Amiranda, Sara (2023) Loss-of-function analysis of Carbonic Anhydrase IX in cellular and animal models. [Tesi di dottorato]

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Abstract

The human carbonic anhydrase IX (CA IX) is a hypoxia-induced transmembrane protein belonging to the α-CAs enzyme family. It has a crucial role in pH regulation in hypoxic cells and acts by buffering intracellular acidosis induced by hypoxia. Indeed, it is frequently expressed in cancer cells, where it contributes to tumour progression. CA IX is also localized in the nucleus, where it contributes to 47S rRNA precursor genes transcription, but the mechanism providing its nuclear translocation remains unclear. Since CA IX is involved in a plethora of physio-pathological processes, we analysed CA IX functions in cellular and animal models more deeply, by implementing different loss-of-function strategies. Firstly, we analysed the results of specific mutations, targeting the C-terminal domain, on CA IX subcellular distribution, focusing on two loss-of-interaction mutants (MUT1 and MUT4) in the SH-SY5Y cell line; secondly, we characterized the impact of CA IX inactivation in ischemic processes, either with knock-out ex vivo murine primary neurons or with enzymatic inhibition in neurons and in rat brain. The mutagenesis showed an altered subcellular localization of mutant forms, both unable to stably reside in the plasma membrane or in the nucleus, suggesting that CA IX nuclear translocation depends on its transit through secretory and endocytic pathways. On the other hand, CA IX inactivation resulted to be protective in all the ischemic models studied. Although further characterization needs to be done to uncover the molecular mechanism underlying this protective role, this could pave the way for possible strategies for ischemic treatment, a condition with still too few therapeutic options.

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