Posttranscriptional Regulation of the Human LDL Receptor by the U2-Spliceosome

Background: The low-density lipoprotein receptor (LDLR) in the liver is the major determinant of LDL-cholesterol levels in human plasma. The discovery of genes that regulate the activity of LDLR helps to identify pathomechanisms of hypercholesterolemia and novel therapeutic targets against atherosclerotic cardiovascular disease.Methods: We performed a genome-wide RNA interference screen for genes limiting the uptake of fluorescent LDL into Huh-7 hepatocarcinoma cells. Top hit genes were validated by in vitro experiments as well as analyses of datasets on gene expression and variants in human populations.Results: The knockdown of 54 genes significantly inhibited LDL uptake. Fifteen of them encode for components or interactors of the U2-spliceosome. Knocking down any one of 11 out of 15 genes resulted in the selective retention of intron 3 of LDLR. The translated LDLR fragment lacks 88% of the full length LDLR and is detectable neither in non-transfected cells nor in human plasma. The hepatic expression of the intron 3 retention transcript is increased in non-alcoholic fatty liver disease as well as after bariatric surgery. Its expression in blood cells correlates with LDL-cholesterol and age. Single nucleotide polymorphisms and three rare variants of one spliceosome gene, RBM25, are associated with LDL-cholesterol in the population and familial hypercholesterolemia, respectively. Compared to overexpression of wild type RBM25, overexpression of the three rare RBM25 mutants in Huh-7 cells led to lower LDL uptake.Conclusions: We identified a novel mechanism of post-transcriptional regulation of LDLR activity in humans and associations of genetic variants of RBM25 with LDL-cholesterol levels.</p

Total synthesis of castanospermine analogues via asymetric aldol reaction of isoserinal

Tematem pracy była synteza polihydroksylowych pochodnych chinolizydyny oraz indolizydyny, kluczowym etapem zaproponowanej metodologii była asymetryczna reakcja aldolowa pomiędzy aldehydem izoserynowym oraz α-hydroksyketonami. W toku badań opracowano dwie efektywne metody syntezy hydroksyketonów pozwalające uzyskać wydajność całkowitą w zakresie 40-60%. Przetestowano szereg potencjalnych katalizatorów w asymetrycznej reakcji aldolowej, w tym chiralne aminy trzeciorzędowe, głównie alkaloidy z kory drzewa chinowego oraz bimetaliczne kompleksy cynku z chiralnym ligandem. Najlepsze wyniki otrzymano dla kompleksów cynku, dla których następnie dobrano optymalny rozpuszczalnik. Uzyskano produkty aldolowe z całkowitą wydajnością w zakresie 45-99% oraz wydajnością głównego izomeru sięgającą 71%. Otrzymane aldole następnie poddano cyklizacji otrzymując dwa monocykliczne iminocukry z wydajnościami około 40% oraz potwierdzono powstanie bicyklicznej pochodnej będącej analogiem kastanosperminy.The topic of this thesis was the synthesis of polyhydroxylated derivatives of quinolizidine and indolizidine. The key step of the proposed methodology was the asymmetric aldol reaction between isoserinal aldehyde and α-hydroxyketones. In the course of the research, two effective methods for the synthesis of hydroxyketones were developed, permitting to obtain them with yields in the range of 40-60%. Several catalysts were tested in an asymmetric aldol reaction, including chiral tertiary amines, mainly cinchona alkaloids, and chiral bimetallic zinc complexes. The best results were obtained for zinc complexes, for which the optimal solvent was then selected. Consequently, the aldol products were obtained with a yield ranging from 45-99% depending on the catalyst and substrate used. Furthermore, the yield of the main isomer was up to 71%. Then, the obtained aldols were cyclized to get two monocyclic iminosugars with an approximate yield of 40%. Moreover, the formation of a bicyclic derivative, which was an analogue of castanospermine, was confirmed

Asymmetric aldol reaction of 2-phenylacetophenon catalyzed by tertiary amine

Tematem pracy było sprawdzenie aktywności katalitycznej chiralnych amin trzeciorzędowych – głównie pochodnych alkaloidów kory drzewa chinowego w asymetrycznej reakcji aldolowej pomiędzy 2-aryloacetofenonem i wybranymi aldehydami. W toku prowadzonych badań uzyskano wydajności w przedziale od 5% do 62% i nadmiary enancjomeryczne od 6% ee do 43% ee w zależności od stosowanego katalizatora. Praca badawcza obejmowała również optymalizację warunków reakcji dla najlepszego katalizatora. W wyniku przeprowadzonej optymalizacji podniesiono enancjoselektwność reakcji do poziomu 30% ee oraz zwiększono wydajność reakcji do 52%, niestety kosztem zmniejszenia diastereoselektywności reakcji syn/anti 4:1 do 2:1.The topic of the thesis was to check the catalytic activity of chiral tertiary amines - mainly derivatives of the cinchona alkaloids in the asymmetric aldol reaction between 2-phenylacetophenon and selected aldehydes. In the course of the research, yields ranged from 5% to 62% and enantiomeric excess from 6% ee to 43% ee, depending on the catalyst used. The research also included optimizing the reaction conditions for the best catalyst. As a result of the optimization, the enantioselectivity of the reaction was increased to 30% ee and the reaction efficiency was increased to 52%, unfortunately at the cost of diastereoselectivity of the syn / anti reaction 4: 1 to 2: 1

Posttranscriptional Regulation of the Human LDL Receptor by the U2-Spliceosome

International audienceBACKGROUND: The LDLR (low-density lipoprotein receptor) in the liver is the major determinant of LDL-cholesterol levels in human plasma. The discovery of genes that regulate the activity of LDLR helps to identify pathomechanisms of hypercholesterolemia and novel therapeutic targets against atherosclerotic cardiovascular disease. METHODS: We performed a genome-wide RNA interference screen for genes limiting the uptake of fluorescent LDL into Huh-7 hepatocarcinoma cells. Top hit genes were validated by in vitro experiments as well as analyses of data sets on gene expression and variants in human populations. RESULTS: The knockdown of 54 genes significantly inhibited LDL uptake. Fifteen of them encode for components or interactors of the U2-spliceosome. Knocking down any one of 11 out of 15 genes resulted in the selective retention of intron 3 of LDLR. The translated LDLR fragment lacks 88% of the full length LDLR and is detectable neither in nontransfected cells nor in human plasma. The hepatic expression of the intron 3 retention transcript is increased in nonalcoholic fatty liver disease as well as after bariatric surgery. Its expression in blood cells correlates with LDL-cholesterol and age. Single nucleotide polymorphisms and 3 rare variants of one spliceosome gene, RBM25, are associated with LDL-cholesterol in the population and familial hypercholesterolemia, respectively. Compared with overexpression of wild-type RBM25, overexpression of the 3 rare RBM25 mutants in Huh-7 cells led to lower LDL uptake. CONCLUSIONS: We identified a novel mechanism of posttranscriptional regulation of LDLR activity in humans and associations of genetic variants of RBM25 with LDL-cholesterol levels