RESUMEN
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.
Asunto(s)
Autofagia , Ferroptosis , Ratones , Animales , Autofagia/fisiología , Hipocalcina/farmacología , Proteómica , Muerte CelularRESUMEN
Hippocalcin is a Ca2+-binding protein that is expressed mainly in pyramidal nerve cells of the hippocampus. However, its functions and mechanism in the brain remain unclear. To elucidate the role of hippocalcin, we used a conditionally immortalized hippocampal cell line (H19-7) and showed that bFGF treatment increased the expression of hippocalcin during bFGF-induced neurite outgrowth of H19-7 cells. Overexpression of hippocalcin dramatically elongated neurites and increased the expression of basic helix-loop-helix transcription factor, that is, NeuroD without bFGF stimulation. Treatment of the cells with hippocalcin siRNA completely blocked bFGF-induced neurite outgrowth and NeuroD expression. bFGF stimulation resulted in activation of phospholipase C-gamma (PLC-gamma) and an increased level of intracellular Ca2+. Hippocalcin expression by bFGF stimulation was fully blocked by both the PLC-gamma inhibitor U73122 and BAPTA-AM, a chelator of intracellular Ca2+, suggesting that hippocalcin expression by bFGF is dependent on PLC-gamma and Ca2+. Moreover, both U73122 and BAPTA-AM completely blocked bFGF-induced neurite outgrowth and NeuroD expression. Taken together, these results suggest for the first time that bFGF induces hippocalcin expression in H19-7 cells through PLC-gamma activation, which leads to neurite outgrowth.
Asunto(s)
Factor 2 de Crecimiento de Fibroblastos/farmacología , Regulación de la Expresión Génica/fisiología , Hipocalcina/farmacología , Neuritas/efectos de los fármacos , Neuronas/citología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Calcio/metabolismo , Línea Celular Transformada , Quelantes/farmacología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Estrenos/farmacología , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Hipocampo/citología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuritas/fisiología , Neuronas/efectos de los fármacos , Fosfolipasa C gamma/metabolismo , Pirrolidinonas/farmacología , ARN Interferente Pequeño/metabolismo , ARN Interferente Pequeño/farmacología , Ratas , Transfección/métodosRESUMEN
The increased expression of markers associated with a differentiated phenotype, such as P-glycoprotein (Pgp), follows treatment with histone deacetylase inhibitors. Because depsipeptide (FR901228, FK228, NSC630176) is a substrate for Pgp, up-regulation of the gene that encodes it, MDR1, would mean that depsipeptide induces its own mechanism of resistance. To examine the effect of depsipeptide on expression of ATP-binding cassette transporters associated with multidrug resistance, the kidney cancer cell lines 108, 121, 127, and 143 were treated with depsipeptide and evaluated by quantitative reverse transcription-PCR. Increased levels of MDR1 (1.3- to 6.3-fold) and ABCG2 (3.2- to 11.1-fold) but not MRP1 (0.9- to 1.3-fold) were observed. The induced Pgp transported the fluorescent substrates rhodamine 123, bisantrene, calcein-AM, BODIPY-vinblastine, and BODIPY-paclitaxel. In normal peripheral blood mononuclear cells (PBMC) and circulating tumor cells obtained from patients receiving depsipeptide, increased levels of histone H3 acetylation were found. We next examined MDR1 levels in normal and malignant PBMCs obtained from 15 patients enrolled in clinical trials with depsipeptide and detected up to a 6-fold increase in normal PBMCs and up to an 8-fold increase in circulating tumor cells after depsipeptide administration. In one patient with Sézary syndrome, increased MDR1 gene expression was accompanied by increased cell surface Pgp expression in circulating Sézary cells as determined by measurement of MRK-16 staining by flow cytometry. These studies suggest that depsipeptide induces its own mechanism of resistance and thus provide a basis for clinical trials evaluating depsipeptide in combination with a Pgp inhibitor.