bims-apauto Biomed News
on Apoptosis and autophagy
Issue of 2021‒11‒28
nine papers selected by
Su Hyun Lee
Seoul National University

  1. Nat Commun. 2021 Nov 22. 12(1): 6783
      The ancestral origins of the lytic cell death mode, necroptosis, lie in host defense. However, the dysregulation of necroptosis in inflammatory diseases has led to widespread interest in targeting the pathway therapeutically. This mode of cell death is executed by the terminal effector, the MLKL pseudokinase, which is licensed to kill following phosphorylation by its upstream regulator, RIPK3 kinase. The precise molecular details underlying MLKL activation are still emerging and, intriguingly, appear to mechanistically-diverge between species. Here, we report the structure of the human RIPK3 kinase domain alone and in complex with the MLKL pseudokinase. These structures reveal how human RIPK3 structurally differs from its mouse counterpart, and how human RIPK3 maintains MLKL in an inactive conformation prior to induction of necroptosis. Residues within the RIPK3:MLKL C-lobe interface are crucial to complex assembly and necroptotic signaling in human cells, thereby rationalizing the strict species specificity governing RIPK3 activation of MLKL.
  2. Autophagy. 2021 Nov 25. 1-21
      ABBREVIATIONS: ALDOA: aldolase A; AMPK: AMP-activated protein kinase; ATG: autophagy related; ATG5: autophagy related 5; ATP: adenosine triphosphate; BMDMs: bone marrow-derived macrophages; CALCOCO2: calcium binding and coiled-coil domain 2; CASP1: caspase 1; CQ: chloroquine; FOXO3: forkhead box O3; IL1B: interleukin 1 beta; LPS: lipopolysaccharide; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; MT: mutant; mtDNA: mitochondrial DNA; MTORC1: mechanistic target of rapamycin kinase complex 1; mtROS: mitochondrial reactive oxygen species; NLRP3: NLR family, pyrin domain containing 3; OPTN: optineurin; PBS: phosphate-buffered saline; PRKN/Parkin: parkin RBR E3 ubiquitin protein ligase; SN: supernatant; SQSTM1/p62: sequestosome 1; STK11/LKB1: serine/threonine kinase 11; TOMM20: translocase of outer mitochondrial membrane 20; ULK1: unc-51 like autophagy activating kinase 1; v-ATPase: vacuolar type H+-ATPase; WT: wild-type.
    Keywords:  ALDOA; AMPK; LYG-202; NLRP3 inflammasome; mitophagy
  3. Autophagy. 2021 Nov 26. 1-21
      Macroautophagy/autophagy is a highly conserved nutrient-recycling pathway that eukaryotes utilize to combat diverse stresses including nutrient depletion. Dysregulation of autophagy disrupts cellular homeostasis leading to starvation susceptibility in yeast and disease development in humans. In yeast, the robust autophagy response to starvation is controlled by the upregulation of ATG genes, via regulatory processes involving multiple levels of gene expression. Despite the identification of several regulators through genetic studies, the predominant mechanism of regulation modulating the autophagy response to subtle differences in nutrient status remains undefined. Here, we report the unexpected finding that subtle changes in nutrient availability can cause large differences in autophagy flux, governed by hitherto unknown post-transcriptional regulatory mechanisms affecting the expression of the key autophagyinducing kinase Atg1 (ULK1/ULK2 in mammals). We have identified two novel post-transcriptional regulators of ATG1 expression, the kinase Rad53 and the RNA-binding protein Ded1 (DDX3 in mammals). Furthermore, we show that DDX3 regulates ULK1 expression post-transcriptionally, establishing mechanistic conservation and highlighting the power of yeast biology in uncovering regulatory mechanisms that can inform therapeutic approaches.
