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Classification and Effect of Correctors on Sitosterolemia Classification and Effect of Correctors on Sitosterolemia Associated Mutants in ABCG8

Brittney Poole

University of Kentucky, brittney.poole@uky.com

Author ORCID Identifier: 

http://orcid.org/0000-0002-0955-8815 Digital Object Identifier: https://doi.org/10.13023/etd.2022.339

Recommended Citation

Poole, Brittney, "Classification and Effect of Correctors on Sitosterolemia-Associated Mutants in ABCG8" (2022). Theses and Dissertations--Medical Sciences. 23. https://uknowledge.uky.edu/medsci_etds/23

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Brittney Poole, Student 

Dr. Gregory A. Graf, Major Professor 

Dr. Melinda Wilson, Director of Graduate Studies

CLASSIFCATION AND EFFECT OF CORRECTORS ON SITOSTEROLEMIA ASSOCIATED MUTANTS IN ABCG8

 ________________________________________

THESIS 

________________________________________

A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in the College of Medicine at the University of Kentucky

By 

Brittney Poole 

Lexington, Kentucky 

Director: Dr. Gregory A. Graf, Professor of Pharmaceutical Sciences 

Lexington, Kentucky

Copyright © Brittney Poole 2022 

http://orcid.org/0000-0002-0955-8815

BSTRACT OF THESIS

CLASSIFICATION AND EFFECT OF CORRECTORS ON SITOSTEROLEMIA ASSOCIATED CYTOSOLIC MUTANTS IN ABCG8

Objective: To classify mutants of ABCG8 identified in subjects with clinically confirmed Sitosterolemia, a rare form of Familial Hypercholesterolemia distinguished by the accumulation of phytosterols in plasma and tissues and determine the effects of correctors and/or regulators of proteostasis on maturation of the ABCG5/ABCG8 sterol transporter.

Methods: Disease-causing missense mutants within the cytosolic domain of ABCG8 were generated through site-directed mutagenesis. Normal and mutant proteins were expressed in human hepatocytes. Cellular proteins were prepared, and maturation was assessed by SDS-PAGE and immunoblotting. Formation of the higher molecular weight, mature form of glycoproteins was used as a bioassay for trafficking the G5G8 complex beyond the Endoplasmic Reticulum. The impact of correctors and regulators of proteostasis on Class II mutant maturation was also determined.

are maturation incompetent. Of those which matured beyond the ER, 60% were not able to traffic to the cell membrane. Of the mutants that did not mature, none were able to be rescued by small molecular chaperones (correctors).

Conclusion: HuH-7 cells are an efficiently transfected cell line that provides a system to manipulate ABCG5 and ABCG8 to make conclusions about protein maturation and trafficking to the cell surface. These experiments gave insight into the complexity of diseases caused by genetic mutations and the underlying mechanism of loss-of-function mutations. Further experimentation would be required to determine the fate of the CFTR correctors and/or regulators of proteostasis in the application in cases of Sitosterolemia.

KEYWORDS: Sitosterolemia, ABC transporters, lipids, correctors, proteostasis

Brittney Poole 

(Name of Student) 

07/27/22

 Date

CLASSIFICATION AND EFFECT OF CORRECTORS ON SITOSTEROLEMIA ASSOCIATED CYTOSOLIC MUTANTS IN ABCG8 

By Brittney Poole

Gregory A. Graf 

Director of Thesis 

Melinda Wilson 

Director of Graduate Studies 

07/27/22 

Date

ACKNOWLEDGMENTS

The following thesis, while an individual work, benefited from the insights and direction of several people. First, my Thesis Chair, Dr. Gregory A. Graf, who provided a space to have a “first day in the lab.” Dr. Graf was not only supportive during the research process, but also challenged me to understand the literature and develop my own scientific thinking. Next, I wish to thank the Thesis Committee: Dr. Scott Gordon and Dr. Ryan Temel. Each individual provided insights that guided and challenged my thinking. Next, I would like to thank all the lab members of the Graf and Helsley lab for their support and expertise in the lab. I would also like to acknowledge the use of BioRender to create the figures in this thesis.

In addition to the assistance mentioned above, I received equally important assistance from family and friends. To my family, Brett, Sonja, and Courtney Poole provided on-going support while navigating living in a new state alone.

