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Vpliv kalijevih ionov in vrste oligomera NLRP3 na aktivacijo inflamasoma
Boršić, Elvira (Author), Hafner Bratkovič, Iva (Mentor) More about this mentor... This link opens in a new window, Gunčar, Gregor (Co-mentor)

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Abstract
Inflamasom NLRP3 je del naravne imunosti, ki omogoča zaznavo številnih patogenih in endogenih molekulskih vzorcev, ki aktivirajo senzorski protein NLRP3. Njegova oligomerizacija omogoči pripenjanje ASC in pro-kaspaze-1, ki postane aktivna in sproži zorenje vnetnih citokinov IL-1β in IL-18 ter cepitev gasdermina D. N-končni del proteina tvori pore v membrani, skozi katere se v izvencelični prostor sprosti citokin IL1β, ki izzove lokalno vnetje ter celično smrt imenovano piroptoza. Neregulirano delovanje inflamasoma NLRP3 je povezano s številnimi boleznimi, kot sta ateroskleroza in nevrodegenerativne bolezni. Zaenkrat začetne stopnje aktivacije NLRP3 še niso pojasnjene, saj je malo verjetno, da strukturno tako različni aktivatorji neposredno aktivirajo NLRP3. Namen našega dela je bil ovrednotiti začetni del poti aktivacije z osredotočenostjo na oligomerizacijo in pripravo sistemov za preučevanje vpliva K+, kot skupnega dejavnika različnih aktivatorjev, na NLRP3. Ker je nejasno, kakšno strukturo tvori NLRP3 ob aktivaciji, nas je zanimalo, ali lahko inflamasom aktiviramo iz struktur, ki tvorijo brez-membranske organele (LLPS). Na NLRP3PYD smo pripeli proteine, ki fazno separirajo, saj lahko z LLPS lokalno povečamo koncentracijo komponent. Pripravljene variante NLRP3PYD smo stabilno izrazili v makrofagih. Ugotovili smo, da je fuzija s TDP-43 najboljša za aktivacijo inflamasoma, kar smo zaznali kot močno izločanje IL-1β in piroptozo. Opazili smo, da je aktivnost inflamasoma odvisna od kompaktnosti nastalega kondenzata. Z metodo vračanja fluorescence po fotobeljenju smo pokazali, da NLRP3PYD in tudi ASC zamrežita strukturo LLPS, tako da ni več dinamična. Za nadaljnje delo smo pripravili dobra kandidata, ki aktivirata inflamasom, in omogočata preučevanje mehanizma zgornjih poti. V drugem delu smo želeli vzpostaviti sistem za preučevanje oligomerizacije in vitro, saj v celičnem okolju ne moremo enoznačno analizirati neposrednih vplivov na NLRP3. Analizirali smo izražanje NLRP3 z dvema različnima evkariontskima kompletoma za transkripcijo in translacijo in vitro, pri čemer smo zgolj z enim uspešno pripravili proteine, ki so nam omogočili izvedbo fluorescenčne korelacijske spektroskopije. Ugotovili smo, da je slabost takšnih reagentov nizek izplen reakcije. Za preučevanje vpliva K+ na aktivacijo inflamasoma v celicah, smo pripravili funkcionalen NLRP3 s pripetim senzorjem za K+, GINKO1, ki bi lahko omogočal vizualno in kvantitativno spremljanje koncentracij K+. V okviru magistrskega dela smo vzpostavili sisteme, ki nam bodo omogočili nadaljnje raziskovanje vpliva iztoka K+ na oligomerizacijo NLRP3, ter pokazali, da brez-membranski organeli omogočajo robustno aktivacijo inflamasoma.

Language:Slovenian
Keywords:NLRP3, PYD, LLPS, kalijevi ioni, transkripcija/translacija in vitro
Work type:Master's thesis/paper (mb22)
Tipology:2.09 - Master's Thesis
Organization:FKKT - Faculty of Chemistry and Chemical Technology
Year:2021
COBISS.SI-ID:72152579 This link opens in a new window
Views:121
Downloads:9
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Secondary language

Language:English
Title:The effect of potassium ions and type of NLRP3 oligomer on inflammasome activation
Abstract:
NLRP3 inflammasome is a part of innate immunity that allows the identification of a plethora of pathogen and endogenous molecular patterns that trigger the activation of NLRP3. Its oligomerization enables the binding of ASC and pro-caspase-1, which becomes active and leads to the maturation of IL-1β and IL-18 and the cleavage of gasdermin D. N-terminal part of the protein forms pores, through which mature cytokines are released into the extracellular space where they cause local inflammation and pyroptotic cell death. It is still unknown how the initial stages of NLRP3 activation proceed, as it is unlikely for structurally different activators to directly activate NLRP3. The purpose of our work was to evaluate the early steps of this pathway with the focus on oligomerization and the preparation of systems to study the influence of K+ as a common factor downstream of different NLRP3 activators. Since it is unclear what structure NLRP3 forms upon activation, we were interested in whether inflammation can be activated from structures that form membrane-less organelles (LLPS). We fused proteins that phase separate with NLRP3PYD as LLPS can locally increase the concentration of components. Prepared variants of NLRP3PYD were stably expressed in macrophages. Fusion with TDP-43 was found to trigger inflammasome activation, which was determined as strong IL-1β secretion as well as pyroptotic cell death. With fluorescence recovery after photobleaching we showed that NLRP3PYD as well as ASC tether the LLPS structure to a less dynamic structure. The compactness of the condensate formed influences the kinetics of inflammasome activation. We prepared good candidates that activate inflammasome and allow further studies of early steps in NLRP3 activation. In the second part, we wanted to establish a system for studying oligomerization in vitro since we cannot analyze the direct effects of K+ efflux on NLRP3 in the complex cellular environment. We analyzed the expression of NLRP3 with two different eukaryotic kits for in vitro translation and transcription, but only one enabled us to prepare proteins for examination with fluorescence correlation spectroscopy. We found that the disadvantage of such reagents is the low yield of the reaction. To study the effect of K+ on inflammasome activation in cells, we developed a functional NLRP3 with an attached K+ sensor, GINKO1, that could allow visual and quantitative monitoring of fluctuations in K+ concentrations. In the master thesis, we established systems that will allow us to further investigate the influence of K+ efflux on the oligomerization of NLRP3 and show that membrane-less organelles allow robust activation of the inflammasome.

Keywords:NLRP3, PYD, LLPS, potassium ions, in vitro transcription/translation

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