Priprava in karakterizacija proteinskih adapterjev AAA+ ATPaze p97, Npl4 in Ufd1

Andoljšek, Maša (avtor), Podobnik, Marjetka (mentor), Pavšič, Miha (komentor)

Poškodbe genomske DNA povzročajo endogeni ali eksogeni dejavniki, kot so reaktivni metaboliti, ultravijolično in ionizirajoče sevanje, kemikalije itd. Nepopravljene poškodbe lahko vodijo do nestabilnega genoma in bolezni, kot so rak, pospešeno staranje ali nevrodegenerativne bolezni. Med poškodbe DNA sodijo tudi kovalentne povezave proteinov z DNA (ang. DNA-protein crosslinks, DPC). Pri človeku odpravljanje DPCjev vključuje proteazo SPRTN, ki razgradi kovalentno vezane proteine do peptidov, nato pa ostali mehanizmi opravijo preostali del popravljanja. V tem procesu s SPRTN sodelujejo tudi proteini p97, Ufd1 in Npl4. p97 ali tudi VCP (ang. valosin-containing protein) je AAA+ ATPaza, ki iz makromolekulskih kompleksov ekstrahira polipeptidne substrate in jih translocira skozi poro v sredini p97 heksamera, kar energijsko poganja hidroliza ATP. Raznolikost delovanja p97 omogočajo partnerski oziroma adapterski proteini; znanih je že več kot 30. Adapterja Npl4 in Ufd1 delujeta v paru in s p97 sodelujeta pri različnih bioloških procesih, kot so kontrola delovanja kromosomov, razgradnji nepravilno zvitih proteinov ter regulaciji kromatina, p97-Npl4/Ufd1 pa skupaj SPRTN sodelujejo tudi pri odpravljanju DPCjev. Razumevanje interakcij p97 z adapterji je ključnega pomena za razumevanje mehanizma delovanja teh molekul in potencialne terapevtske uporabe. Namen magistrske naloge je bila priprava rekombinantnih proteinov Npl4 in Ufd1, njuna karakterizacija ter osnovna analiza interakcij med njima in p97. Delo je vključevalo molekulsko kloniranje, izražanje rekombinantnih proteinov v bakterijskih celicah, izolacijo in čiščenje proteinov s kromatografskimi metodami, biokemijsko in biofizikalno analizo čistosti, stabilnosti ter pravilnega zvitja proteinov, ter preliminarno karakterizacijo proteinskih interakcij. Npl4 in Ufd1 smo uspešno klonirali in izrazili v bakterijskih celicah. Optimizacija pogojev je omogočila, da smo oba proteina pridobili v velikih količinah v topni obliki in z visoko stopnjo čistosti. S preliminarnimi poskusi smo pokazali interakcijo med Npl4 in Ufd1 ter njuno posamično interakcijo s p97. Ugotovili smo tudi, da je Ufd1 bolj termično stabilen v primerjavi z Npl4. Rezultati te naloge bodo pomembna podlaga nadaljnjim raziskavam mehanizma delovanja p97 pri DPCjih skupaj z Npl4, Ufd1 in SPRTN.

• Jezik: Slovenski jezik
• Ključne besede: AAA+ ATPaza p97, adaptorski protein, Ufd1, Npl4, popravljanje DNA, priprava rekombinantnih proteinov, karakterizacija proteinov, molekulske interakcije
• Vrsta gradiva: Magistrsko delo/naloga
• Organizacija: FKKT - Fakulteta za kemijo in kemijsko tehnologijo
• Leto izida: 2024
• Datum objave v RUL: 11.09.2024

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Preparation and characterization of the AAA+ ATPase protein adapters p97, Npl4 and Ufd1

Izvleček

Genomic DNA damage is caused by endogenous or exogenous factors such as reactive metabolites, UV and ionizing radiation, chemicals, etc. Unrepaired damage can lead to genomic instability and cancer, accelerated aging, or neurodegenerative disorders. DNA damage also includes covalent DNA-protein crosslinks (DPC). In humans, the removal of DPCs involves the protease SPRTN, which breaks down covalently bound proteins into peptides, after which other mechanisms complete the repair process. In this process, proteins such as p97, Ufd1, and Npl4 also collaborate with SPRTN. p97, also known as VCP (valosin-containing protein), is an AAA+ ATPase that uses ATP hydrolysis to extract and translocate polypeptides through its central pore. The versatility of p97’s functions is enabled by partner or adapter proteins, with more than 30 known to date. The adapters Npl4 and Ufd1 work together and associate with p97 in various biological processes, such as chromosome activity control, degradation of misfolded proteins, and chromatin regulation. Additionally, the p97-Npl4/Ufd1 complex collaborates with SPRTN in removing DPCs. Understanding the interactions between p97 and its adapters is crucial for comprehending the mechanism of action of these molecules and their potential therapeutic applications. The aim of this master’s thesis was the preparation of recombinant Npl4 and Ufd1 proteins, their characterization, and a preliminary analysis of their interactions with p97. The work involved molecular cloning, expression of recombinant proteins in bacterial cells, protein isolation and purification using chromatographic methods, biochemical and biophysical analysis of protein purity, stability, and proper folding, as well as a preliminary characterization of protein interactions. Npl4 and Ufd1 were successfully cloned and expressed in bacterial cells. Optimization of conditions enabled the production of both proteins in large quantities in a soluble form with a high degree of purity. Preliminary experiments demonstrated the interaction between Npl4 and Ufd1, as well as their individual interactions with p97. It was also found that Ufd1 is more thermally stable compared to Npl4. The results of this thesis will provide an important foundation for further research into the mechanism of p97 function in DPC removal in collaboration with Npl4, Ufd1, and SPRTN.

• Ključne besede: AAA+ ATPase p97, adaptor protein, Ufd1, Npl4, DNA repair, recombinant protein preparation, protein characterization, molecular interactions