Optineurin provides a mitophagy contact site for TBK1 activation
Ontology highlight
ABSTRACT:
SUBMITTER: Koji Yamano
PROVIDER: S-SCDT-10_1038-S44318-024-00036-1 | biostudies-other |
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Tank-binding kinase 1 (TBK1) is a Ser/Thr kinase that is involved in many intracellular processes, such as innate immunity, cell cycle, and apoptosis. TBK1 is also important for phosphorylating the autophagy adaptors that mediate the selective autophagic removal of damaged mitochondria. However, the mechanism by which PINK1-Parkin-mediated mitophagy activates TBK1 remains largely unknown. Here, we show that the autophagy adaptor optineurin (OPTN) provides a unique platform for TBK1 activation. Both the OPTN-ubiquitin and the OPTN-pre-autophagosomal structure (PAS) interaction axes facilitate assembly of the OPTN-TBK1 complex at a contact sites between damaged mitochondria and the autophagosome formation sites. At this assembly point, a positive feedback loop for TBK1 activation is initiated that accelerates hetero-autophosphorylation of the protein. Expression of monobodies engineered here to bind OPTN impaired OPTN accumulation at contact sites, as well as the subsequent activation of TBK1, thereby inhibiting mitochondrial degradation. Taken together, these data show that a positive and reciprocal relationship between OPTN and TBK1 initiates autophagosome biogenesis on damaged mitochondria.
| S-EPMC10907724 | biostudies-literature
Project description:Mitochondria play an essential role in maintaining cellular homeostasis. The removal of damaged or depolarized mitochondria occurs via mitophagy, in which damaged mitochondria are targeted for degradation via ubiquitination induced by PTEN-induced putative kinase 1 (PINK1) and Parkin. Mitophagy receptors, including optineurin (OPTN), nuclear dot 52 kDa protein (NDP52), and Tax1-binding protein 1 (TAX1BP1), are recruited to mitochondria via ubiquitin binding and mediate autophagic engulfment through their association with microtubule-associated protein light chain 3 (LC3). Here, we use live-cell imaging to demonstrate that OPTN, NDP52, and TAX1BP1 are recruited to mitochondria with similar kinetics following either mitochondrial depolarization or localized generation of reactive oxygen species, leading to sequestration by the autophagosome within ?45 min after insult. Despite this corecruitment, we find that depletion of OPTN, but not NDP52, significantly slows the efficiency of sequestration. OPTN is phosphorylated by the kinase TANK-binding kinase 1 (TBK1) at serine 177; we find that TBK1 is corecruited with OPTN to depolarized mitochondria. Inhibition or depletion of TBK1, or expression of amyotrophic lateral sclerosis (ALS)-associated OPTN or TBK1 mutant blocks efficient autophagosome formation. Together, these results indicate that although there is some functional redundancy among mitophagy receptors, efficient sequestration of damaged mitochondria in response to mitochondrial stress requires both TBK1 and OPTN. Notably, ALS-linked mutations in OPTN and TBK1 can interfere with mitophagy, suggesting that inefficient turnover of damaged mitochondria may represent a key pathophysiological mechanism contributing to neurodegenerative disease.
| S-EPMC4914160 | biostudies-literature
Project description:Mitophagy is a key process regulating mitochondrial quality control. Several mechanisms have been proposed to regulate mitophagy, but these have mostly been studied using stably expressed non-native proteins in immortalized cell lines. In skeletal muscle, mitophagy and its molecular mechanisms require more thorough investigation. To measure mitophagy directly, we generated a stable skeletal muscle C2C12 cell line, expressing a mitophagy reporter construct (mCherry-green fluorescence protein-mtFIS1101-152 ). Here, we report that both carbonyl cyanide m-chlorophenyl hydrazone (CCCP) treatment and adenosine monophosphate activated protein kinase (AMPK) activation by 991 promote mitochondrial fission via phosphorylation of MFF and induce mitophagy by ~20%. Upon CCCP treatment, but not 991, ubiquitin phosphorylation, a read-out of PTEN-induced kinase 1 (PINK1) activity, and Parkin E3 ligase activity toward CDGSH iron sulfur domain 1 (CISD1) were increased. Although the PINK1-Parkin signaling pathway is active in response to CCCP treatment, we observed no change in markers of mitochondrial protein content. Interestingly, our data shows that TANK-binding kinase 1 (TBK1) phosphorylation is increased after both CCCP and 991 treatments, suggesting TBK1 activation to be independent of both PINK1 and Parkin. Finally, we confirmed in non-muscle cell lines that TBK1 phosphorylation occurs in the absence of PINK1 and is regulated by AMPK-dependent signaling. Thus, AMPK activation promotes mitophagy by enhancing mitochondrial fission (via MFF phosphorylation) and autophagosomal engulfment (via TBK1 activation) in a PINK1-Parkin independent manner.