    Keywords:  ATG1; Amino acid starvation; DDX3; DED1; RAD53; ULK1; autophagosome; autophagy
  4. Cells. 2021 Nov 11. pii: 3124. [Epub ahead of print]10(11):
      Phosphatidylinositol-3-phosphate (PtdIns(3)P) is essential for cell survival, and its intracellular synthesis is spatially and temporally regulated. It has major roles in two distinctive cellular pathways, namely, the autophagy and endocytic pathways. PtdIns(3)P is synthesized from phosphatidylinositol (PtdIns) by PIK3C3C/VPS34 in mammals or Vps34 in yeast. Pathway-specific VPS34/Vps34 activity is the consequence of the enzyme being incorporated into two mutually exclusive complexes: complex I for autophagy, composed of VPS34/Vps34-Vps15/Vps15-Beclin 1/Vps30-ATG14L/Atg14 (mammals/yeast), and complex II for endocytic pathways, in which ATG14L/Atg14 is replaced with UVRAG/Vps38 (mammals/yeast). Because of its involvement in autophagy, defects in which are closely associated with human diseases such as cancer and neurodegenerative diseases, developing highly selective drugs that target specific VPS34/Vps34 complexes is an essential goal in the autophagy field. Recent studies on the activation mechanisms of VPS34/Vps34 complexes have revealed that a variety of factors, including conformational changes, lipid physicochemical parameters, upstream regulators, and downstream effectors, greatly influence the activity of these complexes. This review summarizes and highlights each of these influences as well as clarifying key questions remaining in the field and outlining future perspectives.
    Keywords:  ATG14L; Beclin 1; PtdIns(3)P; UVRAG; VPS15; VPS34; autophagy; endocytic pathway; lipids; membranes
  5. Autophagy. 2021 Nov 22. 1-19
      Sjögren syndrome (SS) is a chronic and progressive autoimmune disease characterized by dry mouth and dry eyes, and characteristic autoantibodies. Evidence of altered macroautophagy/autophagy and apoptosis has been associated with SS, but a mechanistic understanding of the gene expression changes associated with these abnormal processes has not been realized. Recently, increased LAMP3 (lysosomal associated membrane protein 3) expression was found in a subset of SS patients and was associated with increased apoptosis and autoantigen accumulation and release. To better understand how LAMP3 expression might modulate apoptosis, cell biology, and biochemical studies were used to examine the effect of LAMP3 expression in minor salivary gland cells. LAMP3 expression resulted in degradation of LAMP1 increasing lysosomal membrane permeabilization and relocalization of cathepsins to the cytoplasm, resulting in destabilizing autophagic flux and caspase activation. These findings highlight the central role of LAMP3 expression in the pathogenesis of SS.
    Keywords:  Apoptosis; Sjögren syndrome; autophagy; lysosomal membrane permeabilization; lysosome-associated membrane protein 3; salivary gland
  6. Mol Cell. 2021 Nov 12. pii: S1097-2765(21)00947-3. [Epub ahead of print]
      Deubiquitinases (DUBs) are specialized proteases that remove ubiquitin from substrates or cleave within ubiquitin chains to regulate ubiquitylation and therefore play important roles in eukaryotic biology. Dysregulation of DUBs is implicated in several human diseases, highlighting the importance of DUB function. In addition, many pathogenic bacteria and viruses encode and deploy DUBs to manipulate host immune responses and establish infectious diseases in humans and animals. Hence, therapeutic targeting of DUBs is an increasingly explored area that requires an in-depth mechanistic understanding of human and pathogenic DUBs. In this review, we summarize the multiple layers of regulation that control autoinhibition, activation, and substrate specificity of DUBs. We discuss different strategies to inhibit DUBs and the progress in developing selective small-molecule DUB inhibitors. Finally, we propose a classification system of DUB inhibitors based on their mode of action.
