Inflammasomes are cytosolic multiprotein complexes composed of pattern recognition receptor, adaptor protein, and pro-caspase-1 molecules. Inflammasome receptors are activated by sensing pathogen-associated molecular patterns or damage-associated molecular patterns. This triggers the activation of caspase 1, which in turn cleaves proprotein isoforms into active cytokines IL-1β and IL-18. In addition, caspase 1 catalyzes the site-specific proteolysis of gasdermin D, releasing its N-terminal domain, which upon binding the phospholipids in the cell membrane forms pores, leading to pyroptosis. Pyroptosis is a programmed immunogenic cell death characterized by cell lysis, and the release of inflammatory molecules into the extracellular environment. Pyroptosis protects the host against pathogens. It triggers inflamation and activates immune system, thus pyroptosis and its effector gasdermin D represent promising approach for novel cancer therapy. The aim of the master's thesis was to determine the pyroptotic potential of recombinant constructs located in the nucleus, as this could enhance treatment effectiveness, minimize adverse effects, and potentially overcome drug resistance of cancer cells. In this study, we used designed gasdermin D variants that are activated specifically by the viral TEV protease, enabling precise control over pyroptosis induction. We showed that the addition of a nuclear localization sequence (NLS) resulted in successful delivery of recombinant constructs into the nucleus. Our findings revealed that incorporating nuclear localization signal (NLS) and nuclear export signal (NES) into the designed gasdermin D and TEV protease sequences does not impact pyroptosis efficiency in HEK293 cells. While we tested the system on cancer cell lines N2A and B16F10, we encountered excessive cell toxicity, prompting further optimization of the protocol for the delivery of genetic information encoding recombinant constructs. Alternatively, electroporation provides a promising option for plasmid delivery into cells, given its successful application in clinical practice.
During master's thesis we showed that pyroptosis can be triggered if gasdermin and activating protease are located in the cell nucleus. This enables further development where pyroptosis is only induced when cancer-specific DNA sequence is present, suggesting that designed gasdermin D has notable potential for future application in cancer immunotherapy.
|
