Abstract Background Microtubules acetylation has emerged as a key regulator of cellular homeostasis, but its roles in autophagy remain understudied. Here, we identify α-tubulin acetyltransferase 1 (ATAT1), the enzyme responsible for α-tubulin K40 acetylation, as a critical regulator of NCOA4-mediated ferritinophagy and iron homeostasis in cancer cells. Methods Human cancer cell lines were stably or transiently silenced for ATAT1 expression. Autophagy induction was evaluated by visualizing punctate structures and by analyzing changes in autophagic marker levels. Seahorse and flow cytometry experiments were conducted to study the impact of ATAT1 silencing on cell metabolism. Additionally, analysis of iron homeostasis genes, free iron pool, as well as colocalization of NCO4A and ferritin to autophagosome were analyzed to confirm activation of ferritinophagy. Finally, we treated cells with RSL3 (a ferroptosis inducer) and ferrostatin-1 or chloroquine to understand the connection between ATAT1, autophagy, and ferroptosis-induced cell death. Genetic approaches were used to study the role of NCO4A and K40 acetylation in these pathways. Results We show that ATAT1 silencing induces an oxidative stress response accompanied by a functional autophagic flux. Notably, ATAT1-silenced cells exhibited reduced ATP production and oxygen consumption rate compared with control cells, as well as altered mitochondrial dynamics under both normal and stress conditions. Importantly, ATAT1 loss leads to intracellular iron overload by inducing NCOA4-mediated ferritinophagy, which targets the degradation of the iron storage protein ferritin, thus maintaining intracellular iron homeostasis. Activation of ferritinophagy, in turn, renders ATAT1-silenced lung cancer cells more susceptible to ferroptotic cell death. Notably, the key phenotypes observed in ATAT1-silenced cells are absent in cells with non-acetylatable α-tubulin, demonstrating a direct role for the loss of ATAT1 protein on the induction of a ferroptosis vulnerability phenotype. Conclusions These findings challenge the traditional view of ATAT1 as a simple microtubule modifier and position this acetyltransferase as a central node in redox, metabolic, and autophagic regulation.
Pellegrini et al. (Fri,) studied this question.