bims-apauto Biomed News
on Apoptosis and autophagy
Issue of 2021‒11‒21
nine papers selected by
Su Hyun Lee
Seoul National University

  1. Nat Commun. 2021 Nov 16. 12(1): 6622
      The mammalian target of rapamycin (mTORC1) has been shown to regulate autophagy at different steps. However, how mTORC1 regulates the N-ethylmaleimide-sensitive protein receptor (SNARE) complex remains elusive. Here we show that mTORC1 inhibits formation of the SNARE complex (STX17-SNAP29-VAMP8) by phosphorylating VAMP8, thereby blocking autophagosome-lysosome fusion. A VAMP8 phosphorylation mimic mutant is unable to promote autophagosome-lysosome fusion in vitro. Furthermore, we identify SCFD1, a Sec1/Munc18-like protein, that localizes to the autolysosome and is required for SNARE complex formation and autophagosome-lysosome fusion. VAMP8 promotes SCFD1 recruitment to autolysosomes when dephosphorylated. Consistently, phosphorylated VAMP8 or SCFD1 depletion inhibits autophagosome-lysosome fusion, and expression of phosphomimic VAMP8 leads to increased lipid droplet accumulation when expressed in mouse liver. Thus, our study supports that mTORC1-mediated phosphorylation of VAMP8 blocks SCFD1 recruitment, thereby inhibiting STX17-SNAP29-VAMP8 complex formation and autophagosome-lysosome fusion.
  2. Autophagy. 2021 Nov 19. 1-11
      PINK1 accumulation at the outer mitochondrial membrane (OMM) is a key event required to signal depolarized mitochondria to the autophagy machinery. How this early step is, in turn, modulated by autophagy proteins remains less characterized. Here, we show that, upon mitochondrial depolarization, the proautophagic protein AMBRA1 is recruited to the OMM and interacts with PINK1 and ATAD3A, a transmembrane protein that mediates mitochondrial import and degradation of PINK1. Downregulation of AMBRA1 expression results in reduced levels of PINK1 due to its enhanced degradation by the mitochondrial protease LONP1, which leads to a decrease in PINK1-mediated ubiquitin phosphorylation and mitochondrial PRKN/PARKIN recruitment. Notably, ATAD3A silencing rescues defective PINK1 accumulation in AMBRA1-deficient cells upon mitochondrial damage. Overall, our findings underline an upstream contribution of AMBRA1 in the control of PINK1-PRKN mitophagy by interacting with ATAD3A and promoting PINK1 stability. This novel regulatory element may account for changes of PINK1 levels in neuropathological conditions.
    Keywords:  Autophagy; LONP1; PRKN/PARKIN; TOMM complex; ubiquitin phosphorylation
  3. J Biol Chem. 2021 Nov 11. pii: S0021-9258(21)01212-6. [Epub ahead of print] 101405
      Several amyotrophic lateral sclerosis (ALS)-related proteins such as FUS, TDP-43, and hnRNPA1 demonstrate liquid-liquid phase separation, and their disease-related mutations correlate with a transition of their liquid droplet form into aggregates. Missense mutations in SQSTM1/p62, which have been identified throughout the gene, are associated with ALS, frontotemporal degeneration (FTD), and Paget's disease of bone. SQSTM1/p62 protein forms liquid droplets through interaction with ubiquitinated proteins, and these droplets serve as a platform for autophagosome formation and the anti-oxidative stress response via the LC3-interacting region (LIR) and KEAP1-interacting region (KIR) of p62, respectively. However, it remains unclear whether ALS/FTD-related p62 mutations in the LIR and KIR disrupt liquid droplet formation leading to defects in autophagy, the stress response, or both. To evaluate the effects of ALS/FTD-related p62 mutations in the LIR and KIR on a major oxidative stress system, the Keap1-Nrf2 pathway, as well as on autophagic turnover, we developed systems to monitor each of these with high sensitivity. These methods such as intracellular protein-protein interaction assay, doxycycline-inducible gene expression system and gene expression into primary cultured cells with recombinant adenovirus revealed that some mutants, but not all, caused reduced NRF2 activation and delayed autophagic cargo turnover. In contrast, while all p62 mutants demonstrated sufficient ability to form liquid droplets, all of these droplets also exhibited reduced inner fluidity. These results indicate that like other ALS-related mutant proteins, p62 missense mutations result in a primary defect in ALS/FTD via a qualitative change in p62 liquid droplet fluidity.
