Classification and E Classification and Effect of Corr ect of Correctors on Sit ors on Sitosterolemia- olemiaAssociated Mutants in ABCG8emiaAssociated Mutants in ABCG8 Brittney Poole Univ

University of Kentucky

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2022

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

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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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REVIEW, APPROVAL AND ACCEPTANCE

The document mentioned above has been reviewed and accepted by the student’s advisor, on behalf of the advisory committee, and by the Director of Graduate Studies (DGS), on behalf of

the program; we verify that this is the final, approved version of the student’s thesis including all  changes required by the advisory committee. The undersigned agree to abide by the statements above.

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

ABSTRACT 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.

Results: Approximately 44% of cytosolic, Sitosterolemia-associated mutants in ABCG8 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

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 Helsleylab 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      ImmunofluorescenceMicroscopy ......................................................................... 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

2.5 Experiment 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 CompetentMutants ............... 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 ahead-to-head orientation 1,2,3 . Sitosterolemia results from a lack of

function for ABCG5 or G8 in the absence of theirrespective 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 disease46.

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 factor10.

Phytosterols are not endogenously synthesized de novo and are strictly enter from the diet. Phytosterols are structurally similar to cholesterol, but differ from cholesterol by  the side chain on the D ring of the sterol backbone (Fig. 1). For this reason, chromatography  and/or mass spectrophotometry is required to distinguish phytosterols from cholesterol in the  plasma.  Phytosterols  have  an  observed  toxicity  in  the  body,  as  a  study  of ABCG5/ABCG8 KOmice fed a high-phytosterol showed signs of premature death, cardiac lesions, liver damage, and hepatosplenomegaly11. With a functional ABCG5/8 transporter effectively opposing absorption and excreting of phytosterols into the feces, mice and  humans are not affected by high-phytosterol containing diets11. In atypical lipid panel used to measure cholesterol, cholesterol oxidase attacks the 3ß -hydroxyl group on the A ring of

cholesterol. This structural feature is shared amongsterols (animal/phytosterols) (Fig. 1) 12.

GC-Mass Spectrometry would be required to separate phytosterols from cholesterol in a

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.

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

the intestinal lumen via the gallbladder, ABCB11, and ABCB4, respectively, along with

dietary cholesterol to form mixed micelles. Mixed micelles act as the "cholesterol

acceptor" for dietary sterols16. Free cholesterol and phytosterols are absorbed from the

mixed micelle into the small intestine (duodenum/jejunum) by NPC1L1 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,

cholesterol is esterified by the enzyme ACAT-2 (Acyl-CoA: Cholesterol

Acyltransferase), which preferentially esterifies cholesterol relative tophytosterols20.

Cholesterol is incorporated into chylomicrons, secreted into the lymphatic system, and

enters the plasma compartment via the left subclavian vein. Cholesterol and plant sterols that are not esterified by ACAT are exported by ABCG5/G8 back to the intestinal lumen.

In the plasma compartment, chylomicrons and triglycerides are metabolized by

Lipoprotein lipases (LPL), generating a smaller, cholesterol-rich chylomicron remnant, which can enter the liver via LDL-R or other related receptors. This pathway delivers

dietary cholesterol andre-absorbed biliary cholesterol to the liver.

Another mechanism by which cholesterol can enter the liver is reverse cholesterol transport (RCT). RCT is a pathway in which cholesterol accumulated in peripheral tissue is transported through the plasma to the liver for excretion as neutral sterols or bile acids

in the feces21. Peripheral tissues, such as macrophages, export excess cholesterol by

ABCA1 to a pre-HDL particle composed of a lipidated Apo-A1. This process is

dependent on the Lecithin Cholesterol Acyltransferase (LCAT) enzyme. LCAT esterifies

cholesterol to fatty acids to generate cholesterol esters that get deposited in the core of

HDL particles22. As opposed to ACAT, LCAT is thought to freely esterify phytosterols to

generate mature, spherical HDL. ABCG1, SR-B1, CD36, and other possible sterol transporting enzymes can deliver esterified sterols in the form of HDL to the liver

through Scavenger Receptor Class B I (SR-BI)23.

