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Vpliv nanotelesa M33 na celično smrt povzročeno s proteinom MLKL
ID Černe, Urška (Author), ID Gunčar, Gregor (Mentor) More about this mentor... This link opens in a new window

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Abstract
Psevdokinaza MLKL je pomembna molekula pri procesu nekroptoze, oblike programirane celične smrti z morfološkimi značilnostmi nekroze. Nekroptoza privede do naluknjanja celične membrane in izlitja celične vsebine v okolico sosednjih celic, kar sproži vnetni odziv. Povezujejo jo s številnimi človeškimi vnetnimi boleznimi. Protein MLKL oligomerizira in se prestavi na celično membrano, kjer povzroči celično smrt. Fluorescenčna nanotelesa predstavljajo pomembno orodje za vizualizacijo mnogih antigenov znotraj žive celice. Razvita so bila številna nanotelesa proti proteinom, ki sodelujejo pri vnetju in s tem vplivajo na imunski odziv. V sklopu magistrske naloge smo potrdili vezavo proteina MLKL in fuzijskega nanotelesa M33-mCherry s kromatografijo z ločevanjem po velikosti. Nanotelo M33 se veže na N-končni del proteina MLKL (MLKLN-154). Detekcija proteina MLKL, ki se po predhodni transfekciji izraža v celicah HEK293T, s fuzijskim nanotelesom M33-mCherry ni bila uspešna. Določali smo kolokalizacijo proteinov M33 in MLKLN-154 v celicah HEK293T po predhodni transfekciji z obema konstruktoma. Kolokalizacijo smo opazovali na celični membrani, kamor se translocira protein MLKL za sprožitev celične smrti. Protein MLKLN-154 ne sproži celične smrti in se nahaja v citoplazmi. Mutirana oblika MLKLN-154 D144K povzroči celično smrt, torej se translocira na membrano in je zato primerna za opazovanje kolokalizacije. Določili smo, kdaj se mutiran protein začne izražati, vendar še ne sproži celične smrti. Potrdili smo tudi izražanje fuzijskega proteina M33-mCherry. Kljub temu nismo uspeli zaznati kolokalizacije. Pri vzorcih, ki so vsebovala nanotelo in pri tistih brez nanotelesa, ni bilo opaznih razlik v deležu celične smrti. Na osnovi našega dela smo zaključili, da nanotelo M33 ne zmanjša deleža celične smrti.

Language:Slovenian
Keywords:nekroptoza, MLKL, fluorescenčno nanotelo, mCherry
Work type:Master's thesis/paper
Typology:2.09 - Master's Thesis
Organization:FKKT - Faculty of Chemistry and Chemical Technology
Year:2019
PID:20.500.12556/RUL-109927 This link opens in a new window
COBISS.SI-ID:1538406339 This link opens in a new window
Publication date in RUL:10.09.2019
Views:1076
Downloads:260
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Secondary language

Language:English
Title:Effect of M33 nanobody on cell death caused by MLKL protein
Abstract:
The mixed lineage kinase domain-like (MLKL) pseudokinase is an essential molecular component of necroptosis, a form of programmed cell death with morphological characteristics of necrosis. Necroptosis results in plasma membrane perforation and the release of cellular contents, which elicit an immune response from neighbouring cells. It has an important function in many human inflammatory diseases. To cause cell death, MLKL needs to oligomerize and translocate to the cell membrane. Nanobodies fused with fluorescent proteins are an important tool in the visualization of almost any antigen within the living cell. Nanobodies against proteins involved in inflammation have been developed to modulate an immune response. In our study, we confirmed the binding of the fusion protein M33-mCherry by size-exclusion chromatography. Nanobody M33 recognizes the N-terminal region of MLKL (MLKLN-154). Detection of MLKL protein expressed in HEK293T cells with M33-mCherry fusion protein was unsuccessful. Further on, we determine whether M33-mCherry and MLKLN-154 colocalize in HEK293T cells after transfection with both constructs. Membrane translocation of MLKL is crucial for necroptosis, so colocalization might be observed on the plasma membrane. MLKLN-154 localizes in the cytoplasm and cannot induce necroptosis, but D144K mutant, which mimics activated MLKL, causes cell death. We determined the time point when MLKLN-154 D144K is expressed, but cells are still alive and also confirmed the expression of the M33-mCherry fusion protein. However, we could not observe their colocalization. There was no difference in cell death level between samples with nanobody and without it, so we concluded that in this case, the M33-mCherry nanobody does not decrease the level of cell death.

Keywords:necroptosis, MLKL, fluorescent nanobody, mCherry

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