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

Musille, Paul Michael (2014)

Permanent URL: https://etd.library.emory.edu/concern/etds/t722h908f?locale=es
Published

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

Rights statement
  • Permission granted by the author to include this thesis or dissertation in this repository. All rights reserved by the author. Please contact the author for information regarding the reproduction and use of this thesis or dissertation.
School
Department
Subfield / Discipline
Degree
Submission
Language
  • English
Research Field
Palabra Clave
Committee Chair / Thesis Advisor
Committee Members
Última modificación

Primary PDF

Supplemental Files