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Vnetni odziv v okviru prirojene imunosti je ključnega pomena za uspešno spopadanje z vdorom patogenih organizmov in za regeneracijo poškodovanega tkiva. V ta namen so imunske celice opremljene z receptorji za zaznavanje molekul mikrobnega izvora in različnih nemikrobnih endogenih molekul. Eden najpomembnejših regulatorjev nemikrobnega vnetja v imunskih celicah je proteinski kompleks inflamasom NLRP3, ki se aktivira ob zaznavi molekul, kot sta ATP in mitohondrijska DNA, pa tudi raznovrstnih patoloških delcev, kot so amiloidni plaki pri nevrodegenerativnih boleznih, holesterolni kristali pri aterosklerozi ali azbestna vlakna pri azbestozi. Inflamasomski kompleks NLRP3 je sestavljen iz oligomera aktiviranih receptorjev NLRP3, okoli katerega se preko medproteinskih interakcij v domenah PYD in CARD nanizajo molekule adapterskega proteina ASC in molekule pro-kaspaze-1, ki po avtokatalitskem procesiranju v aktivno kaspazo-1 aktivirajo citokina IL-1ß in IL-18 ter piroptotično smrt. Mehanizem aktivacije senzorskega proteina NLRP3 ostaja neznan, kar otežuje razvoj selektivnih terapevtikov za številne bolezni, ki so povezane z njegovo prekomerno aktivacijo. V pričujočem doktorskem delu smo osvetlili nekatera odprta vprašanja v zvezi z začetnimi stopnjami aktivacije inflamasoma NLRP3. Na podlagi celičnih linij, ki so izražale himerne konstrukte NLRP3, pri katerih smo njegovo interakcijsko domeno PYD zamenjali z drugimi interakcijskimi domenami, smo ugotovili, da je domena NLRP3PYD pomembno vpletena v vzdrževanje neaktivne oblike senzorskega proteina NLRP3, saj se je njena zamenjava z različnimi domenami CARD odražala v konstitutivni aktivnosti inflamasoma. S pomočjo konstruktov, pri katerih smo interakcijsko domeno PYD povezali z različnimi oligomerizacijskimi domenami, smo raziskali tudi stehiometrijo senzorskega proteina NLRP3 in ugotovili, da je že povezava domen NLRP3PYD v trimer zadostna za aktivacijo inflamasoma NLRP3. V nadaljevanju nas je zanimala tudi struktura in razporeditev proteinov znotraj inflamasomskega kompleksa NLRP3. S superresolucijsko mikroskopijo 3D SIM fluorescenčno označenih proteinov znotraj kompleksa v živih celicah smo ugotovili, da oligomerizirani proteini ASC-GFP tvorijo visoko zamreženo strukturo z gosto sredico, v kateri se nahaja NLRP3. Spremljanje dinamike njegove tvorbe v realnem času pa je pokazalo, da se filamenti ASC-GFP v značilno kompaktno obliko kondenzirajo preko intenzivnega razvejanja filamentov, na kar pomembno vpliva domena ASCCARD. Na podlagi strukturnih in molekularno- bioloških študij proteinov, udeleženih v inflamasom NLRP3, ki so identificirale njihove interakcijske površine, smo načrtovali tudi peptidne inhibitorje inflamasoma NLRP3. Identificirali smo pet načrtovanih inhibitorjev, ki so inhibirali sproščanje IL-1ß, aktivacijo kaspaze-1 in oligomerizacijo ASC. Peptid iz segmenta ASCPYD/H2-H3, ki je inflamasom NLRP3 inhibiral selektivno, smo za morebitne terapevtske aplikacije na področju nevrodegenerativnih bolezni opremili s peptidnim zaporedjem Angiopep-2 in pokazali, da je tako opremljena različica inhibitorja ASCPYD/H2-H3 uspešno prehajala krvno-možgansko pregrado, obenem pa ni imela vpliva na njegov inhibitorni potencial. V tem doktorskem delu smo prispevali novo znanje o molekularnem mehanizmu aktivacije inflamasoma NLRP3 in načrtovali set peptidnih inhibitorjev, ki bi lahko predstavljali podlago za nove pristope k terapiji z inflamasomom NLRP3 povezanih bolezni. Innate immunity associated inflammatory responses are crucial for the efficient elimination of the infectious agents and tissue repair. For this purpose immune cells are equipped with receptors capable of sensing molecules of both microbial and endogenous origin. One of the key sterile inflammation regulators in imune cells is the NLRP3 inflammasome. Formation of this multiprotein complex is triggered by the endogenous molecules such as extracellular ATP and mitochondrial DNA as well as various pathological deposits including amyloid plaques in neurodegenerative diseases, cholesterol crystals in atherosclerosis and asbestos fibres in asbestosis. NLRP3 inflammasome assembly is initiated by an oligomer of activated NLRP3 receptors that provides a nucleus for the oligomerisation of the adaptor protein ASC and molecules of pro-caspase-1 via interactions in their PYD and CARD domains. Autocatalytical cleavage of pro-caspase-1 results in the formation of active caspase-1 which in turn cleaves cytokine IL-1β and IL-18 precursors into their active forms as well as initiates pyroptotic cell death. The molecular mechanism of the NLRP3 inflammasome activation remains largely elusive which hinders the development of selective therapeutic strategies for numerous diseases associated with disregulation of NLRP3. In this doctoral dissertation we addressed some of the open questions regarding the early stages of the NLRP3 inflammasome activation. Establishing cell lines that expressed chimeric NLRP3 variants where PYD domain was replaced by other interaction domains we found that NLRP3PYD domain is critically involved in the maintenance of inactive NLRP3 conformation as its replacement with various CARD domains resulted in constitutive inflammasome activation. Using the constructs where NLRP3PYD domain was coupled to various oligomerisation domains we also investigated the minimal active stoichiometry of NLRP3 oligomer and found that a trimer of NLRP3PYD domains was sufficient for the inflammasome activation. In addition, we were interested in the structure and protein arrangement within the complex. 3D SIM superresolution microscopy of fluorescently labelled proteins within the complex revealed that ASC-GFP forms a highly branched network with a dense core containing the NLRP3 oligomer. Monitoring the dynamics of the complex formation in real time further revealed that ASC-GFP filaments are being condensed into a speck through intense branching of filaments, a process supported by ASCCARD domains. Moreover, based on the available protein crystal structures and mutagenesis studies that identified interaction surfaces of inflammasome proteins we also designed peptide inhibitors of the NLRP3 inflammasome. Five of the designed peptides inhibited IL-1β release, caspase-1 activation and ASC oligomerisation. Peptide comprising the ASCPYD/H2-H3 segment selectively inhibited the inflamasom NLRP3. In order to underline its relevance in a neurological setting, it was equipped with Angiopep-2 peptide sequence which enabled its transfer through the brain-blood barrier in the absence of any negative effects on its inhibitory potential. This doctoral thesis provides new mechanistic clues on NLRP3 inflammasome activation as well as a toolbox of designed peptide inhibitors that could represent the basis for the novel therapeutic approaches to treating NLRP3 inflammasome associated diseases. |
