Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Apolipoprotein A-I (apoA-I) and ATP-Binding Cassette A1 (ABCA1) transporter play important roles in nascent high density lipoprotein (nHDL) biogenesis – the first step in the reverse cholesterol transport pathway. Based on the crystal structure of a C-terminally truncated form of apoA-I (apoA-I(1-184)) determined in the laboratory, structurally designed and naturally occurring mutants of apoA-I were conformationally characterized in solution. The function of these mutants in nHDL formation was assessed in ABCA1-transfected HEK293 cells. An apoA-I mutant designed to destabilize the N-terminal helical bundle at the first hinge region, 38/40G, exhibited a locally reduced α-helical content, destabilized overall structure, and increased lipid binding ability in solution, indicating a destabilized N-terminal helical bundle. In the cellular system, 38/40G showed significantly enhanced nHDL forming ability, suggesting that a destabilized N-terminal bundle will facilitate nHDL formation. Other designed N-terminal mutants (Q41A, P66G, G65A, V67P, T68P, 65/67/68P) and the naturally occurring mutants (R153P, L178P, and insertion mutant apoA-INashua) all showed either unchanged or destabilized overall structure, unchanged lipid binding abilities in solution and unchanged nHDL formation and cholesterol efflux promotion from the cells. Mutants designed to progressively extend the C-terminus (1-184, 1-198, 1-209, 1-220, 1-231) yielded progressively increased nHDL formation and cholesterol efflux, suggesting that the C-terminus of apoA-I is critical for these two activities. Central Helix 5 triple glycine mutation (H5 3xG) designed to lock the monomer conformation of apoA-I resulted in reduced nHDL formation but unaffected cholesterol efflux, suggesting that hindering apoA-I monomer to dimer conversion could retard nHDL formation. Remarkably, studies of cholesterol efflux and nHDL particle formation indicated that the two processes might be two uncoupled events. Analysis of the nHDL particles revealed the presence of ganglioside (GM1) in the complexes. Cross-linking data demonstrated binding of apoA-I to ABCA1-expressing cells. The binding level of apoA-I mutants to ABCA1-expressing cells was positively correlated with nHDL forming ability of these mutants. ABCA1 was isolated from FreeStyle™ HEK293-F cells in suspension by detergent solubilization and was shown to have ATPase activity. A direct interaction between apoA-I and amphipol solubilized- ABCA1 in solution was detected for the first time. Furthermore, the successful purification of ABCA1 has laid the foundation of structure determination of this protein in the future.

The ATP-binding cassette transporter, sub-family A, member 1 (ABCA1) mediates the major pathway for cholesterol exit from non-hepatic cells and thereby controls the rate-limiting step in the biogenesis of high density lipoprotein (HDL) particles. In humans,ABCA1 deficiency results in Tangier disease, characterized by low levels of HDL cholesterol, cellular cholesterol accumulation and increased risk for atherosclerosis. More than 100 coding variants have been described in the ABCA1 gene. We attempted to understand how both naturally occurring and engineered mutations in ABCA1 impact its role in cholesterol transport in a variety of in vitro and in vivo systems. We attempted to correlate specific genetic variants in ABCA 1 with phenotypes in patients carrying the sevariants, and used an evolutionary approach to predict which specific variants in ABCA1would impact its function. We then turned to the study of tissue-specific genetic deletion of ABCA1 in mice to study its role in HDL biogenesis, atherosclerosis and glucose metabolism. We found that intestinal ABCA1 is an important site of HDL biogenesis and that activation of intestinal ABCA1 raises HDL levels in vivo. Hepatic ABCA1, which is a major site of HDL biogenesis, was shown to significantly contribute to susceptibility to atherosclerosis. Finally, we show that ABCA1 plays an unsuspected role in B-cell function and insulin secretion. These studies have contributed to our understanding of the impact of genetic variation in ABCA1 on diverse biological and pathological processes, and have identified novel aspects of ABCA 1 function in specific cell types.

