A missense mutation in acyl-CoA synthetase ACSL4 reveals essential residues for catalytic activity in ferroptosis

Ferroptosis is a non-apoptotic cell death characterized by iron-dependent lipid peroxidation and is implicated in renal diseases, including acute kidney injury and diabetic nephropathy. In renal proximal tubular cells, the regulation of ferroptosis is particularly critical for maintaining cellular homeostasis. While inducing ferroptosis in normal rat kidney proximal tubular epithelial (NRK-52E) cells, we observed the emergence of resistant subpopulations and established two ferroptosis-resistant clones, designated clone A and clone B, to investigate the underlying mechanisms. Transcript and immunoblot analyses revealed that clone A lacked acyl-CoA synthetase long-chain family member 4 (ACSL4), and this deficiency conferred ferroptosis resistance. Furthermore, although clone B expressed ACSL4, its enzymatic activity was markedly reduced, leading us to hypothesize that clone B harbors a mutation that impairs ACSL4 function. Therefore, we performed sequence analysis and identified a novel T237A missense mutation in ACSL4. Sequence alignment and structural superposition of rat ACSL4 and long-chain fatty acyl-CoA synthetase from Thermus thermophilus suggested that Thr237 in rat ACSL4 cooperates with Glu429 in Mg²⁺ coordination. Functional assays using ACSL4-deficient cells expressing ACSL4 variants (T237A or E429A) confirmed that both residues are essential for catalytic activity. These findings provide new insights into the structural and functional roles of mammalian ACSL4 and may facilitate the development of ACSL4-targeted therapeutics.