Once cholesterol enters the hepatocyte, it can be excreted in one of two ways. The first pathway is metabolism by cytochrome P450 enzymes (ex. CYP7A1/CYP27A1) into

bile salts. Bile salts are secreted at the canalicular membrane of hepatocytes by the

transporter BSEP (Bile salt export protein, ABCB11)23. The other mechanism by which   cholesterol can be excreted from the liver is as free cholesterol via ABCG5/8,which also functions at the hepatocyte canalicular membrane. However, 95% of bile acids and 80%

of cholesterol secreted will be re-absorbed in the intestine by apical sodium bile acid

transporter (ASBT) and Niemann-Pick C1-Like 1 (NPC1L1)25,26  . Alternatively, sterols

can return to the plasma in the form of VLDL particles along with Apo-B100,

triglycerides, and LDL particles are secreted from the liver a shared mechanism with    chylomicrons and ultimately give rise to the LDL particle. Both chylomicron remnants

and LDL are pro-atherogenicparticles.

The Transintestinal Cholesterol Excretion (TICE) pathway is an alternative

pathway to hepatobiliary excretion of cholesterol from the body. TICE is the process of

cholesterol transport from the bloodstream, across the enterocyte, and directly into the

intestinal lumen27. Cholesterol is taken up by LDL-R on the basolateral membrane of

enterocytes (with possibilities for another pathway)28 . Cholesterol is then translocated

across the enterocyte and secreted via ABCG5/8 at the apical membrane for excretion in

the feces or possible reabsorption by NPC1L128. This alternate pathway is thought to

account for 30-50% of RCT29.

1.4 Sitosterolemia

In 1974, two sisters presented with tuberous xanthomas and plasma plant sterols that accounted for roughly 30% of their total plasma sterols. In atypical human subject,

plant sterols are typically only detected in trace amounts due to poor absorption, only

about 5%4. Both parents of the sisters that unaffected, leading to the conclusion that this rare lipid disease was inherited in an autosomal recessive manner4. It wasn’t until 2000

that ABCG5 and ABCG8 were discovered to be the two genes mutated in clinically

confirmed patients with Sitosterolemia30. In 2003, it was confirmed that ABCG5 and

ABCG8 function as obligate heterodimers, with both proteins being required to reach the

cell surface and transport sterols across the membrane31. In addition to the previously

reported phenotypes of Sitosterolemic patients, subjects with clinically confirmed

Sitosterolemia could also present with hematological abnormalities, including

macrothrombocytopenia, hemolytic anemia, and splenomegaly5.

Currently, the primary treatment(s) for Sitosterolemic patients are dietary

modifications (low sterol diet), Ezetimibe, bile acid-binding resins, ileal bypass surgery, or LDL apheresis. However, these treatments decrease plasma phytosterols and improve

symptoms but fail to return plasma sterols to the range of a normal human subject32.

Ezetimibe is an NPC1L1 inhibitor, reduces cholesterol, and phytosterol absorption by

~50%, and is currently the primary pharmacotherapy used to treat cases of

Sitosterolemia33. While Ezetimibe has shown efficacy in reducing phytosterol

accumulation, the drug does not target ABCG5/G8. This is important because ABCG5/8

promotes sterol, primarily phytosterol, efflux at the enterocyte and hepatocyte apical

surface, whereas NPC1L1 primarily functions in sterol absorption in the enterocyte.

NPC1L1 is highly expressed in both the liver and intestine and oppose biliary sterol

secretion in humans, while there is low expression in mice liver19. Even if phytosterol

uptake into absorption is opposed, there is still a lack of hepatic secretion and an inability

to eliminate phytosterols from plasma and tissues once accumulated (Fig. 1.4- 1).

Figure 1.4- 1 Diagram demonstrating the absorption, excretion, and secretion pathway of

phytosterols and current therapeutics for Sitosterolemia.

Figure 1.4- 1 Pathway for excretion and secretion of phytosterols in the liver and intestine. Diagram also exhibits the mechanism of action for ezetimibe, the most common current treatment management of Sitosterolemia.

In a 10-year follow-up study of two sisters who had a homozygous nonsense

mutation (R446X inABCG5), treatment with Ezetimibe only moderately decreased

phytosterol levels while diet changes had little to no change on phytosterol levels34.

Starting phytosterol (sitosterol and campesterol) levels were greater than 300 µmol/L and

decreased to ~100 µmol/L, however,the normal range for a healthy individual is 10-20

µmol/L34. While Ezetimibe is a form of Sitosterolemia management, these patients still

have far higher cholesterol and phytosterol absorption, which can increase the risk of

cardiovascular events later in life. This demonstrates a need for a more specific

pharmacologic agent that targets the underlying molecular defect of ABCG5 and ABCG8

disease-causing mutants as opposed to disease management through Ezetimibe.

