HSD17B11 maintains FSP1 localization on lipid droplets to support ferroptosis defense

Ferroptosis is a regulated form of cell death driven by iron-dependent lipid peroxidation, and lipid droplets (LDs) are increasingly recognized as important regulators of this process. Consistent with this role, the anti-ferroptotic factor ferroptosis suppressor protein 1 (FSP1) is known localizing on LDs through N-myristoylation-dependent membrane targeting, where it protects LD lipids from peroxidation. Here, we identify the LD protein HSD17B11 as an additional factor required for maintaining both FSP1 localization on LDs and cellular FSP1 abundance. Silver staining followed by mass spectrometry analysis of purified LD proteins identified reduced LD-associated FSP1 in HSD17B11-deficient cells, which was further validated by immunoblotting and imaging analyses. Mechanistically, HSD17B11 physically interacted with FSP1 and was required to preserve FSP1 association with LDs. Mutational analyses further demonstrated that both FSP1 N-myristoylation and an intact HSD17B11 interaction interface are necessary for LD targeting. Correspondingly, HSD17B11 deficiency reduced LD-associated and total cellular FSP1 levels and increased cellular sensitivity to lipid oxidative stress. Together, our findings identify HSD17B11 as a previously unrecognized regulator of LD-associated FSP1 and reveal an additional mechanism controlling compartmentalized ferroptosis defense.