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Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

ATP-binding cassette (ABC) transporters are transmembrane proteins that transport a wide variety of substrates across intra and extra-cellular membranes. A few examples of endo and xenobiotic substrates are metabolic products, lipids, sterols, and drugs. An important function of ABC transporters involved in export is to prevent intracellular the buildup of toxic products. Several ABC transporters have also been associated with drug resistance upon treatment with chemotherapeutic agents. P-glycoprotein (P-GP) and the multidrug resistant (MRP) transporters of the ABC C family are examples of transporters that confer chemo-resistance. We have studied two unique roles of ABC transporters in the liver and the heart. In the liver, maintenance of bile secretion is important during lactation to ensure proper absorption of nutrients for the offspring. Three main ABC transporters are involved in this process: ABCB11 (transports bile acids), ABCB4 (transporters phospholipids), and ABCG5/ABCG8 (transports cholesterol). In the rat, expression of ABCB11 remains the same as the size of the bile acid pool increases. However, the expression of ABCG5/ABCG8 is abolished, preventing excessive export and loss of cholesterol from the liver. The regulation of these transporters during lactation maintains the production of bile acids from cholesterol by decreasing export while preventing toxicity from bile acids by maintaining bile flow. Another protective role of ABC transporters is seen in oxidative stress-induced toxicity of cardiac tissue following treatment with Doxorubicin (DOX), a drug used in cancer treatment. Multidrug resistance protein 1 (Mrp1) can transport toxic products by conjugation with sulfate, glutathione (GSH) or glucuronide. In Mrp1-/- mice, DOX causes advanced cell damage through intracellular edema and increased apoptotic nuclei. However, P-glycoprotein expression increases upon DOX treatment, potentially compensating for the loss of Mrp1. Mrp1 can also transport GSH, GSH disulfide (GSSG), and products of oxidation, like GSH conjugates. In the absence of Mrp1, GSH levels are increased in the heart, providing protection against oxidative stress. Both of these examples in liver and heart show the diversity of ABC transporters and the role they play in preventing cell toxicity. These studies also provide insight into ways to prevent cell toxicity through manipulation of ABC transport proteins.

The heterodimeric ATP-binding cassette (ABC) transporter, ABCG5/G8, is responsible for direct secretion of cholesterol and dietary sterols into the gut lumen and the bile. Inactivating mutations of ABCG5/G8 cause sitosterolemia, a rare autosomal recessive disease characterized by the accumulation of plant sterols in plasma, hypercholesterolemia and development of premature coronary heart disease. Functional and structural characterization of ABCG5/G8 is necessary to understand its mechanism and how the genetic defects impact its function. In this thesis, I expressed seventeen constructs of various disease-causing or catalytically deficient missense mutations in Pichia pastoris yeast. This establishes reagents for in vitro functional and structural studies. Secondly, I focused on two disease mutants (ABCG5-E146Q and ABCG8-R543S) and a sterol binding mutation (ABCG5-A540F) and established large-scale purification of these mutants. Using a cholesterol hemisuccinate (CHS)-dependent ATPase assay, I determined ATP hydrolysis by these three mutants and analyze their kinetic parameters. All missense mutants showed a significantly impaired ATPase activity, but the ability of ATP binding appeared unchanged between the WT and the mutants. This work demonstrates an intimate structure-function relationship in ABCG5/G8 and sheds some light on the mechanistic details of this important cholesterol-regulating ABC transporter.

Global obesity is linked to chronic diseases including hypercholesterolemia, a cardiovascular disease risk factor, thus weight reduction in obesity is a key priority for combatting obesity. The cholesterol transporters ABCG5, ABCG8 and NPC1L1 mediate cholesterol trafficking across the intestinal wall, thus are important in regulating cholesterol metabolism and circulating levels. The objective of this study was to examine if single nucleotide polymorphisms (SNP) of cholesterol transporters ABCG5, ABCG8 and NPC1L1 are associated with changes in cholesterol synthesis and absorption and lipid parameters (LP) subsequent to weight loss (WtL) in overweight individuals. Eighty-nine individuals from two WtL trials (Trial A (n = 54) and Trial B (n = 35)) completed a 20-wk WtL period. After 10% WtL, lipid parameters excluding LDL-C were improved in Trial A, while all lipid parameters were ameliorated after 12% of WtL when Trial A and B were combined. Post-WtL, cholesterol synthesis (CS) was reduced; however, cholesterol absorption was not changed in either Trial A or the combined trials. Polymorphisms in ABCG8 V632A were associated with changes in TC and TG levels after WtL in both trial A and the combined data. SNPs in ABCG5 Q604E, ABCG8 T400K, were associated with changes in CS because of WtL in Trial A; however, the association is no longer seen in combined analysis. In conclusion, cardio-protective changes in LP due to weight loss were mediated by reductions in CS. Additionally, polymorphisms in ABCG8 were associated with amelioration in LP after WtL. Thus, the benefits in CVD risk subsequent to weight loss vary across individuals due to genetic factors associated with cholesterol trafficking.

Global obesity is linked to chronic diseases including hypercholesterolemia, a cardiovascular disease risk factor, thus weight reduction in obesity is a key priority for combatting obesity. The cholesterol transporters ABCG5, ABCG8 and NPC1L1 mediate cholesterol trafficking across the intestinal wall, thus are important in regulating cholesterol metabolism and circulating levels. The objective of this study was to examine if single nucleotide polymorphisms (SNP) of cholesterol transporters ABCG5, ABCG8 and NPC1L1 are associated with changes in cholesterol synthesis and absorption and lipid parameters (LP) subsequent to weight loss (WtL) in overweight individuals. Eighty-nine individuals from two WtL trials (Trial A (n = 54) and Trial B (n = 35)) completed a 20-wk WtL period. After 10% WtL, lipid parameters excluding LDL-C were improved in Trial A, while all lipid parameters were ameliorated after 12% of WtL when Trial A and B were combined. Post-WtL, cholesterol synthesis (CS) was reduced; however, cholesterol absorption was not changed in either Trial A or the combined trials. Polymorphisms in ABCG8 V632A were associated with changes in TC and TG levels after WtL in both trial A and the combined data. SNPs in ABCG5 Q604E, ABCG8 T400K, were associated with changes in CS because of WtL in Trial A; however, the association is no longer seen in combined analysis. In conclusion, cardio-protective changes in LP due to weight loss were mediated by reductions in CS. Additionally, polymorphisms in ABCG8 were associated with amelioration in LP after WtL. Thus, the benefits in CVD risk subsequent to weight loss vary across individuals due to genetic factors associated with cholesterol trafficking.