1.5 ABCG5/G8 Physiology

ABCG5 and ABCG8 (ABCG5/8) are two ABC-half transporters that function as a

heterodimer at the apical membrane of hepatocytes (liver) and enterocytes (small

intestine)30. ABCG5 and ABCG8 at the transcriptional level have been shown to be

regulated by two nuclear receptors, Liver X Receptors (LXR) and Farnesoid X Receptors (FXR). LXR“ and β belong to the family of nuclear receptors that are master regulators

of genes involved in cholesterol elimination pathways and form heterodimers with

Retinoid X Receptors (RXR)35. These transcriptional factors areactivated by cholesterol metabolites, oxysterols36. It was determined that gene expression of ABCG5 and ABCG8was increased when mice were fed a high cholesterol diet in wildtype mice but in LXR-/- ,

indicating the two genes are also regulated by the LXR/RXR transcription factor37.

ABCG5 and ABCG8 can also be upregulated by the FXR pathway, a nuclear receptor

activated in the presence of bile acids37. ABCG5 and ABCG8 are also positively regulated by the transcription factors hepatocyte nuclear factor 4a (HNF4“), Forkhead box protein

O1 (FOXO1), and Liver receptor homolog 1 (LRH1), but the relationships to sterol

homeostasis are not as clear38,39 .

Beyond the nucleus, ABCG5 and ABCG8 are independently translated in the

rough ER. It is unclear how ABCG5 and ABCG8 emerge from the ribosome and find

theirrespective binding partner. Within the ER, ABCG5 and ABCG8 are recognized by

molecular chaperones, such as calnexin, that facilitate folding of the ABCG5/ABCG8

complex in an N-linked glycan-dependent mechanism. Chaperones recognize the

terminal glucose on theN-linked glycan, a tag that sorts proteins for their destination. Proteins that are unfolded are recognized by a glucosyltransferase in the ER and the

protein is either is re-glucosylated or exits the chaperone folding “cycle." If in fact

ABCG5/8 have dimerized, they are sorted to the apical membrane through the cell's

secretory pathways to function as a heterodimer40.

In summary, ABCG5/G8 function as an obligate heterodimer at the apical surface of hepatocytes and enterocytes. In the liver, ABCG5/8 promotes secretion of phytosterols and cholesterol into bile31. In the intestine, ABCG5/G8 opposes phytosterol absorption by NPC1L1. In mice, expression in one organ, either liver or intestine, is sufficient to protect the animal from Sitosterolemia and the downstream effects of phytosterol accumulation41.

However, it is unclear whether this remains true regarding RCT.

1.6 ABC Transporters

ABC transporters are a family of integral proteins that function to translocate

substrates across membranes, a mechanism powered by ATP hydrolysis7. The ABC

transporter superfamily consists of 48 proteins and can be further divided into

subfamilies, A-G42.  The key characteristics of ABC transporters are two nucleotide-

binding domains (NBD), two transmembrane domains (TMD) that have 12 membrane-

spanning alpha-helices, and two ATP-Binding Cassette’s (ABC), however, there are

exceptions among the ABC transporters (Fig. 2)42,43 . Among the 48 transporters in

humans, there are half transporters (ABCG5/8) and full transporters (for example,

ABCB1).  TheNBD of ABC transporters is a domain that is highly conserved and

includes the Walker A motif, Walker B motif, Walker C motif (signature motif), Q-loop,

and H-loop (switch motif)6,7 .

While structurally similar across ABC transporters, the TMD sequence differs

significantly across and within families, likely a reflection of different substrate binding    pockets6. TheNBD binds and hydrolyzes ATP to power the pump6. ATP binds to both of theNBDs which forms a tight dimer and two ATPase active sites44. A helix in the TMD

fits into a groove on theNBD, resulting in the opening and closing of theNBD

simultaneously to transmembrane helices movement44.

Figure 1.6- 1 Structure of ABC transporter families45.

Figure 1.6- 1 Structure of the ABC transporter families showing the Transmembrane domain and Nucleotide Binding domain, as well    as the structural differences between each of the families, most notably differences in whether theNBD resides at theN or C-terminus.

ABC Transporters import (prokaryotes) or export (eukaryotes) substrates via ATP

hydrolysis cycling7.  Substrate translocation begins with the substrate binding to the

TMD and initiating a conformational change from an open to closed conformation. After

the substrate is bound to the TMD, ATP can bind to theNBD, which induces the ATP

"power stroke” and promotes the closed formation of the TMD. ATP is then hydrolyzed

into ADP and inorganic phosphate (Pi), effectively translocating the substrate. The

release of ADP and Pi indicates the end of the ATP hydrolysis cycle. The protein is  restored to its original open conformation and is ready to transport another substrate

across the membrane6.