The ATP-binding cassette transporter, sub-family A, member 1 (ABCA1) mediates the major pathway for cholesterol exit from non-hepatic cells and thereby controls the rate-limiting step in the biogenesis of high density lipoprotein (HDL) particles. In humans,ABCA1 deficiency results in Tangier disease, characterized by low levels of HDL cholesterol, cellular cholesterol accumulation and increased risk for atherosclerosis. More than 100 coding variants have been described in the ABCA1 gene. We attempted to understand how both naturally occurring and engineered mutations in ABCA1 impact its role in cholesterol transport in a variety of in vitro and in vivo systems. We attempted to correlate specific genetic variants in ABCA 1 with phenotypes in patients carrying the sevariants, and used an evolutionary approach to predict which specific variants in ABCA1would impact its function. We then turned to the study of tissue-specific genetic deletion of ABCA1 in mice to study its role in HDL biogenesis, atherosclerosis and glucose metabolism. We found that intestinal ABCA1 is an important site of HDL biogenesis and that activation of intestinal ABCA1 raises HDL levels in vivo. Hepatic ABCA1, which is a major site of HDL biogenesis, was shown to significantly contribute to susceptibility to atherosclerosis. Finally, we show that ABCA1 plays an unsuspected role in B-cell function and insulin secretion. These studies have contributed to our understanding of the impact of genetic variation in ABCA1 on diverse biological and pathological processes, and have identified novel aspects of ABCA 1 function in specific cell types.

The ATP-binding cassette transporter, sub-family A, member 1 (ABCA1) mediates the major pathway for cholesterol exit from non-hepatic cells and thereby controls the rate-limiting step in the biogenesis of high density lipoprotein (HDL) particles. In humans,ABCA1 deficiency results in Tangier disease, characterized by low levels of HDL cholesterol, cellular cholesterol accumulation and increased risk for atherosclerosis. More than 100 coding variants have been described in the ABCA1 gene. We attempted to understand how both naturally occurring and engineered mutations in ABCA1 impact its role in cholesterol transport in a variety of in vitro and in vivo systems. We attempted to correlate specific genetic variants in ABCA 1 with phenotypes in patients carrying the sevariants, and used an evolutionary approach to predict which specific variants in ABCA1would impact its function. We then turned to the study of tissue-specific genetic deletion of ABCA1 in mice to study its role in HDL biogenesis, atherosclerosis and glucose metabolism. We found that intestinal ABCA1 is an important site of HDL biogenesis and that activation of intestinal ABCA1 raises HDL levels in vivo. Hepatic ABCA1, which is a major site of HDL biogenesis, was shown to significantly contribute to susceptibility to atherosclerosis. Finally, we show that ABCA1 plays an unsuspected role in B-cell function and insulin secretion. These studies have contributed to our understanding of the impact of genetic variation in ABCA1 on diverse biological and pathological processes, and have identified novel aspects of ABCA 1 function in specific cell types. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21820号 / 農博第2333号 / 新制||農||1067(附属図書館) / 学位論文||H31||N5192(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 和光, 教授 三芳 秀人, 教授 三上 文三 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第17986号 / 農博第2033号 / 新制||農||1019(附属図書館) / 学位論文||H26||N4811(農学部図書室) / 80830 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 和光, 教授 植田 充美, 教授 三芳 秀人 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18125号 / 医博第3845号 / 新制||医||1001(附属図書館) / 30983 / 京都大学大学院医学研究科医学専攻 / (主査)教授 横出 正之, 教授 柳田 素子, 教授 岩井 一宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

