Uporaba haloalkan dehalogenaze za označevanje variant MLKL v celičnih linijah HEK

Zaveršek, Nika (avtor), Gunčar, Gregor (mentor)

Protein MLKL (angl. mixed-lineage kinase domain-like) je izvršilec nekroptoze, vrste regulirane celične smrti. Sestavljen je iz psevdokinazne domene in domene svežnja štirih vijačnic (4HB), ki ima izvršilno funkcijo. Študije so pokazale, da vijačnica v povezovalnem delu med domenama inhibira domeno 4HB. Uporabili smo štiri variante hMLKL1: celotno hMLKL1, skrajšano varianto hMLKL1 iz N-končnih 154 aminokislinskih ostankov in obe varianti z mutacijo D144K, ki aktivira hMLKL1. Fluorescenčne oznake so uporabno orodje pri opazovanju celične lokalizacije proteinov. Med najbolj znane fluorescenčne oznake sodi EGFP (angl. enhanced green fluorescent protein), ena izmed novejših in zanimivih oznak je HaloTag7, ki veže organska barvila na kloroalkanskem distančniku. Zanimalo nas je, kako proteinski oznaki HaloTag7 in EGFP vplivata na funkcijo hMLKL1 in njegovih variant, ter katera je boljša za opazovanje hMLKL1 in njegovih oznak z mikroskopom. Pripravili smo stabilne celične linije HEK293 z vstavljenimi variantami hMLKL1 in proteinskima oznakama EGFP in HaloTag7. Ugotovili smo, da EGFP in HaloTag7 na funkcijo hMLKL1 in njegovih variant vplivata minimalno, pri čemer je vpliv EGFP manjši. Presodili smo, da je EGFP bolj primeren za opazovanje hMLKL1 v celičnih linijah HEK293 s konfokalnim fluorescenčnim mikroskopom. Pri hMLKL1 variantah z mutacijo in obema proteinskima oznakama je bila očitna translokacija na membrano.

• Jezik: Slovenski jezik
• Ključne besede: MLKL, nekroptoza, HaloTag7, konfokalna fluorescenčna mikroskopija
• Vrsta gradiva: Magistrsko delo/naloga
• Tipologija: 2.09 - Magistrsko delo
• Organizacija: FKKT - Fakulteta za kemijo in kemijsko tehnologijo
• Leto izida: 2022
• PID: 20.500.12556/RUL-141951
• COBISS.SI-ID: 134097155
• Datum objave v RUL: 12.10.2022

Sekundarni jezik

• Jezik: Angleški jezik
• Naslov: Application of haloalkane dehalogenase for tagging of the MLKL variants in HEK cell lines

Izvleček

MLKL (mixed-lineage kinase domain-like) protein is believed to be an effector of a type of regulated cell death, necroptosis. It consists of the pseudokinase domain and the four-helix bundle domain (4HB) that has a necroptosis executive function. It has been suggested that the first connecting helix between both domains inhibits 4HB. We used four variants of hMLKL1: full-length hMLKL1, the truncated variant of hMLKL1 consisting of the N-terminal 154 amino acid residues, and both variants with the D144K mutation, which is known to activate hMLKL1. Fluorescent tags are very useful tools in observing the cellular localization of proteins. EGFP (enhanced green fluorescent protein) is probably the most commonly used fluorescent tag, whereas the protein tag HaloTag7 is part of a new labelling technology, which is based on the binding of organic dyes with reactive chloroalkane linker into a modified version of the enzyme haloalkane dehalogenase. We investigated the influence of both fluorescent tags, HaloTag7 and EGFP, on the function of all hMLKL1 variants. We aimed to estimate which fluorescent tag is more suitable for observing hMLKL1 and its variants by confocal microscopy. We have generated stable HEK293 cell lines with inserted variants of hMLKL1 in combination with chosen fluorescent tags. We found that EGFP and HaloTag7 do not significantly affect the function of hMLKL1 and its variants but that in comparison the EGFP affects it less. We found that EGFP is better suited for observing hMLKL1 in HEK293 cell lines with confocal fluorescence microscopy. In hMLKL1 variants with said mutation, translocation to the membrane was observed using both fluorescent tags.

• Ključne besede: MLKL, necroptosis, HaloTag7, confocal fluorescent microscopy