Autophagy
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The ubiquitination of mitochondrial proteins labels damaged mitochondria for degradation through mitophagy. We recently developed an in vivo system in which mitophagy is slowed by inhibiting mitochondrial division through knockout of Dnm1l/Drp1, a dynamin related GTPase that mediates mitochondrial division. Using this system, we revealed that the ubiquitination of mitochondrial proteins required SQSTM1/p62, but not the ubiquitin E3 ligase PRKN/parkin, during mitophagy. ⋯ These data suggest that mitochondrial ubiquitination is promoted by SQSTM1 independently of PINK1 and PRKN during mitophagy. PINK1 and PRKN appear to control the balance between mitochondrial division and fusion in vivo. Abbreviations: DNM1L/DRP1: dynamin 1-like; KEAP1: kelch-like ECH-associated protein 1; KO: knockout; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MFN1/2: mitofusin 1/2; OPA1: OPA1, mitochondrial dynamin like GTPase; PDH: pyruvate dehydrogenase E1; PINK1: PTEN induced putative kinase 1; PRKN/parkin: parkin RBR E3 ubiquitin protein ligase.
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Comment
Mitochondrial transport proteins RHOT1 and RHOT2 serve as docking sites for PRKN-mediated mitophagy.
The Parkinson disease-associated proteins PINK1 and PRKN coordinate the ubiquitination of mitochondrial outer membrane proteins to tag them either for degradation or for autophagic clearance of the mitochondrion. The proteins include the mitochondrial trafficking proteins RHOT1 and RHOT2, the removal of which may be required for immobilization of mitochondria prior to mitophagy. ⋯ RHOT1, and likely also RHOT2, may act as a docking site for inactive PRKN prior to mitochondrial damage, thus keeping PRKN in close proximity to its potential substrates and thereby facilitating mitophagy. We also show that RHOT1 functions as a calcium-sensing docking site for PRKN, and we suggest that calcium binding to RHOT is a key step in the calcium-dependent activation of mitophagy machinery.
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Cigarette smoke (CS)-induced accumulation of mitochondrial damage has been widely implicated in chronic obstructive pulmonary disease (COPD) pathogenesis. Mitophagy plays a crucial role in eliminating damaged mitochondria, and is governed by the PINK1 (PTEN induced putative protein kinase 1)-PRKN (parkin RBR E3 ubiquitin protein ligase) pathway. Although both increased PINK1 and reduced PRKN have been implicated in COPD pathogenesis in association with mitophagy, there are conflicting reports for the role of mitophagy in COPD progression. ⋯ Conversely PINK1 overexpression failed to recover impaired mitophagy caused by PRKN knockdown, indicating that PRKN protein levels can be the rate-limiting factor in PINK1-PRKN-mediated mitophagy during CSE exposure. These results suggest that PRKN levels may play a pivotal role in COPD pathogenesis by regulating mitophagy, suggesting that PRKN induction could mitigate the progression of COPD. Abbreviations: AD: Alzheimer disease; AEC: airway epithelial cells; BALF: bronchoalveolar lavage fluid; AKT: AKT serine/threonine kinase; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CDKN1A: cyclin dependent kinase inhibitor 1A; CDKN2A: cyclin dependent kinase inhibitor 2A; COPD: chronic obstructive pulmonary disease; CS: cigarette smoke; CSE: CS extract; CXCL1: C-X-C motif chemokine ligand 1; CXCL8: C-X-C motif chemokine ligand 8; HBEC: human bronchial epithelial cells; 4-HNE: 4-hydroxynonenal; IL: interleukin; KO: knockout; LF: lung fibroblasts; LPS: lipopolysaccharide; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; 8-OHdG: 8-hydroxy-2'-deoxyguanosine; OPTN: optineurin; PRKN: parkin RBR E3 ubiquitin protein ligase; PCD: programmed cell death; PFD: pirfenidone; PIK3C: phosphatidylinositol-4:5-bisphosphate 3-kinase catalytic subunit; PINK1: PTEN induced putative kinase 1; PTEN: phosphatase and tensin homolog; RA: rheumatoid arthritis; ROS: reactive oxygen species; SA-GLB1/β-Gal: senescence-associated-galactosidase, beta 1; SASP: senescence-associated secretory phenotype; SNP: single nucleotide polymorphism; TNF: tumor necrosis factor.
