Interakcija med period 2 in konstitutivnim androstanskim receptorjem

Martini, Tomaž (author), Mlinarič-Raščan, Irena (mentor), Rozman, Damjana (comentor)

Protein period 2 (PER2) je eden izmed ključnih akterjev tvorbe cirkadianih ritmov v sesalskih celicah, medtem ko je konstitutivni androstanski receptor (CAR) eden ključnih elementov metabolizma lipofilnih molekul, saj z njimi interagira, kar sproži izražanje citokromov P450, ki so odgovorni za oksidacijo teh lipofilnih molekul. Cilj diplomske naloge je bil ugotoviti, ali obstaja interakcija med proteinoma PER2 in CAR, ki bi povezala metabolizem in cirkadiani ritem. Za dosego cilja smo izvedli kotransfekcije mišjih hepatokarcinomskih Hepa 1-6 celic s plazmidoma, ki kodirata PER2 in CAR, in spremljali spremembe v izražanju izbranega gena cirkadianega ritma – Bmal1. Sprememba izražanja tega gena pomeni spremembo v cirkadianem ritmu.Bmal1 smo sklopili z genom za luciferazo. Encimska aktivnost luciferaze nam je služila kot merilo izražanja Bmal1. Izražanje Bmal1 se je po kotransfekciji celic s Per2 in Car povečalo. Možnost neposredne interakcije med period 2 in konstitutivnim androstanskim receptorjem smo preverili tudi in silico z iskanjem zaporedij, prek katerih bi lahko proteina interagirala. Ker je PER2 koaktivator jedrnih receptorjev, smo analizirali njegovi LXXLL zaporedji, pregledali in analizirali pa smo tudi LXXLL zaporedja znanih koaktivatorjev CAR. Ugotovili smo, da obstaja podobnost med drugim LXXLL zaporedjem PER2 in LXXLL zaporedji znanih koaktivatorjev CAR. Na podlagi tega pregleda in analize smo s pomočjo upoštevanja strukturnih podobnosti LXXLL zaporedij in silico ustvarili LXXLL zaporedje PER2. Ob upoštevanju znanih kristalnih struktur jedrnih receptorjev in koaktivatorjev smo predlagali način neposredne interakcije med proteinoma, predlog pa smo podkrepili tudi z molekulsko dinamiko. Predlagali smo metode za izdelavo in silico modela kompleksa CAR in večjega strukturnega dela PER2 okrog drugega LXXLL zaporedja. Takšne strukture PER2 še ni na voljo. Omenjen model bi lahko tudi dodatno potrdil našo predlagano neposredno interakcijo med PER2 in CAR.

• Language: Slovenian
• Keywords: protein period 2 cirkadiani ritem androstanski receptor encimska aktivnost plazmidi
• Work type: Undergraduate thesis
• Typology: 2.11 - Undergraduate Thesis
• Organization: FFA - Faculty of Pharmacy
• Place of publishing: Ljubljana
• Publisher: [T. Martini]
• Year: 2015
• Number of pages: 60 str.
• PID: 20.500.12556/RUL-121049
• UDC: 576.3:616-074(043.2)
• COBISS.SI-ID: 3947889
• Publication date in RUL: 29.09.2020

Secondary language

• Language: English
• Title: Interaction between period 2 and constitutive androstane receptor

Abstract

Period 2 (PER2) is one of the key proteins in circadian rhythms in mammalian cells while the constitutive androstane receptor (CAR) as one of the key elements in metabolism of lipophilic molecules interacts with these. This causes expression of cytochromes P450 and oxidation of lipophilic molecules.Our goal was to find whether PER2 and CAR interact as this would establish a new connection between the circadian rhythm and metabolism. To achieve this we cotransfected mouse hepatocarcinoma Hepa 1-6 cells with plasmids which code PER2 and CAR and monitored changes in expression of a chosen circadian gene – Bmal1. A change in expression of this gene means that there is a change in the circadian rhytm. Bmal1 was fused with a luciferase gene. The enzyme activity of luciferase served as a measure of expression of Bmal1. The expression was upregulated after the cotransfection with Per2 and Car. The possibility of a direct interaction between PER2 and CAR was examined with in silico methods. We searched for potential structural elements that could explain a protein-protein interaction. Because PER2 is a cofactor of nuclear receptors we analysed PER2's LXXLL motifs. We also analysed LXXLL motifs of known CAR coactivators. A similarity was found between the second LXXLL motif of PER2 and LXXLL motifs of know coactivators of CAR. On the basis of this similarity we created a LXXLL motif of PER2 in silico. After searching through known crystal structures of nuclear receptors and their cofactors we suggested a direct interaction between PER2 and CAR. This model was favoured by molecular dynamics. We also suggested methods for making an in silico model of the complex of CAR and a bigger portion of PER2 that contains the second LXXLL motif. Such a crystal structure of PER2 has not been published yet. This model could also additionally back our suggestion of the interaction between PER2 and CAR.