| S-EPMC7212019 | biostudies-literature
Project description:Damaged mitochondria are detrimental to cellular homeostasis. One mechanism for removal of damaged mitochondria involves the PINK1-PARKIN pathway, which poly-ubiquitylates damaged mitochondria to promote mitophagy. We report that assembly of ubiquitin chains on mitochondria triggers autophagy adaptor recruitment concomitantly with activation of the TBK1 kinase, which physically associates with OPTN, NDP52, and SQSTM1. TBK1 activation in HeLa cells requires OPTN and NDP52 and OPTN ubiquitin chain binding. In addition to the known role of S177 phosphorylation in OPTN on ATG8 recruitment, TBK1-dependent phosphorylation on S473 and S513 promotes ubiquitin chain binding in vitro as well as TBK1 activation, OPTN mitochondrial retention, and efficient mitophagy in vivo. These data reveal a self-reinforcing positive feedback mechanism that coordinates TBK1-dependent autophagy adaptor phosphorylation with the assembly of ubiquitin chains on mitochondria to facilitate efficient mitophagy, and mechanistically links genes mutated in Parkinson's disease and amyotrophic lateral sclerosis in a common selective autophagy pathway.
| S-EPMC4592482 | biostudies-literature
Project description:Removal of damaged mitochondria is orchestrated by a pathway involving the PINK1 kinase and the PARKIN ubiquitin ligase. Ubiquitin chains assembled by PARKIN on the mitochondrial outer membrane recruit autophagy cargo receptors in complexes with TBK1 protein kinase. While TBK1 is known to phosphorylate cargo receptors to promote ubiquitin binding, it is unknown whether TBK1 phosphorylates other proteins to promote mitophagy. Using global quantitative proteomics, we identified S72 in RAB7A, a RAB previously linked with mitophagy, as a dynamic target of TBK1 upon mitochondrial depolarization. TBK1 directly phosphorylates RAB7AS72, but not several other RABs known to be phosphorylated on the homologous residue by LRRK2, in vitro, and this modification requires PARKIN activity in vivo. Interaction proteomics using nonphosphorylatable and phosphomimetic RAB7A mutants revealed loss of association of RAB7AS72E with RAB GDP dissociation inhibitor and increased association with the DENN domain-containing heterodimer FLCN-FNIP1. FLCN-FNIP1 is recruited to damaged mitochondria, and this process is inhibited in cells expressing RAB7AS72A. Moreover, nonphosphorylatable RAB7A failed to support efficient mitophagy, as well as recruitment of ATG9A-positive vesicles to damaged mitochondria. These data reveal a novel function for TBK1 in mitophagy, which parallels that of LRRK2-mediated phosphorylation of the homologous site in distinct RABs to control membrane trafficking.
| S-EPMC6314648 | biostudies-literature
Project description:TANK-binding kinase 1 (TBK1) is a multifunctional kinase with an essential role in mitophagy, the selective clearance of damaged mitochondria. More than 90 distinct mutations in TBK1 are linked to amyotrophic lateral sclerosis (ALS) and fronto-temporal dementia, including missense mutations that disrupt the abilities of TBK1 to dimerize, associate with the mitophagy receptor optineurin (OPTN), autoactivate, or catalyze phosphorylation. We investigated how ALS-associated mutations in TBK1 affect Parkin-dependent mitophagy using imaging to dissect the molecular mechanisms involved in clearing damaged mitochondria. Some mutations cause severe dysregulation of the pathway, while others induce limited disruption. Mutations that abolish either TBK1 dimerization or kinase activity were insufficient to fully inhibit mitophagy, while mutations that reduced both dimerization and kinase activity were more disruptive. Ultimately, both TBK1 recruitment and OPTN phosphorylation at S177 are necessary for engulfment of damaged mitochondra by autophagosomal membranes. Surprisingly, we find that ULK1 activity contributes to the phosphorylation of OPTN in the presence of either wild-type or kinase-inactive TBK1. In primary neurons, TBK1 mutants induce mitochondrial stress under basal conditions; network stress is exacerbated with further mitochondrial insult. Our study further refines the model for TBK1 function in mitophagy, demonstrating that some ALS-linked mutations likely contribute to disease pathogenesis by inducing mitochondrial stress or inhibiting mitophagic flux. Other TBK1 mutations exhibited much less impact on mitophagy in our assays, suggesting that cell-type-specific effects, cumulative damage, or alternative TBK1-dependent pathways such as innate immunity and inflammation also factor into the development of ALS in affected individuals.