The G family of ABC transporters all function as dimers and transport sterols47.

Sterols, and a number of other lipids, are amphipathic and insoluble in water. Sterols

require carriers and transporters to move through the body and plasma membrane.

ABCG1, 2, and 4 are functionally active as homodimers whileABCG5/G8 is functionally

active as an obligate heterodimer. ABCG1/4 have been proposed as candidates for

heterodimerization due to their sequence similarity, although the evidence is lacking48.

ABCG5/8 specifically functions to secrete sterols from hepatocytes into bile and from

enterocytes back to the intestinal lumen for excretion. Both hepatocytes and enterocytes

are polarized cell types each expressing an apical and basolateral membrane that serve

different functions for the cell in addition to different transporter makeup. NBS2 is

thought to be the driver of conformational change with respect to ATP hydrolysis and

sterol transport while NBS1 binds ATP but does not hydrolyze it49. NBS1 and NBS2 are

located within ABCG5 and ABCG849.

Many ABC transporters that are disease-causing, such as ABCC7/Cystic Fibrosis

or ABCB4/PFIC3, have systems to classify mutations50,51 . Shown in Table 1.2 is the

proposed classification system for Sitosterolemia-associated G5/G8 mutations based on

the systems mentioned above already in place52. Class I mutants are

nonsense/frameshift/deletion mutations, whereas classes II-V aremissense mutations.

Class II mutants are defective in maturing beyond the Endoplasmic Reticulum, class III    mutants are ones in which the protein is properly folded but lack functional activity, class IV mutants are unstable, and class V mutants are proposed to be defective in trafficking

at the apical surface.

Table 1.2. Proposed Sitosterolemia Classification System52

Table 1.2. Table of proposed Sitosterolemia classification system in addition to previously published classifications of ABCG5 and G8 mutants.

1.7 Proteostasis Regulation and Roscovitine

Proteostasis (protein homeostasis) refers to the process of maintaining a protein's

quaternary structure and location, typically through transcriptional/translational

modifications53. Proteostasis influences protein synthesis, folding, trafficking,

disaggregation, and degradation53. The ER is responsible for the synthesis, folding,

maturation, and trafficking of transmembrane proteins as well as secretory proteins. In

disease states where proteins are misfolded, proteostasis pathways detect and rapidly

degrade themisfolded proteins via the Endoplasmic Reticulum-Associated Degradation

(ERAD) pathway. Disruptions in protein folding trigger of ER Stress and activate the

Unfolded Protein Response (UPR). Conditions that disrupt protein homeostasis and

activate UPR are changes in temperature, calcium concentration disturbance, mutations,

redox disruptions, and changes in cellular pH54.

The unfolded protein response (UPR) is activated when the cell’sprotein folding

machinery becomes overwhelmed and misfolded proteins accumulate in the ER55.

Disruption of protein homeostasis occurs in conditions as previously mentioned,

including mutant proteins, alterations in gene expression, or permanently when there are

mutations that affect protein maturation56. The ERAD (ER-Associated Degradation)

pathway is an essential step in newly synthesized protein quality control57.  Chaperones  recognize proteins such as calnexin/calreticulin and heat-shock proteins, which facilitate

folding. Transmembrane proteins are glycosylated with an N-linked glycan and the

terminal glucose acts a tag that the protein is unfolded and thus recognized by

calnexin/calreticulin. Proteins that are unfolded are recognized by aglucosyltransferase in the ER and the protein is either re-glucosylated or exits the chaperon folding "cycle."

When prolonged, themisfolded protein is targeted by the retro-translocon and/or E3

ligases58. Themisfolded protein is then retrotranslocated to the cytoplasm, ubiquitinated

by E3 ligases, recognized by the 19S cap of the 26S proteasome, and degraded58.

Roscovitine is a small molecule pharmacologic that arrests cell cycle progression by inhibiting cyclin-dependent kinases (CDK) 1, 2, 5, 7, and 959. Roscovitine was first    studied in ABC transporters in its rescue of an ABCC7 (CFTR) mutant, F508del60. The

group suggested that the mechanism of action of Roscovitine F508del correction was

likely independent of inhibition of CDK's and, more generally in inhibition of kinases as

well as directly inhibiting activity of the proteasome60. This led to the proposal of

Roscovtine's rescue ofF508del through modulation of proteostasis, specifically the

ERQC (ER quality control) and ERAD system independently60. The research done by

this group influenced another group (Falguières) to test Roscovitine on three ABCB4

mutants in an attempt to achieve similar outcomes61. The three mutants tested were class  II (maturation deficient) mutations61. Because ATP transporters are well conserved across ABC transporter families, there are potential compounds successfully used in other ABC

transporter families that could be repurposed to treat Sitosterolemia-associated ABCG8

mutations.

