20.500.12556/RUL-182813 Mehanizem aktivacije inflamasoma NLRP3 in priprava umetnih inflamasomov Mechanism of NLRP3 inflammasome activation and design of synthetic inflammasomes Inflamasomi so večproteinski kompleksi, ki nastanejo kot odziv na okužbe ali poškodbe tkiva. Med njimi izstopa inflamasom NLRP3, saj se odziva na izjemno raznolike aktivatorje, kot so mikrobi, amiloidi, azbestna vlakna in ionsko neravnovesje. Po zaznavi aktivatorja NLRP3 oligomerizira, veže adapter ASC in prokaspazo 1, ki tvorijo inflamasom in omogočajo aktivacijo prokaspaze 1. Slednja cepi vnetna citokina interlevkin (IL)-1β in IL-18 ter protein gasdermin D (GSDMD), čigar N-končna domena (NtGSDMD) tvori pore v plazmalemi in povzroči litično celično smrt, imenovano piroptoza. Nenadzorovana aktivacija inflamasoma NLRP3 je vpletena v pogosta obolenja, kot so sladkorna bolezen, bolezni srca in ožilja, nevrodegenerativne bolezni ter rak. Kljub intenzivnim raziskavam ostaja še veliko nepojasnjenih vprašanj v začetnih in poznih korakih aktivacije inflamasoma. V prvem delu doktorske naloge nas je zanimalo, kako lahko isti aktivator sproži aktivacijo NLRP3 na različnih znotrajceličnih lokacijah. S sintezno biološkim pristopom smo ugotovili, da se lahko NLRP3 aktivira na različnih membranah in tudi večjih proteinskih ogrodjih. Pokazali smo, da NLRP3 tvori skupke na teh ogrodjih, kar vodi v tvorbo inflamasoma. Predpostavljamo, da najrazličnejši aktivatorji povzročijo motnje celične homeostaze in ultrastrukturne spremembe, ki vodijo do nastanka ogrodij, ki jih NLRP3 zazna in se nanje veže, kar vodi do nastanka aktivnih oligomerov NLRP3 in posledično do tvorbe inflamasoma. Vnetni odziv, ki je posledica inflamasomov, določa tudi piroptoza, ki velja za imunogeno celično smrt. Pokazali smo, da različni stresorji povzročijo oksidativno okolje, ki pospešuje delovanje NtGSDMD in da zaznavanje redoks sprememb poteka preko cisteina na mestu 192. S sintezno bioloških pristopom smo zasnovali umetne inflamasome, ki temeljijo na oligomerizaciji signalnih komponent ter sproščanju vnetnih citokinov in celične smrti. Motive adapterjev prirojene imunosti MAVS, TRIF in MyD88, zaslužne za signaliziranje, smo vezali na ogrodni protein, ki omogoča spontano oligomerizacijo, in dosegli aktivacijo specifičnih signalnih poti. Ko smo to sklopili še s posrednikom litične celične smrti, GSDMD ali MLKL, smo ustvarili umetne inflamasome, ki v celicah izzovejo tvorbo citokinov in celično smrt. Delovanje umetnih inflamasomov smo preverili tudi v rakavih celicah, kjer so sprožili izražanje citokinov, kar nakazuje na potencialno uporabo v imunoterapiji raka za spreminjanje tumorskega mikrookolja v takšno, ki spodbuja imunski odziv proti tumorju. V doktorskem delu smo opisali nove mehanistične vpoglede v aktivacijo NLRP3 in regulacijo piroptoze, hkrati pa s sintezno biološkim pristopom razvili minimalne vnetne module. Inflammasomes are multiprotein complexes that assemble in response to infection or tissue damage. Among them, the NLRP3 inflammasome stands out as it responds to an exceptionally wide range of stimuli, including microbes, amyloids, asbestos fibers, and ionic imbalance. Upon sensing such signals, NLRP3 oligomerizes and recruits the adaptor ASC and pro-caspase-1, which together form the inflammasome and enable the activation of pro-caspase-1. Active caspase-1 cleaves pro-inflammatory cytokines IL-1β and IL-18, as well as gasdermin D (GSDMD), whose N-terminal fragment (NtGSDMD) inserts into the plasma membrane and induces lytic cell death known as pyroptosis. Uncontrolled NLRP3 inflammasome activity contributes to common diseases such as diabetes, cardiovascular disorders, neurodegenerative diseases and cancer. Despite intensive research, many aspects of the upstream and downstream signaling pathways that govern inflammasome activation remain poorly understood. In the first part of this doctoral thesis, we addressed how the same stimulus can trigger NLRP3 activation at distinct subcellular locations. Using a synthetic biology-based approach we found that NLRP3 can be activated at various membranes and even at protein scaffolds. We showed that NLRP3 forms clusters at these scaffolds, which promote inflammasome assembly. We propose that diverse NLRP3 activators induce disturbances in cellular homeostasis and ultrastructural changes that lead to scaffold formation, where NLRP3 can bind to, leading to active NLRP3 oligomer formation and inflammasome assembly. Inflammasome-driven inflammation is also shaped by pyroptosis, an immunogenic form of cell death. We showed that an oxidative environment induced by various stressors enhanced NtGSDMD activity, and that redox sensing occurs through cysteine 192. Using synthetic biology approach, we designed synthetic inflammasomes combining the proinflammatory cytokine release and cell death through oligomerization and activation of key components. By fusing signaling motifs of innate immune adaptors MAVS, TRIF, and MyD88 to an oligomerizing scaffold protein, we achieved the activation of specific signaling pathways. Coupling these modules with effectors of lytic cell death, GSDMD or MLKL, enabled construction of synthetic inflammasomes that induced cytokine production and cell death. We tested these synthetic inflammasomes in cancer cells, where they triggered cytokine expression, indication potential application in cancer immunotherapy for modulating immune responses within the tumor microenvironment. This doctoral work provides new mechanistic insights into NLRP3 activation and regulation of pyroptosis, while also establishing a synthetic biology-based strategy for building minimal inflammatory modules. inflamasom NLRP3 gasdermin D molekularni mehanizem signalosomi umetni inflamasomi NLRP3 inflammasome gasdermin D molecular mechanism signalosomes synthetic inflammasomes true false false Slovenski jezik Angleški jezik Doktorsko delo/naloga 2026-05-24 07:16:08 2026-05-24 07:16:24 2026-05-25 04:35:59 0000-00-00 00:00:00 2026 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 2027-05-13 39129 9011.pdf 9011.pdf 1 751ECDA10DB0FFC5A92C6205A5817EC1 7697c9b55c9e64774b82b78e64adf5a2a32eaa848886bb79ddba8d5c9e267147 67ac3757-572f-11f1-b0ab-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=234352 Medicinska fakulteta 0 0 0