bims-apauto Biomed News
on Apoptosis and autophagy
Issue of 2021‒08‒01
ten papers selected by
Su Hyun Lee
Seoul National University

  1. Mol Cell. 2021 Jul 16. pii: S1097-2765(21)00543-8. [Epub ahead of print]
      HECT ubiquitin ligases play essential roles in metazoan development and physiology. The HECT ligase HUWE1 is central to the cellular stress response by mediating degradation of key death or survival factors, including Mcl1, p53, DDIT4, and Myc. Although mutations in HUWE1 and related HECT ligases are widely implicated in human disease, our molecular understanding remains limited. Here we present a comprehensive investigation of full-length HUWE1, deepening our understanding of this class of enzymes. The N-terminal ∼3,900 amino acids of HUWE1 are indispensable for proper ligase function, and our cryo-EM structures of HUWE1 offer a complete molecular picture of this large HECT ubiquitin ligase. HUWE1 forms an alpha solenoid-shaped assembly with a central pore decorated with protein interaction modules. Structures of HUWE1 variants linked to neurodevelopmental disorders as well as of HUWE1 bound to a model substrate link the functions of this essential enzyme to its three-dimensional organization.
    Keywords:  E3; HECT; HUWE1; cryo-EM; modular assembly; protein degradation; ubiquitin; ubiquitin ligase
  2. J Biol Chem. 2021 Jul 22. pii: S0021-9258(21)00805-X. [Epub ahead of print] 101003
      Autophagy is an evolutionarily conserved pathway mediating the breakdown of cellular proteins and organelles. Emphasizing its pivotal nature, autophagy dysfunction contributes to many diseases; nevertheless, development of effective autophagy modulating drugs is hampered by fundamental deficiencies in available methods for measuring autophagic activity, or flux. To overcome these limitations, we introduced the photoconvertible protein Dendra2 into the MAP1LC3B locus of human cells via CRISPR/Cas9 genome editing, enabling accurate and sensitive assessments of autophagy in living cells by optical pulse labeling. We used this assay to perform high throughput drug screens of four chemical libraries comprising over 30,000 diverse compounds, identifying several clinically relevant drugs and novel autophagy modulators. A select series of candidate compounds also modulated autophagy flux in human motor neurons modified by CRISPR/Cas9 to express GFP-labeled LC3. Using automated microscopy, we tested the therapeutic potential of autophagy induction in several distinct neuronal models of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). In doing so, we found that autophagy induction exhibited discordant effects, improving survival in disease models involving the RNA binding protein TDP-43, while exacerbating toxicity in neurons expressing mutant forms of UBQLN2 and C9ORF72 associated with familial ALS/FTD. These studies confirm the utility of the Dendra2-LC3 assay, while illustrating the contradictory effects of autophagy induction in different ALS/FTD subtypes.
    Keywords:  ALS; FTD; autophagy; high-content; high-throughput; neurodegeneration; photoconvertible; repurposing; screen; stem cell
  3. Methods Cell Biol. 2021 ;pii: S0091-679X(20)30211-9. [Epub ahead of print]165 139-152
      Autophagy is a major protein degradation pathway responsible for the removal of primarily long-lived and misfolded proteins, contributing to cellular homeostasis. Autophagy dysfunction has been associated with the onset of various human pathologies. Visualizing key proteins that govern autophagy pathway activity, the molecular machinery and cargo is essential to elucidate roles and mechanisms of autophagy function. Although multiple fluorescence-based microscopy approaches exist to assess autophagy, the limit of resolution associated with light microscopy makes precise intracellular protein localization, interaction and molecular distribution challenging. Here we describe a detailed protocol for both super-resolution structured illumination microscopy (SR-SIM) as well as direct stochastic optical reconstruction microscopy (dSTORM) for the visualization of key proteins associated with the autophagy molecular machinery and cargo. The presented method enables to achieve increased resolving power to assess localization and molecular density profiles, typically not achievable with standard confocal or wide field fluorescence microcopy.
