Vloga Cyp51 v biosintezi holesterola pri miših in preslikava na človeka : [diplomska naloga]

Kos, Špela (author), Mlinarič-Raščan, Irena (mentor), Rozman, Damjana (comentor)

Lanosterol 14α-demetilaza (CYP51) je encim, ki katalizira odstranitev 14α-metilne skupine s sterolnega obroča v sintezi holesterola in sodeluje pri zorenju spolnih celic pri sesalcih. Uvrščamo ga v naddružino citokromov P450, med katerimi je evolucijsko najbolj ohranjen in malo polimorfen, kot smo dokazali z uporabo računalniških metod in prosto dostopnih zbirk podatkov. S popolnim izbitjem gena Cyp51 mišji zarodki odmrejo po prvih dneh razvoja, kar kaže na pomembno vlogo gena že v zgodnji embriogenezi. Odsotnost gena Cyp51 tako ni združljiva z življenjem. Vpliv izbitja le enega alela tega gena na sintezo in homeostazo holesterola pa smo preučevali na pri nas razvitem mišjem modelu Cyp51+/-. Z merjenjem nivoja izražanja genov z uporabo kvantitativne verižne reakcije s polimerazo in analizo proteinov z metodo prenosa western smo pokazali, da se učinek števila kopij gena kaže s polovičnim prepisom mRNA in polovično količino proteina CYP51 pri heterozigotih, ne pa tudi s spremenjenim fenotipom. Iz tega lahko sklepamo, da je polovična količina encima dovolj za sintezo zadostnih količin holesterola za nemoteno delovanje organizma. Večji vpliv genotipa se izkaže šele po obremenitvi miši s hrano bogato z lipidi, z ali brez holesterola. V obeh primerih imajo heterozigotne miši zmanjšano sposobnost uravnavanja homeostaze holesterola, kar vodi do povišanih plazemskih koncentracij holesterola. Iz tega lahko sklepamo, da Cyp51 ni udeležen samo v sintezi, ampak tudi pri celostnem uravnavanju holesterola v telesu. Bistvo študij na mišjih modelih je pojasniti vpliv izbitja gena, izsledke pa preslikati na človeka, s čimer lahko izboljšamo razvoj novih zdravil in pojasnimo bolezni ter tveganja, ki lahko pri ljudeh privedejo do enakega fenotipa, kot ga opažamo pri mišjem modelu Cyp51+/-. Tveganje predstavlja prisotnost polimorfizmov gena CYP51A1 in gena POR, zdravljenje z inhibitorji sinteze holesterola, pomanjkanje holesterola v nosečnosti in neustrezna prehrana. Mišji model Cyp51+/- odpira še številne možnosti nadaljnjega dela, ki bi pojasnile vpliv genotipa in vlogo gena Cyp51 pri sesalcih.

• Language: Slovenian
• Keywords: holesterol homeostaza holesterola sinteza holesterola Cyp51 analiza proteinov izolacija proteinov genotipizacija miši
• Work type: Undergraduate thesis
• Typology: 2.11 - Undergraduate Thesis
• Organization: FFA - Faculty of Pharmacy
• Place of publishing: Ljubljana
• Publisher: [Š. Kos]
• Year: 2013
• Number of pages: IX, 79 f.
• PID: 20.500.12556/RUL-121137
• UDC: 616-074(043.2)
• COBISS.SI-ID: 3476593
• Publication date in RUL: 30.09.2020

Secondary language

• Language: English
• Title: Role of Cyp51 in mice cholesterol biosynthesis and reflection to humans

Abstract

Lanosterol 14α-demethylase enzyme (CYP51) catalyzes the oxidative removal of the 14α-methyl group of lanosterol and is involved in cholesterol biosynthesis and maturation of germ cells in mammals. It is the most evolutionarily conserved member of CYP superfamily and according to our in silico results it is less polymorphic compared to non-essential genes from the same family. Cyp51-/- knock-out mice provided evidence that Cyp51 is essential for embryogenesis and complete absence of gene leads to lethality at early stages of embryonic development. A mouse Cyp51+/- model was developed to investigate the effect of one missing allele on homeostasis and biosynthesis of cholesterol. The aim of our study was to investigate potencial difference on gene expression level between heterozygous and wild-type animals. The qPCR results supported by western blot analysis indicate 50% decrease of Cyp51 mRNA and half of the present amount of CYP51 protein in heterozygotes compared to wild-type animals, althougt no significant difference in mice phenotype has been demonstrated. Mice heterozygous for the Cyp51 mutation appear normal in their development and cholesterol biosynthesis, suggesting that one copy of the Cyp51 produces sufficient CYP51 protein for the hepatic cholesterol homeostasis similar to the wild-type animals with two copies of Cyp51. Higher influence of genotype is observed after challenging mice with extra cholesterol in the diet and after diet with zero cholesterol. In both cases heterozygous mice loss the ability to regulate cholesterol homeostasis, what leads to accumulation of cholesterol in the body. These findings suggest that Cyp51 is not involved just in cholesterol synthesis but also in its regulation. Mouse knockout models represent an informative in vivo resorce for new drug development because they mimic the systemic effect of drugs that target a particular enzyme and cause lowering or ablation of its activity and additonally, they can predict the risk of diseases caused by genetic mutations in humans. The risk factors due to lower expression of CYP51A1 and lower activity of CYP51 are polymorphisms of CYP51A1 and POR, treatment with inhibitors of CYP51, deficiency of maternal cholesterol and unhealthy western diet. Our research work enables further possibilities to investigate Cyp51+/- mice model to deepen the knowledge on the role of Cyp51 in mammals.

• Keywords: cholesterol biosynthesis Lanosterol 14α-demethylase (CYP51) quantitative polymerase chain reaction western diet evolutionary conservation p olymorphism