Mutations in class II that get retained in the ER would make a compound like

Roscovitine a viable target for "rescuing" defective ABCG5/G8 transporters, as it has

shown the ability to modulate proteostasis60. It has been well established that ABCG5/G8

are poor folding proteins, as only about 40-50% of the protein matures beyond the ER,

which strengthens the argument for potential stabilization31.  Roscovitine has significant

cellular toxicity. Therefore, 11 analogs were synthesized in an attempt to lower CDK

inhibition and cytotoxic activity (Table 1.3)61. Some of these analogs partially rescue  mutants of ABCB4, which strengthens the hypothesis that Roscovitine rescue works    independently of CDK inhibition in ABCC7 and ABCB4. The efficacy of Roscovitine across multiple ABC transporters (ABCB4/ABCC7) and their structural similarities to

ABCG5/G8 is the scientific premise of this project.

Table 1.3. Roscovitine and Analogs Structure.

Table 1.3. Table of Roscovitine and 11 analogs synthesized by ManRos Therapeutics61.

1.8 Ivacaftor and ABCC7 Potentiation

Cystic Fibrosis (CF) is a disease in which the Cystic Fibrosis Transmembrane

Regulator (CFTR),ABCC7, fails to flux chloride ions leading to an accumulation of

chloride ions in the epithelial cells and in turn a change in mucus viscosity in the

lungs62,63 . Ivacaftor is an FDA-approved Cystic Fibrosis treatment that potentiates

(increased channel open probability) CFTR and partially restores chloride ion transport.

ABCC7 differs from most ABC transporters as it is an ion channel that utilizes ATP

hydrolysis, whereas ABCG5/8 is a protein transporter that uses energy from ATP to

efflux sterols64. Ivacaftor potentiator function in CFTR has therapeutic benefits in

patients with CFTR gating (class III) defective mutations50,65 . The mechanism of action

of Ivacaftor is binding to the PKA-phosphorylated ABCC7 in an ATP-independent

fashion66. Ivacaftor functions mainly for mutations that disrupt gating due to disruption to the ATP-dependent binding site66. It has not been reported that regulation of ABCG5 and

ABCG8 is PKA-dependent and ABCG5/G8 does not operate as an ion channel.

Therefore, there is not a strong foundation for why Ivacaftor would be able to stabilize

mutants of ABCG8.

However, in maturation, defective mutants of CFTR are “rescued” by small

molecule correctors of the Ivacaftor-family (Table 1.4), which may facilitate folding of

ABCG5/G8. Elexacaftor, Tezacaftor, and Lumacaftor are compounds that were

formulated after Ivacaftor for CF mutants that were not gating defective. They are small molecular "correctors" as they correct themisfolding of proteins that fail to exit the ER. The correctors' mechanism of action is directly binding to CFTR, but how they function

as a protein folding chaperone to escape degradation remains unclear. Rescued CFTR

mutants localize to the cell surface67. This suggests that escaping ERAD is sufficient for activity. In more recent studies, looking at the cryo-EM structure of CFTR in complex

with Lumacaftor or Tezacaftor demonstrates that the drugs bind to CFTR in the

hydrophobic pocket of TMD- 1, which ultimately stabilized the domain and prevented

degradation68. Lumacaftor and Tezacaftor bind within the hydrophobic pocket at a Lysine

and Arginineresidue before the start of the TMD- 168. ABCG5 is structurally similar in

that it too has a Lysine and Arginine residue prior to the start of the first TM domain49.

One could hypothesize that because of this structural similarity there is potential for

Lumacaftor or Tezacaftor binding to ABCG5 at the apical membrane.

There is amuch larger family of CFTR modulators (including correctors) that are

being investigated in vitro that have demonstrated stabilization in mutants of multiple

ABC transporter systems. Most notably, correction of ABCG2 mutants involved in Gout

by Cor-4a and due to sequence homology may too be effective in stabilizing

Sitosterolemia-associated mutants69. Stabilization with these and other compounds could

give reason to believe that these compounds could be used in Class II mutants across a

wide range of ABC transporter-related diseases.

Table 1.4. Ivacaftor and Correctors Structure