| S-EPMC8214690 | biostudies-literature
Project description:ER-mitochondria contact sites (EMCSs) are important for mitochondrial function. Here, we have identified a EMCS complex, comprising a family of uncharacterised mitochondrial outer- membrane proteins, TraB1, TraB2 and the ER protein, VAP27-1. In Arabidopsis, there are three TraB isoforms and the trab1a/trab2 double mutant exhibits abnormal mitochondrial morphology, strong starch accumulation and impaired energy metabolism, indicating that these proteins are essential for normal mitochondrial function. Moreover, TraB1 and TraB2 proteins also interact with ATG8 in order to regulate mitochondrial degradation (mitophagy). The turnover of depolarised mitochondria is significantly reduced in both trab1/trab2 and VAP27 mutants (vap27-1,3,4,6) under mitochondrial stress conditions, with an increased population of dysfunctional mitochondria present in the cytoplasm. Consequently, plant recovery after stress is significantly perturbed, suggesting that TraB1 regulated mitophagy and ER-mitochondrial interaction are two closely related processes. Taken together, we ascribe a dual role to TraB1 which is a component of the EMCS complex in eukaryotes, regulating both interaction of the mitochondria to the ER and mitophagy.
2022-08-30 | PXD036285 | Pride
Project description:Contact activation refers to the process of surface-induced activation of factor XII (FXII), which initiates blood coagulation and is captured by the activated partial thromboplastin time (aPTT) assay. Here, we show the mechanism and diagnostic implications of FXII contact activation. Screening of recombinant FXII mutants identified a continuous stretch of residues Gln317-Ser339 that was essential for FXII surface binding and activation, thrombin generation and coagulation. Peptides spanning these 23 residues competed with surface-induced FXII activation. Although FXII mutants lacking residues Gln317-Ser339 were susceptible to activation by plasmin and plasma kallikrein, they were ineffective in supporting arterial and venous thrombus formation in mice. Antibodies raised against the Gln317-Ser339 region induced FXII activation and triggered controllable contact activation in solution leading to thrombin generation by the intrinsic pathway of coagulation. The antibody-activated aPTT allows for standardization of particulate aPTT reagents and for sensitive monitoring of coagulation factors VIII, IX, XI.
| S-EPMC8458485 | biostudies-literature
Project description:Cerebral ischemia is one of the leading causes of human mortality and disability worldwide. The treatment of cerebral ischemia is refractory due to its short therapeutic window and lack of effective clinical drugs. Mitophagy, the autophagic elimination of damaged mitochondria, attenuates neuronal injury in cerebral ischemia, indicating the potential of mitophagy inducers as therapies for cerebral ischemia. We previously determined that, by enhancing autophagy flux, the steroidal alkaloid tomatidine can function as a neuroprotective agent against ischemic injury. However, its effects on mitophagy remain unknown. For this purpose, neuroblastoma cell lines Neuro-2a and SH-SY5Y were subjected to ischemic injury induced by oxygen-glucose deprivation/reperfusion (OGD/R) and then treated with tomatidine. OGD/R induced a general decrease of cellular contents, and this study revealed that tomatidine had no impact on mitophagy. In addition, tomatidine did not affect mitochondrial contents, including translocase of outer mitochondrial membrane 20 and voltage-dependent anion channel 1, in either OGD/R-treated or intact SH-SY5H cells. Our results indicate that tomatidine exhibits its neuroprotective effects by enhancing autophagy, but in a potentially mitophagy-independent manner, and provide insights for further investigation into its mechanism(s) and potential therapeutic use against cerebral ischemia.
| S-EPMC8409302 | biostudies-literature
Project description:Idiopathic pulmonary fibrosis (IPF) results in scarring of the lungs by excessive extracellular matrix (ECM) production. Resident fibroblasts are the major cell type involved in ECM deposition. The biochemical pathways that facilitate pathological fibroblast activation leading to aberrant ECM deposition are not fully understood. Tank binding protein kinase-1 (TBK1) is a kinase that regulates multiple signaling pathways and was recently identified as a candidate regulator of fibroblast activation in a large-scale small-interfering RNA (siRNA) screen. To determine the effect of TBK1 on fibroblast activation, TBK1 was inhibited pharmacologically (MRT-68601) and genetically (siRNA) in normal and IPF human lung fibroblasts. Reducing the activity or expression of TBK1 led to reduction in α-smooth muscle actin stress fiber levels by 40-60% and deposition of ECM components collagen I and fibronectin by 50% in TGF-β-stimulated normal and IPF fibroblasts. YAP and TAZ are homologous mechanoregulatory profibrotic transcription cofactors known to regulate fibroblast activation. TBK1 knockdown or inhibition decreased the total and nuclear protein levels of YAP/TAZ. Additionally, low cell-cell contact and increased ECM substrate stiffness augmented the phosphorylation and activation of TBK1, consistent with cues that regulate YAP/TAZ. The action of TBK1 toward YAP/TAZ activation was independent of LATS1/2 and canonical downstream TBK1 signaling mediator IRF3 but dependent on proteasomal machinery of the cell. This study identifies TBK1 as a fibrogenic activator of human pulmonary fibroblasts, suggesting TBK1 may be a novel therapeutic target in pulmonary fibrosis.
| S-EPMC7272740 | biostudies-literature