Structural and Functional Characterization of the Asprosin and Protein Tyrosine Phosphatase Receptor Type D (PTPRD) Interaction Restricted; Files Only

Lute, Hannah (Fall 2023)

Permanent URL: https://etd.library.emory.edu/concern/etds/gf06g3962?locale=pt-BR
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Abstract

By the year 2030, 50% of Americans are expected to be obese. Difficulty in treating obesity comes from an inability to control appetite and modulate hepatic glucose release in patients. A hormone, named asprosin, was recently discovered to stimulate an increase in appetite through its interaction with the protein tyrosine phosphatase receptor type D (PTPRD) in the hypothalamus. This interaction has considerable potential as a therapeutic target that can be harnessed to treat obesity through modulation of appetite. The goal of this project is to characterize the asprosin-PTPRD complex structurally and functionally as a basis for future pharmacological design.

Table of Contents

Table of Contents

Chapter One: Regulation of Appetite Through the Melanocortin System.. 1

Introduction: 1

The melanocortin system and appetite regulation. 1

Agouti-related protein (AgRP)/ Neuropeptide Y (NPY)-expressing neurons. 2

Proopiomelanocortin (POMC)-expressing neurons. 2

Key players: leptin, insulin, and ghrelin. 3

Asprosin, a new player in the game. 5

Figures. 6

Figure 1.1: Opposing actions of AgRP and POMC neurons within the ARH. 6

Figure 1.2: Mechanisms of hormone signaling in the melanocortin system. 7

References. 8

Chapter Two: Expression and Purification of Asprosin. 11

Introduction. 11

Results. 12

Bacterial Expression Constructs. 12

Mammalian Expression Constructs. 18

Discussion. 22

Bacterial Expression Constructs. 22

Mammalian Expression Constructs. 23

Figures. 25

Figure 2.1: Asprosin in pET-28a(+) test expressions. 25

Table 2.1: Dialysis conditions for stepwise refold of asprosin from inclusion bodies. 26

Figure 2.2: Asprosin inclusion body stepwise refold. 27

Figure 2.3: Alphafold generated model of asprosin. 28

Figure 2.4: Nickel affinity purification of truncated asprosin cleavable MBP construct. 29

Figure 2.5: Post-TEV cleavage of truncated asprosin cleavable MBP construct. 30

Figure 2.6: SEC of truncated asprosin cleavable MBP post-TEV protease cleavage. 31

Figure 2.7: Amylose-affinity purification of truncated asprosin rigid MBP construct. 32

Figure 2.8: Test expressions of truncated asprosin MBP-fusion constructs co-expressed with molecular chaperones. 33

Figure 2.9: Validation of truncated asprosin cleavable MBP expression with molecular chaperones. 34

Figure 2.10: Purification of truncated asprosin with cleavable MBP from molecular chaperone co-expression. 35

Figure 2.11: Expression of asprosin-pLVX mammalian expression construct. 36

Table 2.2: Primers used for amplification and addition of Gibson arms to asprosin fragment. 37

Figure 2.12: Mammalian expression of full-length (FL) asprosin Fc-fusion and Truncated (Trunc) asprosin Fc-fusion. 38

. 39

Figure 2.13: Purification of asprosin Fc-fusion constructs using nickel-affinity chromatography. 39

Figure 2.14: Asprosin-Fc fusion representations. 40

Materials and Methods. 41

Asprosin in pET-28a(+) 41

Refolding asprosin from inclusion bodies. 41

Mammalian Expression. 43

References. 44

Chapter Three: Expression and Purification of Protein Tyrosine Phosphatase Receptor Type D (PTPRD) 46

Introduction. 46

Results. 48

PTPRD Domains. 48

Full-Length PTPRD Extracellular Domain. 48

Crystallizable PTPRD. 49

Discussion. 50

Figures. 52

Table 3.1: Primers used for generation of PTPRD Expression Plasmids. 52

Figure 3.1: PTPRD Truncation Expression Plasmids. 53

Figure 3.2: Purification scheme for PTPRD-Fn. 54

Figure 3.3: Purification Scheme of PTPRD-IgG. 55

Figure 3.4: Purity of PTPRD domains following size-exclusion chromatography. 56

Table 3.2: Primers used for generation of crystallizable PTPRD construct. 57

Figure 3.5: PCR amplification of crystallizable PTPRD. 58

Materials and Methods. 59

Cloning. 59

Protein Expression and Purification. 60

References. 63

Chapter Four: Characterization of the Asprosin-PTPRD Interaction. 66

Introduction. 66

Results. 67

Negative Stain Electron Microscopy. 67

Cryogenic Electron Microscopy. 68

Bio-Layer Interferometry. 68

Discussion. 69

Figures. 72

Figure 4.1: Reconstruction of the PTPRD-asprosin complex by negative-stain EM. 72

Figure 4.2: 2D Classes of the Asprosin-PTPRD Complex. 73

Figure 4.3: Binding Kinetics of Asprosin and full-length extracellular domain of PTPRD Determined Using BLI. 74

Methods/Materials. 75

Negative Stain Electron Microscopy. 75

Cryogenic Electron Microscopy. 75

Bio-Layer Interferometry. 76

References. 77

Chapter Five: Future Considerations for the Asprosin Project 78

Deep Mutational Scanning. 78

Site-Directed Mutagenesis. 79

Asprosin Clinical Trial 80

Placensin and Gonascin. 80

Figure 5.1: Multiple sequence alignment of asprosin and IL1RAPL1. 82

Figure 5.2: Sandwich ELISA used for plasma asprosin detection. 83

Figure 5.3: 3D modeling of asprosin, placensin, and gonascin. 84

References. 85

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