TABLE OF CONTENTS

CLASSIFCATION AND EFFECT OF CORRECTORS ON SITOSTEROLEMIA 

ASSOCIATED MUTANTS IN ABCG8 ................................................................................. i

 ABSTRACT OF THESIS ..................................................................................................... ii

 ACKNOWLEDGMENTS .................................................................................................... iii 

TABLE OF CONTENTS ..................................................................................................... iv 

LIST OF TABLES ............................................................................................................... vi 

LIST OF FIGURES ........................................................................................................... vii 

CHAPTER 1. Introduction................................................................................................. 1 

1.1 Background.............................................................................................................................. 1 

1.2 Cholesterol vs. Phytosterols ..................................................................................................... 4 

1.3 Sterol Absorption and Excretion ............................................................................................... 5 

1.4 Sitosterolemia .......................................................................................................................... 8 

1.5 ABCG5/G8 Physiology .......................................................................................................... 10 

1.6 ABC Transporters .................................................................................................................. 11 

1.7 Proteostasis Regulation and Roscovitine................................................................................. 15

 1.8 Ivacaftor and ABCC7 Potentiation ......................................................................................... 19 

1.9 Statement of Hypothesis ......................................................................................................... 22 

CHAPTER 2. Materials and Methods ............................................................................. 23

 2.1 Materials and Methods ........................................................................................................... 23 

2.1.1 Reagents ................................................................................................................................. 23 

2.1.2 Cell Culture ............................................................................................................................ 23 

2.1.3 GFP Assay ............................................................................................................................. 24 

2.1.4 Western Botting Analysis ...................................................................................................... 24 

2.1.5 In-vitro Bioassay .................................................................................................................... 25 

2.1.6 Densitometric Analysis .......................................................................................................... 25 

2.1.7 Immunofluorescence Microscopy ......................................................................................... 26 

2.1.8 Statistical Analysis................................................................................................................. 26 

2.2 Experiment I- Generation of Sitosterolemia Associated ABCG8 Cytosolic Mutants ................ 27

2.3 Experiment II- Optimization of Transient Transfection of Human Cell Lines .......................... 30

2.4 Experiment III- Mutant Maturation Assay .............................................................................. 31 

2.5Experiment IV- Native ABCG5/G8 Complex Compound Screening ....................................... 33 

2.6 Experiment V- Roscovitine Toxicity ...................................................................................... 34 

2.7 Experiment VI- Corrector Testing of Class II Mutants ............................................................ 34 

2.8 Experiment VII- Immunofluorescence of Maturation Competent Mutants .............................. 35

CHAPTER 3. Results ....................................................................................................... 36 

3.1 Experiment I- Generation of Sitosterolemia-Associated ABCG8 Cytosolic Mutants ............... 36 

3.2 Experiment II- Optimization of Transient Transfection of Human Cell Lines .......................... 39 

3.3 Experiment III- Mutant Maturation Assay .............................................................................. 41 

3.4 Experiment IV-Corrector Testing of Class II Mutants ............................................................. 45 

3.5 Experiment V- Testing Regulators of Proteostasis on Native ABCG58 ................................... 46 

3.6 Experiment IV- Roscovitine Dose-Response .......................................................................... 48 

3.7 Experiment VI- Immunofluorescence Trafficking Assay ........................................................ 49 

CHAPTER 4. Discussion ................................................................................................. 54 

4.1 Limitations............................................................................................................................. 57 

4.2 Future Directions ................................................................................................................... 58 

REFERENCES .................................................................................................................. 60 

VITA………….. .................................................................................................................. 68

LIST OF TABLES 

TABLE 1.1 ABC TRANSPORTERS AND THEIR ASSOCIATED DISEASE46. .............................................................. 3 

TABLE 1.2. PROPOSED SITOSTEROLEMIA CLASSIFICATION SYSTEM52 ............................................................ 15 

TABLE 1.3. ROSCOVITINE AND ANALOGS STRUCTURE. .................................................................................. 18 

TABLE 1.4. IVACAFTOR AND CORRECTORS STRUCTURE ................................................................................ 21 

TABLE 2.5. SITOSTEROLEMIA-ASSOCIATED MUTANTS GENERATED BY SITE-DIRECTED MUTAGENESIS IN 

THE CYTOSOLIC DOMAIN OF ABCG8 ......................................................................................................... 29 