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The thyroid hormone triiodothyronine (T3) activates thermogenesis by uncoupling electron transport from ATP synthesis in brown adipose tissue (BAT) mitochondria. Although T3 can induce thermogenesis by sympathetic innervation, little is known about its cell autonomous effects on BAT mitochondria. We thus examined effects of T3 on mitochondrial activity, autophagy, and metabolism in primary brown adipocytes and BAT and found that T3 increased fatty acid oxidation and mitochondrial respiration as well as autophagic flux, mitophagy, and mitochondrial biogenesis. ⋯ In summary, T3 has direct effects on mitochondrial autophagy, activity, and turnover in BAT that are essential for thermogenesis. Stimulation of BAT activity by thyroid hormone or its analogs may represent a potential therapeutic strategy for obesity and metabolic diseases. Abbreviations: ACACA: acetyl-Coenzyme A carboxylase alpha; AMPK: AMP-activated protein kinase; Acsl1: acyl-CoA synthetase long-chain family member 1; ATG5: autophagy related 5; ATG7: autophagy related 7; ATP: adenosine triphosphate; BAT: brown adipose tissue; cKO: conditional knockout; COX4I1: cytochrome c oxidase subunit 4I1; Cpt1b: carnitine palmitoyltransferase 1b, muscle; CQ: chloroquine; DAPI: 4',6-diamidino-2-phenylindole; DIO2: deiodinase, iodothyronine, type 2; DMEM: Dulbecco's modified Eagle's medium; EIF4EBP1: eukaryotic translation initiation factor 4E binding protein 1; Fabp4: fatty acid binding protein 4, adipocyte; FBS: fetal bovine serum; FCCP: carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone; FGF: fibroblast growth factor; FOXO1: forkhead box O1; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; Gpx1: glutathione peroxidase 1; Lipe: lipase, hormone sensitive; MAP1LC3B: microtubule-associated protein 1 light chain 3; mRNA: messenger RNA; MTORC1: mechanistic target of rapamycin kinase complex 1; NAD: nicotinamide adenine dinucleotide; Nrf1: nuclear respiratory factor 1; OCR: oxygen consumption rate; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; PPARGC1A: peroxisome proliferative activated receptor, gamma, coactivator 1 alpha; Pnpla2: patatin-like phospholipase domain containing 2; Prdm16: PR domain containing 16; PRKA: protein kinase, AMP-activated; RPS6KB: ribosomal protein S6 kinase; RFP: red fluorescent protein; ROS: reactive oxygen species; SD: standard deviation; SEM: standard error of the mean; siRNA: small interfering RNA; SIRT1: sirtuin 1; Sod1: superoxide dismutase 1, soluble; Sod2: superoxide dismutase 2, mitochondrial; SQSTM1: sequestosome 1; T3: 3,5,3'-triiodothyronine; TFEB: transcription factor EB; TOMM20: translocase of outer mitochondrial membrane 20; UCP1: uncoupling protein 1 (mitochondrial, proton carrier); ULK1: unc-51 like kinase 1; VDAC1: voltage-dependent anion channel 1; WAT: white adipose tissue.
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Macroautophagy/autophagy is emerging as an important process in adult muscle stem cells functions: it regulates metabolic reprogramming during activation from a quiescent state, maintains stemness and prevents senescence. We now show that autophagy is specifically required for neonatal myogenesis and muscle development. Specific deletion of Atg7 in PAX7+ (paired box 7) precursors led in mice to a dwarf phenotype, with an effect restricted to the neonatal phase of muscle development. ⋯ Lower levels of DDIT3 were responsible for reduced GHR expression leading to impaired local production of IGF1. Our results conclusively identify a novel autophagy-dependent pathway that regulates nSC behavior and indicate that autophagy is required for skeletal muscle development in the neonatal phase. Abbreviations: AKT/protein kinase B: Thymoma viral proto-oncogene; ASCs: adult stem cells; ATF4: activating transcription factor 4; ATG7: autophagy related 7; BAT: brown adipose tissue; BMP: bone morphogenetic protein; CEBPB: CCAAT/enhancer binding protein (C/EBP), beta; CSA: cross sectional area; CTNNB1: catenin (cadherin associated protein), beta 1; DDIT3: DNA-damage inducible transcript 3; DM: differentiation medium; E: embryonic stage; EIF2AK3/PERK; EIF4EBP1: eukaryotic translation initiation factor 2 alpha kinase 3; eukaryotic translation initiation factor 4E binding protein 1; ER: endoplasmic reticulum; FGF21: fibroblast growth factor 21; GH: growth hormone; GHR: growth hormone receptor; HSCs: hematopoietic stem cells; IGF1: insulin-like growth factor 1; ITGAM: integrin alpha M; KEAP1: kelch-like ECH-associated protein 1; LY6A/Sca-1; MAP1LC3: lymphocyte antigen 6 complex, locus A; microtubule-associated protein 1 light chain 3; MAPK1/ERK2: mitogen-activated protein kinase 1; MAPK3/ERK1: mitogen-activated protein kinase 3; miRNAs: microRNAs; MSCs: mesenchymal stem cells; MTOR: mechanistic target of rapamycin kinase; mtUPR: mitochondrial unfolded protein response; MYF5: myogenic factor 5; MYH: myosin, heavy polypeptide; MYOD1: myogenic differentiation 1; MYOG: myogenin; NFE2L2: nuclear factor, erythroid derived 2, like 2; nSC: neonatal satellite cells; NSCs: neuronal stem cells; P: postnatal day; PAX7: paired box 7; PECAM1: platelet/endothelial cell adhesion molecule 1; PPARG: peroxisome proliferator activated receptor gamma; PTPRC: protein tyrosine phosphatase, receptor type, C; ROS: reactive oxygen species; RPS6: ribosomal protein S6; SCs: adult satellite cells; SQSTM1: sequestosome 1; STAT5: signal transducer and activator of transcription 5; TGFB1: transforming growth factor beta 1; WAT: white adipose tissue; WT: wild type.