    Keywords:  COVID-19; DUB inhibitors; cancer; enzyme regulation; infectious disease; polyubiquitin; signal transduction; structural biology; ubiquitin signaling; ubiquitylation
  7. Autophagy. 2021 Nov 25. 1-21
      Alphaherpesvirus infection results in severe health consequences in a wide range of hosts. USPs are the largest subfamily of deubiquitinating enzymes that play critical roles in immunity and other cellular functions. To investigate the role of USPs in alphaherpesvirus replication, we assessed 13 USP inhibitors for PRV replication. Our data showed that all the tested compounds inhibited PRV replication, with the USP14 inhibitor b-AP15 exhibiting the most dramatic effect. Ablation of USP14 also influenced PRV replication, whereas replenishment of USP14 in USP14 null cells restored viral replication. Although inhibition of USP14 induced the K63-linked ubiquitination of PRV VP16 protein, its degradation was not dependent on the proteasome. USP14 directly bound to ubiquitin chains on VP16 through its UBL domain during the early stage of viral infection. Moreover, USP14 inactivation stimulated EIF2AK3/PERK- and ERN1/IRE1-mediated signaling pathways, which were responsible for VP16 degradation through SQSTM1/p62-mediated selective macroautophagy/autophagy. Ectopic expression of non-ubiquitinated VP16 fully rescued PRV replication. Challenge of mice with b-AP15 activated ER stress and autophagy and inhibited PRV infection in vivo. Our results suggested that USP14 was a potential therapeutic target to treat alphaherpesvirus-induced infectious diseases.
    Keywords:  Alphaherpesvirus; ER stress; PRV VP16; USP14; selective autophagy
  8. PLoS One. 2021 ;16(11): e0259313
      Lysosomes are terminal, degradative organelles of the endosomal pathway that undergo repeated fusion-fission cycles with themselves, endosomes, phagosomes, and autophagosomes. Lysosome number and size depends on balanced fusion and fission rates. Thus, conditions that favour fusion over fission can reduce lysosome numbers while enlarging their size. Conversely, favouring fission over fusion may cause lysosome fragmentation and increase their numbers. PIKfyve is a phosphoinositide kinase that generates phosphatidylinositol-3,5-bisphosphate to modulate lysosomal functions. PIKfyve inhibition causes an increase in lysosome size and reduction in lysosome number, consistent with lysosome coalescence. This is thought to proceed through reduced lysosome reformation and/or fission after fusion with endosomes or other lysosomes. Previously, we observed that photo-damage during live-cell imaging prevented lysosome coalescence during PIKfyve inhibition. Thus, we postulated that lysosome fusion and/or fission dynamics are affected by reactive oxygen species (ROS). Here, we show that ROS generated by various independent mechanisms all impaired lysosome coalescence during PIKfyve inhibition and promoted lysosome fragmentation during PIKfyve re-activation. However, depending on the ROS species or mode of production, lysosome dynamics were affected distinctly. H2O2 impaired lysosome motility and reduced lysosome fusion with phagosomes, suggesting that H2O2 reduces lysosome fusogenecity. In comparison, inhibitors of oxidative phosphorylation, thiol groups, glutathione, or thioredoxin, did not impair lysosome motility but instead promoted clearance of actin puncta on lysosomes formed during PIKfyve inhibition. Additionally, actin depolymerizing agents prevented lysosome coalescence during PIKfyve inhibition. Thus, we discovered that ROS can generally prevent lysosome coalescence during PIKfyve inhibition using distinct mechanisms depending on the type of ROS.
  9. Cells. 2021 Oct 20. pii: 2814. [Epub ahead of print]10(11):
      Autophagy is an evolutionarily conserved pathway, in which cytoplasmic components are sequestered within double-membrane vesicles called autophagosomes and then transported into lysosomes or vacuoles for degradation. Over 40 conserved autophagy-related (ATG) genes define the core machinery for the five processes of autophagy: initiation, nucleation, elongation, closure, and fusion. In this review, we focus on one of the least well-characterized events in autophagy, namely the closure of the isolation membrane/phagophore to form the sealed autophagosome. This process is tightly regulated by ESCRT machinery, ATG proteins, Rab GTPase and Rab-related proteins, SNAREs, sphingomyelin, and calcium. We summarize recent progress in the regulation of autophagosome closure and discuss the key questions remaining to be addressed.
    Keywords:  autophagosome; autophagy; closure; isolation membrane