Strukturna karakterizacija histon - vezujućih proteina

master's thesis

• Title (english): Structural characterization of histone binding proteins
• Author: Petra Vizjak
• Mentor: Mario Halić (mentor)
• Mentor: Vlatka Zoldoš (mentor)
• Committee member: Vlatka Zoldoš (predsjednik povjerenstva)
• Committee member: Dijana Škorić (član povjerenstva)
• Committee member: Damjan Franjević (član povjerenstva)
• Committee member: Nataša Bauer (član povjerenstva)
• Granter: University of Zagreb; Faculty of Science; (Department of Biology); Zagreb
• Defense date and country: 2015-02-26, Croatia
• Scientific / art field, discipline and subdiscipline: NATURAL SCIENCES; Biology

Abstract

Histoni su osnovna proteinska komponenta kromatina. Histonski šaperon Nap1, zajedno s karioferinom Kap114, transportira H2A-H2B iz citoplazme u jezgru. Djelovanje metiltransferaze Nop1, u kompleksu s proteinom Nop56, ključno je za nastanak metilacije H2AQ105, prve epigenetičke oznake posvećene specifičnoj RNA-polimerazi. Cilj ovog rada bio je strukturno karakterizirati komplekse histona s tim histon-vezujućim proteinima. Korištene su standardne metode molekularnog kloniranja. Proteini su pročišćavani afinitetnom kromatografijom, ionizmjenjivačkom kromatografijom i kromatografijom isključenjem. Proteini su analizirani SDS-poliakrilamidnom gel-elektroforezom i masenom spektrometrijom. Pokazali smo da Nap1 (H. sapiens) tvori kompleks s dimerom H2A-H2B (X. laevis). Terminalne domene Nap1 nisu nužne za nastanak kompleksa. Dijelovi kompleksa koji su važni za njegovu stabilnost, zaštićeni su od djelovanja proteaze. Potvrđene su interakcije Nap1-H2A-H2B s Kap114 (S. pombe). Kompleksi Nap1-H2A-H2B i Nap1-H2A-H2B-Kap114 su pročišćeni i postavljene su pretkristalizacijske probe na kristalizacijskom robotu. Kompleks Nop1-Nop56 (K. lactis) dobiven je in vitro. N-terminalna domena Nop1 nije potrebna za interakciju s Nop56. Kompleks tvori topljive agregate što otežava njegovo pročišćavanje. Na temelju ovih rezultata, razvit će se nove strategije za pročišćavanje ciljnih proteina. Daljnja istraživanja doprinjet će razumijevanju biologije kromatina i bolesti povezanih s histonskim šaperonima.

Abstract (english)

Histones are the chief protein components of chromatin. Histone chaperone Nap1, together with karyopherin Kap114, shuttles H2A-H2B from cytosol to the nucleus. Nop1 methyltransferase in complex with Nop56 protein is responsible for H2AQ105 methylation, the first histone epigenetic mark dedicated to a specific RNA polymerase. The aim of this project was to structurally characterize histones in complex with these histone binding proteins. Standard molecular cloning techniques were used. Proteins were purified by affinity, ion exchange and size exclusion chromatography. Proteins were analyzed by SDS-poliacrylamide gel-electrophoresis and mass sprectrometry. We have shown that Nap1 (H. Sapiens) forms a complex with H2A-H2B (X. laevis) dimer. Nap1 terminal domains are not necessary for complex formation. Regions important for complex stability are protected from protease cleavage. Interaction between Nap1-H2A-H2B and Kap114 (S. Pombe) was confirmed. Complexes Nap1-H2A-H2B and Nap1-H2A-H2B-Kap114 were purified and precrystallization screenings were set up on crystallization robot. Nop1-Nop56 (K. lactis) complex was formed in vitro. Nop1 N-terminal domain is dispensable for interaction with Nop56. Complex forms soluble aggregates which has made purification difficult. Based on obtained results, new purification strategies will be developed. Further research will contribute to understanding of chromatin biology and the human diseases associated with histone chaperones.

• Keywords: fibrilarin; import H2; Kap114; metilacija glutamina H2A; Nap1; Nop56
• Keywords (english): fibrillarin; H2 import; H2A glutamine methylation; Kap114; Nap1; Nop56
• Language: croatian
• URN:NBN: urn:nbn:hr:217:066939
• Study programme: Title: Molecular Biology; Study programme type: university; Study level: graduate; Academic / professional title: magistar/magistra molekularne biologije (magistar/magistra molekularne biologije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Access restricted to students and staff of home institution
• Created on: 2017-04-25 08:39:31