Identification of HPCAL1 as a specific autophagy receptor involved in ferroptosis

Selective macroautophagy/autophagy maintains cellular homeostasis through the lysosomal degradation of specific cellular proteins or organelles. The pro-survival effect of selective autophagy has been well-characterized, but the mechanism by which it drives cell death is still poorly understood. Here, we use a quantitative proteomic approach to identify HPCAL1 (hippocalcin like 1) as a novel autophagy receptor for the selective degradation of CDH2 (cadherin 2) during ferroptosis. HPCAL1-dependent CDH2 depletion increases susceptibility to ferroptotic death by reducing membrane tension and favoring lipid peroxidation. Site-directed mutagenesis aided by bioinformatic analyses revealed that the autophagic degradation of CDH2 requires PRKCQ (protein kinase C theta)-mediated HPCAL1 phosphorylation on Thr149, as well as a non-classical LC3-interacting region motif located between amino acids 46–51. An unbiased drug screening campaign involving 4208 small molecule compounds led to the identification of a ferroptosis inhibitor that suppressed HPCAL1 expression. The genetic or pharmacological inhibition of HPCAL1 prevented ferroptosis-induced tumor suppression and pancreatitis in suitable mouse models. These findings provide a framework for understanding how selective autophagy promotes ferroptotic cell death.Abbreviations: ANXA7: annexin A7; ARNTL: aryl hydrocarbon receptor nuclear translocator like; CCK8: cell counting kit-8; CDH2: cadherin 2; CETSAs: cellular thermal shift assays; CPT2: carnitine palmitoyltransferase 2; DAMP, danger/damage-associated molecular pattern; DPPH: 2,2-diphenyl-1-picrylhydrazyl; DFO: deferoxamine; EBNA1BP2: EBNA1 binding protein 2; EIF4G1: eukaryotic translation initiation factor 4 gamma 1; FBL: fibrillarin; FKBP1A: FKBP prolyl isomerase 1A; FTH1: ferritin heavy chain 1; GPX4: glutathione peroxidase 4; GSDMs: gasdermins; HBSS: Hanks’ buffered salt solution; HMGB1: high mobility group box 1; HNRNPUL1: heterogeneous nuclear ribonucleoprotein U like 1; HPCAL1: hippocalcin like 1; H1-3/HIST1H1D: H1.3 linker histone, cluster member; IKE: imidazole ketone erastin; KD: knockdown; LDH: lactate dehydrogenase; LIR: LC3-interacting region; MAGOH: mago homolog, exon junction complex subunit; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; MDA: malondialdehyde; MLKL: mixed lineage kinase domain like pseudokinase; MPO: myeloperoxidase; MTOR: mechanistic target of rapamycin kinase; OE: overexpressing; OSTM1: osteoclastogenesis associated transmembrane protein 1; PRKC/PKC: protein kinase C; PRKAR1A: protein kinase cAMP-dependent type I regulatory subunit alpha; PRDX3: peroxiredoxin 3; PTGS2: prostaglandin-endoperoxide synthase 2; ROS: reactive oxygen species; SLC7A11: solute carrier family 7 member 11; SLC40A1: solute carrier family 40 member 1; SPTAN1: spectrin alpha, non-erythrocytic 1; STS: staurosporine; UBE2M: ubiquitin conjugating enzyme E2 M; ZYX: zyxin.