    Keywords:  NRF2; amyotrophic lateral sclerosis; autophagy; liquid droplet; p62
  4. Mol Cell. 2021 Nov 18. pii: S1097-2765(21)00931-X. [Epub ahead of print]
      Autophagy is a conserved intracellular degradation pathway exerting various cytoprotective and homeostatic functions by using de novo double-membrane vesicle (autophagosome) formation to target a wide range of cytoplasmic material for vacuolar/lysosomal degradation. The Atg1 kinase is one of its key regulators, coordinating a complex signaling program to orchestrate autophagosome formation. Combining in vitro reconstitution and cell-based approaches, we demonstrate that Atg1 is activated by lipidated Atg8 (Atg8-PE), stimulating substrate phosphorylation along the growing autophagosomal membrane. Atg1-dependent phosphorylation of Atg13 triggers Atg1 complex dissociation, enabling rapid turnover of Atg1 complex subunits at the pre-autophagosomal structure (PAS). Moreover, Atg1 recruitment by Atg8-PE self-regulates Atg8-PE levels in the growing autophagosomal membrane by phosphorylating and thus inhibiting the Atg8-specific E2 and E3. Our work uncovers the molecular basis for positive and negative feedback imposed by Atg1 and how opposing phosphorylation and dephosphorylation events underlie the spatiotemporal regulation of autophagy.
    Keywords:  Atg8 lipidation; autophagy; metabolism; phosphorylation; protein kinases; protein phosphatases; signaling; ubiquitin-like proteins
  5. Proc Natl Acad Sci U S A. 2021 Nov 23. pii: e2110755118. [Epub ahead of print]118(47):
      Nondegradative ubiquitin chains attached to specific targets via Lysine 63 (K63) residues have emerged to play a fundamental role in synaptic function. The K63-specific deubiquitinase CYLD has been widely studied in immune cells and lately also in neurons. To better understand if CYLD plays a role in brain and synapse homeostasis, we analyzed the behavioral profile of CYLD-deficient mice. We found that the loss of CYLD results in major autism-like phenotypes including impaired social communication, increased repetitive behavior, and cognitive dysfunction. Furthermore, the absence of CYLD leads to a reduction in hippocampal network excitability, long-term potentiation, and pyramidal neuron spine numbers. By providing evidence that CYLD can modulate mechanistic target of rapamycin (mTOR) signaling and autophagy at the synapse, we propose that synaptic K63-linked ubiquitination processes could be fundamental in understanding the pathomechanisms underlying autism spectrum disorder.
    Keywords:  CYLD; autism spectrum disorder; autophagy; mTOR signaling; synapse
  6. Inflammopharmacology. 2021 Nov 18.
      The ubiquitin-proteasome pathway is responsible for the turnover of different cellular proteins, such as transport proteins, presentation of antigens to the immune system, control of the cell cycle, and activities that promote cancer. The enzymes which remove ubiquitin, deubiquitylating enzymes (DUBs), play a critical role in central and peripheral immune tolerance to prevent the development of autoimmune diseases and thus present a potential therapeutic target for the treatment of autoimmune diseases. DUBs function by removing ubiquitin(s) from target protein and block ubiquitin chain elongation. The addition and removal of ubiquitin molecules have a significant impact on immune responses. DUBs and E3 ligases both specifically cleave target protein and modulate protein activity and expression. The balance between ubiquitylation and deubiquitylation modulates protein levels and also protein interactions. Dysregulation of the ubiquitin-proteasome pathway results in the development of various autoimmune diseases such as inflammatory bowel diseases (IBD), psoriasis, multiple sclerosis (MS), systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). This review summarizes the current understanding of ubiquitination in autoimmune diseases and focuses on various DUBs responsible for the progression of autoimmune diseases.