A ATP-binding cassette transporter A1 (ABCA1) é uma proteína transmembrana responsável pelo efluxo celular de colesterol e fosfolipídeos, que é um passo essencial para o transporte reverso do colesterol e para a biogênese da HDL. Polimorfismos do gene ABCA1 foram associados com risco de doença arterial coronariana, variações no perfil lipídico e diferenças na resposta a fármacos hipolipemiantes. Com a finalidade de avaliar os efeitos de polimorfismos do ABCA1 sobre a expressão gênica e a resposta a vastatinas, foram selecionados indivíduos normolipidemicos (NL, n=143) e hipercolesterolêmicos (HC, n=224). A resposta a atorvastatina (10 mg/dia/4 semanas) foi avaliada pelo perfil lipídico sérico em 141 indivíduos do grupo HC (ATORVA). DNA e RNA total foram extraídos de amostras de sangue periférico. Os polimorfismos de nucleotídeo único (SNP) G70943A (R219K), C-14T e C-105T, uma variante nova do ABCA1, foram detectados por PCR-RFLP e confirmados por seqüenciamento de DNA. A expressão de RNAm do ABCA1 em células mononucleares do sangue periférico (CMSP) foi analisada por PCR-duplex e PCR em tempo real, utilizando o gene GAPD como referência endógena. A freqüência do alelo -105T foi 1,4% em NL e 2,0% em HC. O alelo 70943A (genótipos GA+AA) foi associado com maior concentração sérica basal de apoAI (NL), de HDL-c (ATORVA) e com menores concentrações basais de triglicerídeos e VLDL-c e menor índice TG/HDL-c (HC e ATORVA) em comparação com o genótipo 70943GG (p<0,05). O polimorfismo C-105T está em desequilíbrio de ligação com o SNP C-14T (p=0,006). Portadores do alelo -105T (genótipos CT+TT), quando comparados aos portadores do genótipo -105CC, tiveram menores valores basais de triglicerídeos e VLDL-c, maior concentração de HDL-c e menor índice TG/HDL-c nos grupos HC e ATORVA e também maiores concentrações de apoAI e menor índice apoB/apoAI no grupo ATORVA (p<0,05). Nos grupos HC e ATORVA, os portadores do haplótipo -14CT+TT/-105CT+TT tiveram menores valores de triglicerídeos e VLDL-c basais, maiores concentrações de HDL-c e menor índice TG/HDL-c quando comparados aos portadores dos outros haplótipos (p<0,05). A expressão basal do ABCA1 foi menor nos HC que nos NL independentemente da taxa de expressão alta (GM1) ou baixa (GM2). Este efeito foi associado com os SNPs C-14T e G70943A SNPs. Após o tratamento com atorvastatina, a expressão de RNAm foi reduzida nos HC portadores do alelo - 14T em comparação com os portadores de alelo -14C. Esses resultados são sugestivos de que ABCA1 SNPs estão envolvidos na variação do perfil lipídico sérico e na expressão de RNAm em resposta a atorvastatina. / The ATP-binding cassette transporter A1 (ABCA1) is a transmembrane protein involved on cholesterol and phospholipid cellular efflux, which is an essential step for the reverse cholesterol transport and HDL biogenesis. Single nucleotide polymorphisms (SNPs) in the ABCA1 gene have been associated with increased risk of coronary heart disease, differences on serum lipid profile and response to lowering-cholesterol drugs. We have evaluated the influence of ABCA1 SNPs on mRNA expression and lipid-lowering response to atorvastatin. Normolipidemic (NL, n=143) hypercholesterolemic (HC, n=224) individuals were enrolled in this study and the response to atorvastatin (10 mg/day/4 weeks) was evaluated in HC individuals (ATORVA, n=141). Blood samples were collected for biochemical analyses, genomic DNA and total RNA extraction. SNPs G70943A (R219K), C-14T and C-105T, a novel variant of ABCA1, were detected by PCR-RFLP and confirmed for DNA sequencing. ABCA1 mRNA expression in peripheral blood mononuclear cells (PBMC) was analysed by PCR-duplex and Real Time PCR, using the GAPD as the endogenous reference. In HC and NL, the frequency of -105T allele was 2.0% and 1.4%, respectively. The 70943A allele (genotypes GA+AA) was associated with higher basal concentrations of apoAI (NL) and HDL-c (ATORVA) and lower triglyceride and VLDL-c and TG/HDL-c ratio (HC and ATORVA) than the 70943GG genotype (p<0.05). We found a linkage disequilibrium between C-14T and C-105T SNPs in HC group (p=0.006). Individuals carrying -105T allele (CT/TT genotypes), when compared with -105CC carriers, had lower basal concentrations of triglyceride and VLDL-c, higher concentration of HDL-c and lower TG/HDL-c ratio in HC and ATORVA groups and also higher concentration of apoAI and lower apoB/apoAI ratio in ATORVA group (p<0.05). In HC and ATORVA, individuals with -14CT+TT/-105CT+TT haplotype had lower basal values of triglyceride and VLDL-c, higher concentration of HDL-c and lower TG/HDL-c ratio than carries of others haplotypes (p<0,05). ABCA1 mRNA basal expression was lower in HC when compared to NL independently of high (GM1) or low (GM2) basal expression rate. This effect was associated with C-14T and G70943A SNPs. After atorvastatin treatment, mRNA expression was reduced in HC individuals carrying -14T allele in comparison with the -14C allele carriers. These results are sugestive that ABCA1 SNPs are involved on variation of serum lipid profile and mRNA expression in response to atorvastatin.

