The Role of Fatty Acid Binding Protein 5 (FABP5) in Peroxisome Proliferator-Activated Receptor β/δ (PPARβ/δ)-Mediated Fatty Acid and Retinoic Acid Signaling: A Structural Perspective Public
Armstrong, Eric (2014)
Published
Abstract
The fatty acid binding proteins (FABPs) are a group of intracellular lipid-binding proteins (iLBPs) found throughout the animal kingdom, nine of which have been characterized in mammals, including humans. Consisting of a 10-stranded β-barrel capped by two α-helices, these ~15 kDa calycins were initially believed to assist in the solubilization of their lipid ligands. More recent studies, however, have expanded the role of certain FABPs as specific mediators of vital signaling pathways. FABP5, like its family members, displays a promiscuous binding profile, being able to form a complex with numerous long chain fatty acids of varying degrees of saturation, as well as fatty acid metabolites, retinoids, and synthetic probes and drugs. Interestingly, only a portion of those tested, such as the ω-6 polyunsaturated linoleic acid and the Vitamin A metabolite all-trans retinoic acid (atRA), had been demonstrated as "activators," whose binding results in the protein's translocation from the cytoplasm to the nucleus, where it is then able to deliver its cargo to the nuclear peroxisome proliferator-activated receptor β/δ (PPARβ/δ), thereby enhancing the receptor's target gene transcription. However, the molecular mechanisms underlying both FABP5's nuclear import as well as its activating ligand selectivity remained unclear.
The work contained herein has established the existence of a tertiary nuclear localization signal (NLS) located within the α-helical cap of FABP5. Formation of the NLS has been found to be dependent upon the interplay between the protein's α2 helix and β2 loop. These dynamics are in turn governed by the conformation of complexed fatty acid, in which a higher degree of alkyl tail curvature within the protein's binding pocket results in FABP5's adoption of the activated state. This model implicates cis bonded polyunsaturated fatty acids as an entire class of potential FABP5 activating ligands. In contrast, the highly planar atRA was not found to bind appreciably to either FABP5 or PPARβ/δ in vitro. Thus, it is proposed that one or more of RA's cis isomers might actually be responsible for the FABP5, PPARβ/δ-mediated retinoid signaling pathway.
Table of Contents
CHAPTER 1: GENERAL INTRODUCTION 1
1.1 Introduction 2
1.1.1 Intracellular Lipid Binding Proteins
2
1.2.1 Metabolism of Vitamin A 9
1.2.2 The Retinoic Acid Receptors 17
1.2.3 Cellular Retinoic Acid-Binding Protein II 22
1.3 The FABP5-PPARβ/δ Signaling Pathway 261.3.1 Long-Chain Fatty Acids 26
1.3.2 The Peroxisome Proliferator-Activated Receptors 29
1.3.3 PPARβ/δ 33
1.3.4 FABP5 35
1.4 Objectives of the
Dissertation 39
CHAPTER 2: STRUCTURAL BASIS FOR LIGAND REGULATION OF
THE FATTY ACID BINDING PROTEIN 5, PEROXISOME PROLIFERATOR-ACTIVATED
RECEPTOR B/Δ (FABP5-PPARB/Δ) SIGNALING
PATHWAY 40
2.1 Introduction
41
2.2 Methods 43
2.3 Results 50
2.3.1 Overall Structure and
Oligomerization Status of Apo and Holo FABP5
50
2.3.2 Linoleic Acid Binds FABP5 In
Two Distinct Conformations 53
2.3.3 Conformation of the Bound Fatty Acid Dictates
Activation of FABP5 55
2.3.4 FABP5 Contains a Ligand-Sensitive NLS Within Its
α-Helical Lid 62
2.3.5 FABP5's NES Equivalent Residues Are Necessary for
Protein Stability 65
2.3.6 Ligand-Specific Dynamics Between β2
Loop and α2 Helix Drives Tertiary NLS
Formation 67
2.4 Discussion 73
CHAPTER 3: VIABILITY OF A STRUCTURE BASED INVESTIGATION
OF NON-CLASSICAL ALL-TRANS RETINOIC ACID (ATRA)
SIGNALING 77
3.1 Introduction 78
3.2 Methods 80
3.3 Results 88
3.3.1 hFABP5 Does Not Bind atRA at Levels Tractable for
Crystallography 88
3.3.2 hFABP5 Interaction With Yeast Importin
α Occurs With Weak Affinity
98
3.3.3 The Transient FABP5-PPARβ/δ
Complex Can Be Captured With Crosslinking
100
3.3.4 hPPARβ/δ Does Not
Appreciably Bind atRA In Vitro
102
3.4 Discussion 104
CHAPTER 4: DISCUSSION
108
4.1 Summary of Results
109
4.1.1 Chapter 2 109
4.1.2 Chapter 3 110
4.2 Future Directions
112
4.2.1 Chapter 2 112
4.2.2 Chapter 3 115
4.3 Conclusion 115
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