A STRUCTURAL AND BIOCHEMICAL INVESTIGATION OF HOW LIPID MESSENGERS ACT THROUGH LIPID TRANSFER PROTEINS TO REGULATE METABOLISM AND LONGEVITY Open Access
Tillman, Matthew (Summer 2020)
Abstract
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
LIPID BINDING PROTEIN 2 AND LIID BINDING PROTEIN 3 EXTEND LIFESPAN IN CAENORHABDITIS ELEGANS THROUGH CARRYING POLYUNSATURATED FATTY ACIDS TO NEURONAL TISSUE 172
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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