TABLE 4.6. UPDATED SITOSTEROLEMIA CLASSIFICATION SYSTEM FOR MUTANTS52. ..................................... 57

LIST OF FIGURES FIGURE 

1.2-1 CHOLESTEROL AND PHYTOSTEROL STRUCTURE. ........................................................................ 5 

FIGURE 1.4-1 DIAGRAM DEMONSTRATING THE ABSORPTION, EXCRETION, AND SECRETION PATHWAY 

OF PHYTOSTEROLS AND CURRENT THERAPEUTICS FOR SITOSTEROLEMIA. ................................................... 9 

FIGURE 1.6-1 STRUCTURE OF ABC TRANSPORTER FAMILIES45. ...................................................................... 13 

FIGURE 2.2-1 EXAMPLE OF SITE-DIRECTED MUTAGENSIS RESTRICTION ENZYME DIGEST AND SEQUENCE 

VERIFICATION (GENERATED USING SNAPGENE SOFTWARE VERSION 4.3.11). ....................................... 29 

FIGURE 2.4-1 GRAPHICAL REPRESENTATION OF IN VITRO MATURATION ASSAY. ............................................ 32 

FIGURE 3.1-1 ABCG8 MUTATION DIAGRAM ................................................................................................. 37 

FIGURE 3.1-2. ABCG8 SEQUENCE CONSERVATION AMONG SPECIES.............................................................. 38 

FIGURE 3.1-3. ABCG8 SEQUENCE CONSERVATION AMONG ABCG FAMILY MEMBERS. ................................ 38 

FIGURE 3.2-1 COMPARISON OF DIFFERENT TRANSFECTION REAGENTS IN THE HUH-7 CELL LINE. ................. 40 

FIGURE 3.2-2 TRANSFECTION OPTIMIZATION IN HUH-7 CELLS. ..................................................................... 41 

FIGURE 3.3-1. PROTEIN MATURATION BIOASSAY DEMONSTRATING ABCG8 CYTOSOLIC MUTANTS CO 

TRANSFECTED WITH ABCG5-MYC. ...................................................................................................... 42 

FIGURE 3.3-2. PROTEIN STABILITY WESTERN BLOTS OF MUTANTS IN ABCG8. ............................................. 43

 FIGURE 3.3-3. RESTRICTION ENZYME DIGEST ON CLASS II MUTANTS. ........................................................... 44 

FIGURE 3.3-4. GEL ELECTROPHORESIS OF CDNA FROM HUH-7 LYSATES ..................................................... 45 

FIGURE 3.4-1 PROTEIN MATURATION BIOASSAY WITH TREATMENT WITH PUBLISHED CONCENTRATIONS OF 

CFTR CORRECTORS68. .......................................................................................................................... 46 

FIGURE 3.5-1. ROSCOVITINE SCREENING ON NATIVE ABCG5/G8 COMPLEX IN HUH-7 CELLS AT 100 UM. ... 47 

FIGURE 3.5-2. ROSCOVITINE SCREENING ON NATIVE ABCG5/G8 COMPLEX IN HEPG2 CELLS AT 100 UM. ... 48 

FIGURE 3.6-1 DOSE-RESPONSE DATA ON ROSCOVITINE IN HUH-7 CELLS. ..................................................... 49 

FIGURE 3.7-1. IMMUNOFLUORESCENCE AND IMAGES FROM THE CYCLOHEXIMIDE TIME COURSE 

EXPERIMENT. .............................................................................................................................................................. 50 

FIGURE 3.7-2. TIME-COURSE WITH 50 AND 100 UG/ML CHX. ........................................................................ 51 

FIGURE 3.7-3 IMMUNOFLUORESCENCE IMAGES OF ABCG8 MUTANTS........................................................... 52

CHAPTER 1. INTRODUCTION

1.1 Background

Sitosterolemia is a rare form of familial hypercholesterolemia (FH) caused by two mutations in either the ATP-Binding Cassette protein (ABC) G5 or G8 gene, which is in close proximity of 375 bp between the initiation codons and share a common promotor that function in a head-to-head orientation 1,2,3. Sitosterolemia results from a lack of function for ABCG5 or G8 in the absence of their respective binding partner, which is distinguished from other forms of FH. Sitosterolemia is an autosomal recessive inherited disease that affects about 1 in 200,000 individuals; however, it is unclear how often this disease is misdiagnosed. Clinical laboratory assays fail to distinguish cholesterol from phytosterols. Consequently, plasma from a patient with Sitosterolemia would present with what appears to be elevated total plasma cholesterol in clinical lab testing. Gas or liquid chromatography is required to distinguish phytosterols from cholesterol, a technique and instrumentation often unavailable in clinical laboratories.