A ATP-binding cassette transporter A1 (ABCA1) é uma proteína transmembrana responsável pelo efluxo celular de colesterol e fosfolipídeos, que é um passo essencial para o transporte reverso do colesterol e para a biogênese da HDL. Polimorfismos do gene ABCA1 foram associados com risco de doença arterial coronariana, variações no perfil lipídico e diferenças na resposta a fármacos hipolipemiantes. Com a finalidade de avaliar os efeitos de polimorfismos do ABCA1 sobre a expressão gênica e a resposta a vastatinas, foram selecionados indivíduos normolipidemicos (NL, n=143) e hipercolesterolêmicos (HC, n=224). A resposta a atorvastatina (10 mg/dia/4 semanas) foi avaliada pelo perfil lipídico sérico em 141 indivíduos do grupo HC (ATORVA). DNA e RNA total foram extraídos de amostras de sangue periférico. Os polimorfismos de nucleotídeo único (SNP) G70943A (R219K), C-14T e C-105T, uma variante nova do ABCA1, foram detectados por PCR-RFLP e confirmados por seqüenciamento de DNA. A expressão de RNAm do ABCA1 em células mononucleares do sangue periférico (CMSP) foi analisada por PCR-duplex e PCR em tempo real, utilizando o gene GAPD como referência endógena. A freqüência do alelo -105T foi 1,4% em NL e 2,0% em HC. O alelo 70943A (genótipos GA+AA) foi associado com maior concentração sérica basal de apoAI (NL), de HDL-c (ATORVA) e com menores concentrações basais de triglicerídeos e VLDL-c e menor índice TG/HDL-c (HC e ATORVA) em comparação com o genótipo 70943GG (p<0,05). O polimorfismo C-105T está em desequilíbrio de ligação com o SNP C-14T (p=0,006). Portadores do alelo -105T (genótipos CT+TT), quando comparados aos portadores do genótipo -105CC, tiveram menores valores basais de triglicerídeos e VLDL-c, maior concentração de HDL-c e menor índice TG/HDL-c nos grupos HC e ATORVA e também maiores concentrações de apoAI e menor índice apoB/apoAI no grupo ATORVA (p<0,05). Nos grupos HC e ATORVA, os portadores do haplótipo -14CT+TT/-105CT+TT tiveram menores valores de triglicerídeos e VLDL-c basais, maiores concentrações de HDL-c e menor índice TG/HDL-c quando comparados aos portadores dos outros haplótipos (p<0,05). A expressão basal do ABCA1 foi menor nos HC que nos NL independentemente da taxa de expressão alta (GM1) ou baixa (GM2). Este efeito foi associado com os SNPs C-14T e G70943A SNPs. Após o tratamento com atorvastatina, a expressão de RNAm foi reduzida nos HC portadores do alelo - 14T em comparação com os portadores de alelo -14C. Esses resultados são sugestivos de que ABCA1 SNPs estão envolvidos na variação do perfil lipídico sérico e na expressão de RNAm em resposta a atorvastatina. / The ATP-binding cassette transporter A1 (ABCA1) is a transmembrane protein involved on cholesterol and phospholipid cellular efflux, which is an essential step for the reverse cholesterol transport and HDL biogenesis. Single nucleotide polymorphisms (SNPs) in the ABCA1 gene have been associated with increased risk of coronary heart disease, differences on serum lipid profile and response to lowering-cholesterol drugs. We have evaluated the influence of ABCA1 SNPs on mRNA expression and lipid-lowering response to atorvastatin. Normolipidemic (NL, n=143) hypercholesterolemic (HC, n=224) individuals were enrolled in this study and the response to atorvastatin (10 mg/day/4 weeks) was evaluated in HC individuals (ATORVA, n=141). Blood samples were collected for biochemical analyses, genomic DNA and total RNA extraction. SNPs G70943A (R219K), C-14T and C-105T, a novel variant of ABCA1, were detected by PCR-RFLP and confirmed for DNA sequencing. ABCA1 mRNA expression in peripheral blood mononuclear cells (PBMC) was analysed by PCR-duplex and Real Time PCR, using the GAPD as the endogenous reference. In HC and NL, the frequency of -105T allele was 2.0% and 1.4%, respectively. The 70943A allele (genotypes GA+AA) was associated with higher basal concentrations of apoAI (NL) and HDL-c (ATORVA) and lower triglyceride and VLDL-c and TG/HDL-c ratio (HC and ATORVA) than the 70943GG genotype (p<0.05). We found a linkage disequilibrium between C-14T and C-105T SNPs in HC group (p=0.006). Individuals carrying -105T allele (CT/TT genotypes), when compared with -105CC carriers, had lower basal concentrations of triglyceride and VLDL-c, higher concentration of HDL-c and lower TG/HDL-c ratio in HC and ATORVA groups and also higher concentration of apoAI and lower apoB/apoAI ratio in ATORVA group (p<0.05). In HC and ATORVA, individuals with -14CT+TT/-105CT+TT haplotype had lower basal values of triglyceride and VLDL-c, higher concentration of HDL-c and lower TG/HDL-c ratio than carries of others haplotypes (p<0,05). ABCA1 mRNA basal expression was lower in HC when compared to NL independently of high (GM1) or low (GM2) basal expression rate. This effect was associated with C-14T and G70943A SNPs. After atorvastatin treatment, mRNA expression was reduced in HC individuals carrying -14T allele in comparison with the -14C allele carriers. These results are sugestive that ABCA1 SNPs are involved on variation of serum lipid profile and mRNA expression in response to atorvastatin.