bims-apauto Biomed News
on Apoptosis and autophagy
Issue of 2022‒04‒17
four papers selected by
Su Hyun Lee
Seoul National University

  1. Autophagy. 2022 Apr 13. 1-19
      Externalization of the phospholipid cardiolipin (CL) to the outer mitochondrial membrane has been proposed to act as a mitophagy trigger. CL would act as a signal for binding the LC3 macroautophagy/autophagy proteins. As yet, the behavior of the LC3-subfamily members has not been directly compared in a detailed way. In the present contribution, an analysis of LC3A, LC3B and LC3C interaction with CL-containing model membranes, and of their ability to translocate to mitochondria, is described. Binding of LC3A to CL was stronger than that of LC3B; both proteins showed a similar ability to colocalize with mitochondria upon induction of CL externalization in SH-SY5Y cells. Besides, the double silencing of LC3A and LC3B proteins was seen to decrease CCCP-induced mitophagy. Residues 14 and 18 located in the N-terminal region of LC3A were shown to be important for its recognition of damaged mitochondria during rotenone- or CCCP-induced mitophagy. Moreover, the in vitro results suggested a possible role of LC3A, but not of LC3B, in oxidized-CL recognition as a counterweight to excessive apoptosis activation. In the case of LC3C, even if this protein showed a stronger CL binding than LC3B or LC3A, the interaction was less specific, and colocalization of LC3C with mitochondria was not rotenone dependent. These results suggest that, at variance with LC3A, LC3C does not participate in cargo recognition during CL-mediated-mitophagy. The data support the notion that the various LC3-subfamily members might play different roles during autophagy initiation, identifying LC3A as a novel stakeholder in CL-mediated mitophagy. Abbreviations: ACTB/β-actin: actin beta; Atg8: autophagy-related 8; CL: cardiolipin; CCCP: carbonyl cyanide m-chlorophenyl hydrazone; DMSO: dimethyl sulfoxide; DOPE: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DTT: DL-dithiothreitol; FKBP8: FKBP prolyl isomerase 8; GABARAP: GABA type A receptor associated protein; GABARAPL1: GABA type A receptor associated protein like 1; GABARAPL2: GABA type A receptor associated protein like 2; GFP: green fluorescent protein; IMM: inner mitochondrial membrane; LUV/LUVs: large unilamellar vesicle/s; MAP1LC3A/LC3A: microtubule associated protein 1 light chain 3 alpha; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAP1LC3C/LC3C: microtubule associated protein 1 light chain 3 gamma; NME4/NDPK-D/Nm23-H4: NME/NM23 nucleoside diphosphate kinase 4; O/A: oligomycin A + antimycin A; OMM: outer mitochondrial membrane; PA: phosphatidic acid; PC: phosphatidylcholine; PG: phosphatidylglycerol; PINK1: PTEN induced putative kinase 1; PtdIns4P: phosphatidylinositol-4-phosphate; Rho-PE: lissamine rhodamine phosphatidylethanolamine; SUV/SUVs: small unilamellar vesicle/s.
    Keywords:  Atg8; LC3/GABARAP-protein family; autophagosome; autophagy cargo recognition; lipid oxidation; lipid-protein interaction; membrane curvature; mitochondria; negatively charged phospholipids
  2. Autophagy. 2022 Apr 10. 1-21
      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.
    Keywords:  Autophagy; degradation; ferroptosis; inhibitor; mechanotransduction; pancreas; phosphorylation
  3. Cell Death Dis. 2022 Apr 14. 13(4): 348
      TNF receptor-associated factor 6 (TRAF6)-BECN1 signaling axis plays a pivotal role in autophagy induction through ubiquitination of BECN1, thereby inducing lung cancer migration and invasion in response to toll-like receptor 4 (TLR4) stimulation. Herein, we provide novel molecular and cellular mechanisms involved in the negative effect of ubiquitin-specific peptidase 15 (USP15) on lung cancer progression. Clinical data of the TCGA and primary non-small cell lung cancer (NSCLC) patients (n = 41) revealed that the expression of USP15 was significantly downregulated in lung cancer patients. Importantly, USP15-knockout (USP15KO) A549 and USP15KO H1299 lung cancer cells generated with CRISPR-Cas9 gene-editing technology showed increases in cancer migration and invasion with enhanced autophagy induction in response to TLR4 stimulation. In addition, biochemical studies revealed that USP15 interacted with BECN1, but not with TRAF6, and induced deubiquitination of BECN1, thereby attenuating autophagy induction. Notably, in primary NSCLC patients (n = 4) with low expression of USP15, 10 genes (CCNE1, MMP9, SFN, UBE2C, CCR2, FAM83A, ETV4, MYO7A, MMP11, and GSDMB) known to promote lung cancer progression were significantly upregulated, whereas 10 tumor suppressor genes (FMO2, ZBTB16, FCN3, TCF21, SFTPA1B, HPGD, SOSTDC1, TMEM100, GDF10, and WIF1) were downregulated, providing clinical relevance of the functional role of USP15 in lung cancer progression. Taken together, our data demonstrate that USP15 can negatively regulate the TRAF6-BECN1 signaling axis for autophagy induction. Thus, USP15 is implicated in lung cancer progression.
  4. Sci Rep. 2022 Apr 11. 12(1): 6063
      The E3 ubiquitin ligase HERC2 has been linked to neurological diseases and cancer, however it remains a poorly characterized human protein. Here, we show that the ZZ domain of HERC2 (HERC2ZZ) recognizes a mimetic of the Nt-R cargo degradation signal. NMR titration experiments and mutagenesis results reveal that the Nt-R mimetic peptide occupies a well-defined binding site of HERC2ZZ comprising of the negatively charged aspartic acids. We report the crystal structure of the DOC domain of HERC2 (HERC2DOC) that is adjacent to HERC2ZZ and show that a conformational rearrangement in the protein may occur when the two domains are linked. Immunofluorescence microscopy data suggest that the stimulation of autophagy promotes targeting of HERC2 to the proteasome. Our findings suggest a role of cytosolic HERC2 in the ubiquitin-dependent degradation pathways.