Individuals diagnosed with FH and Sitosterolemia similarly present with xanthomas and premature coronary artery disease. The two diseases are distinguished by dominant vs. recessive genetics, clinical presentation, and sterol composition in the plasma. FH patients present with elevated LDL cholesterol, while Sitosterolemia patients present with increased plasma phytosterol, decreased excretion of phytosterols and cholesterol, and hemolytic and blood disorders 4,5. The underlying genetic causes of FH and Sitosterolemia differ. In cases of FH, the mutations affect either LDL-R, its ligand Apolipoprotein B (ApoB100), or the machinery required for LDL/LDL-R internalization,LDL Receptor Related Protein-Associated Protein 1 (LRPAP1) or proprotein convertase subtilisin/kexin type 9 (PCKS9) while Sitosterolemia results from genetic mutations in ABCG5 and/or ABCG8.

Many ABC transporters are associated with diseases such as; Cystic Fibrosis (ABCC7), Progressive familial intrahepatic cholestasis type 3 (ABCB4), among others (Table 1.1)6. Investigation and FDA-approved drugs (Roscovitine; CFTR modulators) partially restore function to ABCC7 and ABCB4 mutants based on their underlying molecular defect. The rationale behind testing these modulators on cytosolic mutants of ABCG8 is that if these correctors and regulators of proteostasis are effective in multiple ABC transporters, then due to the evolutionarily conserved nature of the ABC transporters (Fig. 1.2-1), these may also be effective for mutants of ABCG8⁷.

Table 1.1 ABC Transporters and their associated disease_⁴⁶.

Table 1.1   shows  different ABC transporters and their  associated diseases, demonstrating a wide range of diseases caused due to mutations in ABC transporters.

We hypothesize that small molecule correctors will enhance the

maturation of native G5/G8 complex and Class II, maturation deficient mutants of ABCG8.

1.2 Cholesterol vs. Phytosterols

Sterols  are  an  essential  cellular  component  of  eukaryotic membranes,  with cholesterol in animal cells and phytosterols in plant cells. Cholesterol creates rigidity and curvature to the plasma membrane of animal cells. Cholesterol is acquired through diet or is generated by de novo synthesis. Cholesterol biosynthesis is a tightly regulated process that demonstrates negative feedback inhibition. When cholesterol is in excess, it can be toxic to the cell, while cells depleted of cholesterol cannot undergo normal physiological responses,  for  example, receptor  signaling8.  The transcription  factor  sterol regulatoryelement-binding protein 2 (SREBP2) regulates gene expression of the enzymes responsible for  cholesterol  biosynthesis and  uptake  to maintain  homeostasis⁹. While  endogenous cholesterol  synthesis  is  tightly  regulated,  dietary   cholesterol  ranges due  to   dietary

preferences and maybe a pro-atherogenic factor_¹⁰.

plasma sample.

It has been proposed that phytosterols help reduce LDL cholesterol by competing with cholesterol for intestinal absorption resulting in a modest reduction (30-50%) of cholesterol absorption and a 10% decrease in total plasma cholesterol13. The mechanism by which phytosterols compete with cholesterol for intestinal absorption is by reducing the solubilization into the mixed micelle. However, other studies suggest phytosterols promote

cholesterol secretion13,14,15 .

Figure 1.2- 1 Cholesterol and Phytosterol structure.

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Figure 1.2- 1  Chemical structure of cholesterol and 4 of the most common phytosterols showing the differences in side chains as well as the common 3ß-Hydroxyl group. The red circle indicates common 3ß -Hydroxyl amongst sterols and ring structures are labelled in  cholesterol.

1.3 Sterol Absorption and Excretion

As previously mentioned, cholesterol can enter the body in one of two ways.Cholesterol-esters and phytosterols that enter the body exogenously are metabolized by enzymes (pancreatic lipases) and emulsified with bile salts and phospholipids that enter he intestinal lumen via the gallbladder, ABCB11, and ABCB4, respectively, along with ietary cholesterol to form mixed micelles. Mixed micelles act as the "cholesterol cceptor" for dietary sterols16. Free cholesterol and hytosterols are absorbed from the ixed micelle into the small intestine (duodenum/jejunum) by PC1L1 17,18 . NPC1L1 Niemann-Pick C1 Like 1) functions at the apical surface of enterocytes and mediates the internalization of cholesterol and phytosterol to promote absorption19. In the enterocyte, holesterol is esterified by the enzyme ACAT-2 (Acyl-CoA: Cholesterol 

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