    Keywords:  Autophagosome; Autophagy; Direct stochastic optical reconstruction; Fluorescence microscopy; Lysosome; Structured illumination
  4. Methods Cell Biol. 2021 ;pii: S0091-679X(20)30193-X. [Epub ahead of print]165 153-161
      Selective elimination of damaged mitochondria via macroautophagy (mitophagy) is a conserved cellular process that plays an important role in organismal health. In recent years mitophagy has been studied in parallel to the more general, non-selective autophagy pathway induced in response to amino acid starvation with important similarities and differences noted between the two. The elaborate sequence of membrane rearrangements that give rise to autophagosomes in the non-selective pathway have their counterpart in mitophagy, but with the addition of other factors, such as a ubiquitin mark and mitophagy receptors, which mediate cargo recognition. In some types of mitophagy such as the one induced by ivermectin, the forming autophagosomal structure contains six different elements: the targeted mitochondrial fragment, a section of endoplasmic reticulum that provides a cradle, a ubiquitin layer, the mitophagy receptors and the early and late autophagosomal proteins/membranes. Super-resolution microscopy is ideally suited to investigate the spatial relationships between these elements that converge together but retain some distinctive localization, and we provide here a general protocol that can be used for mammalian cells.
    Keywords:  Autophagy; Endoplasmic reticulum; Ivermectin; Mitochondria; Mitophagy; Structured illumination microscopy
  5. Autophagy. 2021 Jul 27. 1-2
      Macroautophagy/autophagy is a sophisticated quality control program that limits cellular damage and maintains homeostasis, being an essential part of several lifespan-promoting interventions. However, autophagy is also necessary for full establishment of cellular senescence, a causal factor for many age-related diseases and aging. What lies ahead of us to unravel such a paradoxical role of autophagy in senescence is to identify specific targets degraded by autophagy during senescence and determine their importance in the senescence regulatory network. Recently, we developed the "Selective autophagy substrates Identification Platform (SIP)" to advance these goals, providing a rich set of autophagy substrate proteins involved in senescence. Our study demonstrated that selective autophagy coordinates the stress support networks in senescent cells by degrading multiple regulatory components, echoing its homeostatic roles in normal cells. Targeting this type of selective autophagy might provide a unique opportunity to develop non-senescence addiction-based therapeutic strategies for senotherapy by disturbing the homeostatic state of senescent cells.
    Keywords:  Autophagy interactome; cellular senescence; inflammation; oxidative stress; proteostasis; regulated protein stability; selective autophagy; stress support networks
  6. Autophagy. 2021 Jul 27. 1-19
      ABBREVIATIONS: Aβ: β-amyloid; AD: Alzheimer disease; AIF1/IBA1: allograft inflammatory factor 1; ALP: autophagy-lysosomal pathway; APP: amyloid beta precursor protein; ATP6V1B1/V-ATPase V1b1: ATPase H+ transporting V1 subunit B1; AVs: autophagy vacuoles; BAF: bafilomycin A1; CFC: contextual/cued fear conditioning assay; CHX: Ca2+/H+ exchanger; CTF-β: carboxy-terminal fragment derived from β-secretase; CTSD: cathepsin D; fAD: familial Alzheimer disease; GFAP: glial fibrillary acidic protein; LAMP1: lysosomal associated membrane protein 1; LTP: long-term potentiation; MCOLN1/TRPML1: mucolipin 1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAPT: microtubule associated protein tau; MWM: Morris water maze; NFT: neurofibrillary tangles; PFC: prefrontal cortex; PSEN1: presenilin 1; SQSTM1/p62: sequestosome 1; TBS: theta burst stimulation; TEM: transmission electronic microscopy; TPCN2/TPC2: two pore segment channel 2; WT: wild-type; V-ATPase: vacuolar type H+-ATPase.
    Keywords:  Alzheimer disease; autophagy-lysosomal pathway; calcium ion; presenilin-1; two pore segment channel 2
  7. Autophagy. 2021 Jul 27. 1-17
      PROPPINs are conserved PtdIns3P-binding proteins required for autophagosome biogenesis that fold into a characteristic group of seven-bladed beta-propellers. Mutations in WDR45/WIPI4, a human member of this family, lead to BPAN, a rare form of neurodegeneration. We have generated mutants for the two PROPPIN proteins present in the model system Dictyostelium discoideum (Atg18 and Wdr45l) and characterized their function. Lack of Wdr45l greatly impairs autophagy, while Atg18 only causes subtle defects in the maturation of autolysosomes. The strong phenotype of the Wdr45l mutant is strikingly similar to that observed in Dictyostelium cells lacking Vmp1, an ER protein required for omegasome formation. Common phenotypes include impaired growth in axenic medium, lack of aggregation, and local enrichment of PtdIns3P as determined by the use of lipid reporters. In addition, Vmp1 and Wdr45l mutants show a chronically active response to ER stress. For both mutants, this altered PtdIns3P localization can be prevented by the additional mutation of the upstream regulator Atg1, which also leads to recovery of axenic growth and reduction of ER stress. We propose that, in addition to an autophagy defect, local autophagy-associated PtdIns3P accumulation might contribute to the pathogenesis of BPAN by disrupting ER homeostasis. The introduction of BPAN-associated mutations in Dictyostelium Wdr45l reveals the impact of pathogenic residues on the function and localization of the protein.
