTER 2: STRUCTURAL CHARACTERIZATION OF LIFE-EXTENDING CAENORHABITIS ELEGANS LIPID BINDING PROTEIN 8                                                                                                                                  33

    Abstract                                                                                                                                   34

    Introduction                                                                                                                            35

    Results                                                                                                                                      37

         Overall structure of apo-LBP-8 and general comparison with other FABPs                      37

              Figure 1. Structural overview of LBP-8.                                                                        38

              Table 1: X-ray data collection and refinement statistics.                                              39

         The lipid sensing portal region                                                                                            40

               Figure 2. The portal region of LBP-8 contains a hydrophobic patch for                    42       

               interacting with membranes and conserved nuclear localization signal. 

         LBP-8 binds to a range of fatty acids with preference for monounsaturated fatty acids    43

             Figure 3. LBP-8 binds to a diverse array of saturated and unsaturated long-chained  44

             fatty acids. 

              Table 2: Identification and relative quantification of lipids co-purified with LBP-8    45

              via MS.

         Analysis of the ligand binding pocket                                                                                 46

              Table 3: Interior cavity surface area and volume of human FABPs and LBP-8.          48

              Figure 4. Comparison of ligand binding pocket of LBP-8 with FABPs.                       48

         Mutational analysis of LBP-8 ligand binding pocket                                                          51

             Figure 5. Analysis of ligand binding pocket mutants.                                                    53

    Discussion                                                                                                                                54

    Materials and Methods                                                                                                          57

    References                                                                                                                               63

CHAPTER 3: ALLOSTERIC REGULATION OF THIOESTERASE SUPERFAMILY MEMBER1 BY FREE FATTY ACIDS AND LYSOPHOSPHATIDYLCHOLINE                                                                                         69

    Abstract                                                                                                                                   70

    Introduction                                                                                                                            71

    Results                                                                                                                                      73

         Them1 StarD binds long-chain fatty acids                                                                          73

              Figure 1. StarD of Them1 binds to long-chain fatty acids.                                            74

              Table 1. Affinity of fatty acids for Them1 START domain determined by MST.            75

         Fatty acids bind within the hydrophobic pocket of Them1’s StarD.                                  76

              Figure 2. Fatty acids fit within crystal structure of Them1 StarD.                                78

              Table 2. Myristic Acid—Them1 START domain X-ray data collection and                  79

              refinement statistics.

         Them1 StarD binds to lysophosphatidylcholine                                                                81

         Fatty acids enhance while 18:1 LPC inhibits Them1 acyl-CoA thioesterase activity        82

               Figure 3. Them1 StarD domain binds to lysophosphatidylcholine.                              83

              Figure 4. Fatty acids enhance while lysophosphatidylcholine inhibits Them1             84

              activity in a StarD-dependent manner.

         Them1 forms homotrimer containing a thioesterase domain core flanked by                    85

         mobile StarDs

               Figure 5. Them1 forms homotrimer with thioesterase domain core and                      86

              flanking StarDs.

         Them1 StarD stabilizes the thioesterase domains                                                               87

         Fatty acids stabilize while 18:1 LPC destabilizes StarD                                                     88

              Figure 6. Fatty acids stabilize while 18:1 LPC destabilizes the StarD.                        89

         18:1 LPC reverses Them1-mediated suppression of fatty acid oxidation                           90

              Figure 7. LPC 18:1 inhibits Them1 mediated suppression of thermogenesis               91

              in brown adipocytes.

         Them1 StarD is necessary for localization to the lipid droplet                                           93

              Figure 8. Them1 StarD drives localization to the lipid droplet and is not                    94

              necessary for Them1 mediated suppression of thermogenesis.

    Discussion                                                                                                                                95

    Experimental Procedure                                                                                                        98

    References                                                                                                                             111

    Supplemental Material                                                                                                        118

         Supplemental Table 1.                                                                                                       118

         Supplemental Figure 1. Structure of Them1 StarD suggests small lipids bind.                120

         Supplemental Figure 2. Negative stain single particle electron microscopy                    121

         of Them1.

         Supplemental Figure 3. HDX-MS heatmap of Them1 and thioesterase domains.            122

CHAPTER 4: BIOCHEMICAL CHARACTERIZATION OF THEM1 ENZYMATIC ACTIVITY AND REGULATION BY SMALL MOLECULES                                                                                                                           125

    Abstract                                                                                                                                 126

    Introduction                                                                                                                          127

    Results                                                                                                                                    128

         Enzymatic Mechanism of Them1                                                                                     128

              Figure 1. Them1 contains two putative active sites.                                                    130

              Figure 2. Asp74 and N232 are essential for catalysis of myristoyl-CoA.                    131

              Figure 3. Enzymatic mechanism of Them1.                                                                 132

         ADP and ATP bind to Them1 to regulate activity                                                            133

              Figure 4. ADP/ATP directly bind and differentially stabilize Them1.                         134

              Figure 5. ADP/ATP binding site in Them1 is conserved.                                            135

         Preliminary crystals of Them1 thioesterase domains                                                        136

         Preliminary Cryo-EM structure of Them1                                                                        137

              Figure 6. Purification and crystallization of trimeric Thio-MBP.                               138

              Table 1. Crystallization conditions that yield Thio-MBP crystals.                              139

              Figure 7. Preliminary cryo-EM image of Them1 homogenously spread                     139

              across grid.

    Discussion                                                                                                                              140

    Methods                                                                                                                                 142

    References                                                                                                                             147

CHAPTER 5: DISCUSSION                                                                                                    150

    Lipid Chaperone the Extends Life                                                                                         151

         Figure 1. LBP-8 extends lifespan in C. elegans through carrying lysosomal fatty          152

         acids to nuclear receptors.

    Additional Lipid Chaperones that Extend Life                                                                      153

    Lipid Sensor that Regulates Thermogenesis                                                                          155

         Figure 2. Schematic of the multiple layers of regulation of Them1 activity.                    156

    Additional Lipid Sensors that Regulate Thioesterase Activity                                              158

    Future Directions of the Field                                                                                                159

         Figure 3. LTPs interact with a diverse array of effector proteins.                                   161

    Utility of LTPS as Drug Targets                                                                                            162

         Figure 4. PCTP suppresses PPAR- transcriptional activity                                           163

    Final Thoughts                                                                                                                        164

    References                                                                                                                              165

APPENDIX                                                                                                                                171


    Introduction                                                                                                                          173

    Results & Discussion                                                                                                            174

         LBP-2 and LBP-3 extend lifespan                                                                                    174

         LBP-2 and LBP-3 bind to C20-PUFAs                                                                             174

              Figure 1. LBP-2 and LBP-3 extend lifespan of C. elegans.                                         175

              Figure 2. LBP-2 and LBP-3 mediate induction of EGL-21 by LIPL-4                        175

              overexpression to extend lifespan.

              Figure 3. LBP-2 and LBP-3 bind to C20-PUFAs.                                                       177

              Figure 4. C20-PUFAs are required for induction of EGL-21 to extend lifespan.       178

         Structural Insights in LBP-3                                                                                              179

              Figure 5. Crystal structure of LBP-3 reveals dimer unable to bind fatty acid.           180

              Figure 6. LBP-3 purifies as a monomer.                                                                      181

    Materials & Methods                                                                                                           183

    References                                                                                                                             188

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