    Keywords:  Autoimmune diseases; Deubiquitination; Deubiquitylating enzyme; E3 ligases; Ubiquitin proteasome system; Ubiquitination
  7. Autophagy. 2021 Nov 15. 1-3
      ER-specific autophagy (reticulophagy) has emerged as a critical degradative route for misfolded secretory proteins. Our previous work showed that RTN3 (reticulon 3) drives reticulophagic clearance of disease-causing mutant prohormones. How RTN3, a protein residing on the cytosolic leaflet of the ER bilayer, recruits these lumenally-localized cargos has remained a mystery. To address this question, we used an unbiased proteomics approach to identify RTN3-interacting partners. We discovered that RTN3 recruits misfolded prohormones for lysosomal degradation through the ER transmembrane protein PGRMC1. RTN3 complexes with PGRMC1, which directly binds to misfolded prohormones via its distal ER lumenal domain. Cargos for the RTN3-PGRMC1 degradative axis include mutant POMC (proopiomelanocortin) and proinsulin, each of which oligomerizes in the ER during misfolding, entrapping their wild-type counterparts, leading to secretion defects. Although reticulophagy is thought to degrade large protein aggregates, PGRMC1 instead selectively recruits and promotes degradation of only small oligomers of the mutant prohormones. Of physiological importance, genetic or pharmacological inactivation of PGRMC1 in pancreatic β-cells expressing both wild-type and mutant proinsulin impairs mutant proinsulin turnover and promotes trafficking of wild-type proinsulin. These findings pinpoint PGRMC1 as a possible intervention point for diseases caused by ER protein retention.
    Keywords:  MIDY; Reticulophagy; diabetes; endoplasmic reticulum; protein misfolding; protein trafficking
  8. Autophagy. 2021 Nov 15. 1-16
      Modulation of the host cell cycle has emerged as a common theme among the pathways regulated by bacterial pathogens, arguably to promote host cell colonization. However, in most cases the exact benefit ensuing from such interference to the infection process remains unclear. Previously, we have shown that Salmonella actively induces G2/M arrest of host cells, and that infection is severely inhibited in cells arrested in G1. In this study, we demonstrate that Salmonella vacuolar replication is inhibited in host cells blocked in G1, whereas the cytosolic replication of the closely related pathogen Shigella is not affected. Mechanistically, we show that cells arrested in G1, but not cells arrested in G2, present dysregulated endolysosomal trafficking, displaying an abnormal accumulation of vesicles positive for late endosomal and lysosomal markers. In addition, the macroautophagic/autophagic flux and degradative lysosomal function are strongly impaired. This endolysosomal trafficking dysregulation results in sustained activation of the SPI-1 type III secretion system and lack of vacuole repair by the autophagy pathway, ultimately compromising the maturation and integrity of the Salmonella-containing vacuole. As such, Salmonella is released in the host cytosol. Collectively, our findings demonstrate that the modulation of the host cell cycle occurring during Salmonella infection is related to a disparity in the permissivity of cells arrested in G1 and G2/M, due to their intrinsic characteristics.
    Keywords:  Autophagy; G1 arrest; Salmonella; Salmonella cytosolic replication; Salmonella-containing vacuole; cell cycle; endolysosomal trafficking; type III secretion system
  9. Nat Commun. 2021 Nov 19. 12(1): 6750
      The multispanning membrane protein ATG9A is a scramblase that flips phospholipids between the two membrane leaflets, thus contributing to the expansion of the phagophore membrane in the early stages of autophagy. Herein, we show that depletion of ATG9A does not only inhibit autophagy but also increases the size and/or number of lipid droplets in human cell lines and C. elegans. Moreover, ATG9A depletion blocks transfer of fatty acids from lipid droplets to mitochondria and, consequently, utilization of fatty acids in mitochondrial respiration. ATG9A localizes to vesicular-tubular clusters (VTCs) that are tightly associated with an ER subdomain enriched in another multispanning membrane scramblase, TMEM41B, and also in close proximity to phagophores, lipid droplets and mitochondria. These findings indicate that ATG9A plays a critical role in lipid mobilization from lipid droplets to autophagosomes and mitochondria, highlighting the importance of ATG9A in both autophagic and non-autophagic processes.