    Keywords:  Atg18; autophagosome; membrane contact site; omegasome; proppin proteins
  8. J Drug Target. 2021 Jul 28. 1-61
      Autophagy is a multi-step lysosomal degradation process, which regulates energy and material metabolism and has been used to maintain homeostasis. Autophagy has been shown to be involved in the regulation of health and disease. But at present, there is no consensus on the relationship between autophagy and tumor, and we consider that it plays a dual role in the occurrence and development of tumor. That is to say, under certain conditions, it can inhibit the occurrence of tumor, but it can also promote the process of tumor. Therefore, autophagy could be used as a target for tumor treatment. The regulation of autophagy plays a synergistic role in the radiotherapy, chemotherapy, phototherapy and immunotherapy of tumor, and nano drug delivery system provides a promising strategy for improving the efficacy of autophagy regulation. This review summarized the progress in the regulatory pathways and factors of autophagy as well as nanoformulations as carriers for the delivery of autophagy modulators.
    Keywords:  autophagy; autophagy regulation; cancer; nanoparticle; phototherapy immunotherapy
  9. EMBO J. 2021 Jul 26. e107336
      During tumor growth-when nutrient and anabolic demands are high-autophagy supports tumor metabolism and growth through lysosomal organelle turnover and nutrient recycling. Ras-driven tumors additionally invoke non-autonomous autophagy in the microenvironment to support tumor growth, in part through transfer of amino acids. Here we uncover a third critical role of autophagy in mediating systemic organ wasting and nutrient mobilization for tumor growth using a well-characterized malignant tumor model in Drosophila melanogaster. Micro-computed X-ray tomography and metabolic profiling reveal that RasV12 ; scrib-/- tumors grow 10-fold in volume, while systemic organ wasting unfolds with progressive muscle atrophy, loss of body mass, -motility, -feeding, and eventually death. Tissue wasting is found to be mediated by autophagy and results in host mobilization of amino acids and sugars into circulation. Natural abundance Carbon 13 tracing demonstrates that tumor biomass is increasingly derived from host tissues as a nutrient source as wasting progresses. We conclude that host autophagy mediates organ wasting and nutrient mobilization that is utilized for tumor growth.
    Keywords:   Drosophila ; autophagy; cancer cachexia; muscle; tumor; wasting
  10. Nat Commun. 2021 07 27. 12(1): 4536
      Despite the substantial impact of post-translational modifications on programmed cell death 1 ligand 1 (PD-L1), its importance in therapeutic resistance in pancreatic cancer remains poorly defined. Here, we demonstrate that never in mitosis gene A-related kinase 2 (NEK2) phosphorylates PD-L1 to maintain its stability, causing PD-L1-targeted pancreatic cancer immunotherapy to have poor efficacy. We identify NEK2 as a prognostic factor in immunologically "hot" pancreatic cancer, involved in the onset and development of pancreatic tumors in an immune-dependent manner. NEK2 deficiency results in the suppression of PD-L1 expression and enhancement of lymphocyte infiltration. A NEK binding motif (F/LXXS/T) is identified in the glycosylation-rich region of PD-L1. NEK2 interacts with PD-L1, phosphorylating the T194/T210 residues and preventing ubiquitin-proteasome pathway-mediated degradation of PD-L1 in ER lumen. NEK2 inhibition thereby sensitizes PD-L1 blockade, synergically enhancing the anti-pancreatic cancer immune response. Together, the present study proposes a promising strategy for improving the effectiveness of pancreatic cancer immunotherapy.