Structural and Functional Studies of the Orphan Nuclear Receptor LRH-1 Öffentlichkeit

Abstract

The NR5A subfamily family of nuclear receptors are important regulators of pluripotency, lipid and glucose homeostasis, and steroidogenesis. Liver receptor homologue 1 (LRH-1; NR5A2) has therapeutic potential for the treatment of metabolic and neoplastic disease; however, a poor understanding of its ligand regulation has hampered the pursuit of these proteins as pharmaceutical targets. It was previously thought that LRH-1 was an intractable therapeutic target due to its orphan classification, but structural and biochemical studies combined with extensive small molecule screening have since shown that LRH-1 is a tractable drug target. LRH-1 is known to bind phospholipids (PLs) but the role of PLs in controlling LRH-1 activation remains highly debated. An improved understanding of the molecular determinants of LRH-1 activation may enhance efforts to target LRH-1 with therapeutics. The data presented here use a variety of structural and biochemical techniques to probe how phospholipid ligands, coregulator proteins and evolutionary changes alter LRH-1's activation state

Table of Contents

Chapter 1: Introduction.................................................................................................... 1

Introduction.......................................................................................................................... 2 Phospholipids................................................................................................................... 2

Nuclear Receptors: lipid regulated transcription factors.................................................... 4

Case Studies........................................................................................................................... 6

SF-1................................................................................................................................. 9 PPARs............................................................................................................................ 11 USP................................................................................................................................ 13

PL transport and PL dependent coactivation...................................................................... 13

PPAR and PC-TP........................................................................................................... 14

Structural Analysis of PL binding proteins........................................................................... 14

What does it take to bind to PLs as a ligand?.................................................................. 14

Shuttlers versus transcription factors............................................................................... 15

Parallels in the immune system....................................................................................... 15

Comparison to the PL PI/PC transporter Sec14.............................................................. 17

PL presentation as a model for PL dependent signaling.................................................. 17

Closing Remarks................................................................................................................. 18

LRH-1 Knowledge Gaps...................................................................................................... 20

Chapter 2: Antidiabetic phospholipid-nuclear receptor complex reveals the mechanism for phospholipid-driven gene regulation 22

Introduction........................................................................................................................ 23

Experimental Procedures.................................................................................................... 24

Results................................................................................................................................ 30

Structure of the activated LRH-1-DLPC-TIF2 NR box 3 complex................................... 30

Phospholipid tail composition drives differential receptor dynamics............................... 34

Generating apo LRH-1................................................................................................... 37

Ligand Binding Alters Co-regulator Preference............................................................... 37

β-sheet-helix 6 mobility is essential for PL driven transactivation.................................... 42

Structure of the apo LRH-1-SHP NR Box1 complex....................................................... 42

Empty receptor binds with high affinity to SMRT............................................................. 47

Discussion........................................................................................................................... 47

Chapter 3: Divergent sequence tunes ligand sensitivity in phospholipid-regulated hormone receptors 50

Introduction........................................................................................................................ 51

Experimental Procedures.................................................................................................... 54

Results................................................................................................................................ 58 Overall Structure............................................................................................................. 58

Apo mlLRH-1 adopts a destabilized active orientation.................................................... 61

Sequence divergence allows to NR5A receptors adopt multiple conformations to achieve the active state 63

Do mLRH-1 and Ftz-F1 bind PLs despite their ability to crystallize empty?.................... 63

mlLRH-1 is specifically stabilized versus hLRH-1............................................................ 67

Discussion........................................................................................................................... 70

Chapter 4: Unexpected Allosteric Network Drives Nuclear Receptor-Phospholipid Signaling 74

Introduction........................................................................................................................ 75

Experimental Procedures.................................................................................................... 77

Results................................................................................................................................ 80

Structure of the apo LRH-1 LBD-TIF complex............................................................... 80

Structure of the LRH-1 LBD-E. coli PL-TIF2 complex................................................... 80

Co-regulator binding interactions are altered by ligand status........................................ 84

Discussion........................................................................................................................... 85

Chapter 5: Discussion..................................................................................................... 87

Phospholipids as signaling molecules................................................................................... 88

LRH-1 is an untapped pharmacological target..................................................................... 88

LRH-1's mode of activation is unique among NRs............................................................... 88

Sequence Divergence throughout evolution has altered the structural mechanisms driving LRH-1 activation 89

LRH-1 structures as invaluable tools to understand the detailed molecular mechanism driving receptor transactivation and transrepression. 90

Future Directions................................................................................................................ 90

Concluding Remarks............................................................................................................ 91

Appendix: Structure of Glycerol Dehydrogenase From Serratia................................ 94

Introduction........................................................................................................................ 95

Experimental Procedures.................................................................................................... 96

Results and Discussion....................................................................................................... 105

References...................................................................................................................... 114

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Molecular & Systems Pharmacology
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Health Sciences, Pharmacology
Stichwort	LRH-1 Nuclear Receptor Phospholipids
Committee Chair / Thesis Advisor	Ortlund, Eric, Emory University
Committee Members	Chen, Jing, Emory University Murphy, T J, Emory University Nahta, Rita, Emory University Conn, Graeme L, Emory University

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