Vpliv lokacije proteina NLRP3 na tvorbo in delovanje inflamasoma

Alič, Barbara (author), Benčina, Mojca (mentor), Hafner Bratkovič, Iva (co-mentor)

Inflamasom NLRP3 je multiproteinski kompleks, ki ima pomembno vlogo pri naravni imunosti. Za njegovo delovanje se mora sestaviti in aktivirati. Kanonična aktivacija poteka v dveh stopnjah, najprej mora priti do aktivacije poti NF-κB z vezavo liganda na receptor TLR, drugo stopnjo pa aktivirajo različni aktivatorji. Rezultat aktivacije inflamasoma je aktivacija kaspaze-1 in posledično izločanje zrelih citokinov IL-1β in IL-18, vodi lahko tudi v celično smrt, imenovano piroptoza. Na katerem mestu v celici pride do njegove aktivacije, še vedno ni pojasnjeno. V magistrski nalogi smo ugotavljali vpliv lokacije inflamasoma NLRP3 na aktivacijo inflamasoma. S kloniranjem smo ustvarili konstrukte NLRP3 v fuziji z rumenim fluorescenčnim proteinom in značko za lokalizacijo na specifične celične organele. Konstrukte smo vnesli v vektor pRetroX-TRE3G, nato pa jih z metodo retrovirusne transdukcije vstavili v mišje makrofage, ki ne vsebujejo lastnega NLRP3 in pripravili stabilne celične linije. Celične linije smo izpostavili LPS ter različnim aktivatorjem inflamasoma NLRP3. S konfokalno mikroskopijo smo pokazali, da nam je uspelo lokalizirati protein na specifične dele v celici. Z encimskoimunskim testom, testom laktat dehidrogenazne aktivnosti pa smo ugotovili, da lokacija NLRP3 vpliva na potek aktivacije inflamasoma.

• Language: Slovenian
• Keywords: Imunologija, inflamasom NLRP3, lokalizacija, aktivacija NLRP3
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Publisher: [B. Alič]
• Year: 2020
• PID: 20.500.12556/RUL-117756
• UDC: 602.68:577.27:57.083.3(043.2)
• COBISS.SI-ID: 23522563
• Publication date in RUL: 24.07.2020

Secondary language

• Language: English
• Title: Role of NLRP3 localization for the inflammasome assembly and activation

Abstract

NLRP3 inflammasome is a multiprotein complex that plays an important role in the innate immunity. Canonical activation requires two steps. In the first step, NF κB pathway is activated then follows the second step, NLRP3 inflammasome assembly, which is initiated by different activators. The result is activaton of caspase-1 and the release of mature cytokines IL-1β in IL-18 and cell death called pyroptosis can also occur. How and where in the cell NLRP3 is activated is a subject of intense research. In this thesis we studied the importance of NLRP3 protein location for inflammasome activation. We designed NLRP3 constructs consisting yellow fluorescent protein and tags for specific cell localization. Contructs were then cloned into the vector pRetroX-TRE3G and stable cell lines were prepared by retroviral transduction of NLRP3-deficient mouse macrophages. Cell lines were then exposed to LPS and different NLRP3 inflammasome activators. As judged from imaging by confocal microscopy we managed to localise NLRP3 protein to the specific cell organelles. By ELISA test and lactate dehydrogenase release measurement we showed that NLRP3 location is important for inflammasome activation.

• Keywords: Immunology, NLRP3 inflammasome, localization, NLRP3 activation