bims-apauto Biomed News
on Apoptosis and autophagy
Issue of 2021‒11‒07
ten papers selected by
Su Hyun Lee
Seoul National University
  1. A gene toolbox for monitoring autophagy transcription.
  2. SHISA5/SCOTIN restrains spontaneous autophagy induction by blocking contact between the ERES and phagophores.
  3. Extracellular vesicles originating from autophagy mediate an antibody-resistant spread of classical swine fever virus in cell culture.
  4. FAIM regulates autophagy through glutaminolysis in lung adenocarcinoma.
  5. Competitive binding of E3 ligases TRIM26 and WWP2 controls SOX2 in glioblastoma.
  6. The SQSTM1/p62 UBA domain regulates Ajuba localisation, degradation and NF-κB signalling function.
  7. Emerging roles of ATG7 in human health and disease.
  8. Ubiquitination of G3BP1 mediates stress granule disassembly in a context-specific manner.
  9. Ubiquitination is essential for recovery of cellular activities after heat shock.
  10. Beclin1 controls caspase-4 inflammsome activation and pyroptosis in mouse myocardial reperfusion-induced microvascular injury.
  1. Cell Death Dis. 2021 Nov 02. 12(11): 1044
    A gene toolbox for monitoring autophagy transcription.
    Matteo Bordi, Rossella De Cegli, Beatrice Testa, Ralph A Nixon, Andrea Ballabio, Francesco Cecconi.
      Autophagy is a highly dynamic and multi-step process, regulated by many functional protein units. Here, we have built up a comprehensive and up-to-date annotated gene list for the autophagy pathway, by combining previously published gene lists and the most recent publications in the field. We identified 604 genes and created main categories: MTOR and upstream pathways, autophagy core, autophagy transcription factors, mitophagy, docking and fusion, lysosome and lysosome-related genes. We then classified such genes in sub-groups, based on their functions or on their sub-cellular localization. Moreover, we have curated two shorter sub-lists to predict the extent of autophagy activation and/or lysosomal biogenesis; we next validated the "induction list" by Real-time PCR in cell lines during fasting or MTOR inhibition, identifying ATG14, ATG7, NBR1, ULK1, ULK2, and WDR45, as minimal transcriptional targets. We also demonstrated that our list of autophagy genes can be particularly useful during an effective RNA-sequencing analysis. Thus, we propose our lists as a useful toolbox for performing an informative and functionally-prognostic gene scan of autophagy steps.
    DOI:  https://doi.org/10.1038/s41419-021-04121-9
  2. Autophagy. 2021 Oct 31. 1-16
    SHISA5/SCOTIN restrains spontaneous autophagy induction by blocking contact between the ERES and phagophores.
    Jee-Eun Lee, Nari Kim, Minkyo Jung, Ji-Young Mun, Joo-Yeon Yoo.
      The phagophore expands into autophagosomes in close proximity to endoplasmic reticulum (ER) exit sites (ERESs). Here, we propose that a single-pass ER transmembrane protein, SHISA5/SCOTIN, acts as an autophagy suppressor under basal condition by blocking the contact between the phagophore and ERES. HeLa cells lacking SHISA5 displayed higher levels of macroautophagy/autophagy. The enhanced autophagy in SHISA5 KO cells requires class III phosphatidylinositol 3-kinase complex I (PtdIns3K-C1) activity and functional assembly of ERES, but not ULK1 activity. A proximity ligation assay (PLA) of SEC16A (Sec16 homolog A, endoplasmic reticulum export factor)-WIPI2 (WD repeat domain, phosphoinositide interacting 2) and SEC31A (Sec31 homolog A, COPII coat complex component)-MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 beta) demonstrated that contact between the ERES and phagophore increased in SHISA5 KO cells, and the cytosolic domain of SHISA5 was sufficient to rescue this phenotype. Close proximity between ERES and phagophore in SHISA5 KO cells was also visualized by performing an ultrastructure correlative image analysis of SEC31A associated with LC3-positive membranes. Furthermore, we observed that SHISA5 was located near ERES under basal conditions, but displaced away from ERES under autophagy-inducing conditions. These data suggest that SHISA5 functions to block spontaneous contact between ERES and phagophore, and the blockage effect of SHISA5 should be relieved for the proper induction of autophagy.
    Keywords:  Constitutive autophagy; SHISA5/SCOTIN; endoplasmic reticulum exit sites; membrane contact; phagophore
    DOI:  https://doi.org/10.1080/15548627.2021.1994297
  3. Autophagy. 2021 Nov 05. 1-17
    Extracellular vesicles originating from autophagy mediate an antibody-resistant spread of classical swine fever virus in cell culture.
    Tao Wang, Liang Zhang, Wulong Liang, Shanchuan Liu, Wen Deng, Yangruiyu Liu, Yaru Liu, Mengzhao Song, Kangkang Guo, Yanming Zhang.
      Free spread is a classical mode for mammalian virus transmission. However, the efficiency of this transmission approach is generally low as there are structural barriers or immunological surveillances in the extracellular environment under physiological conditions. In this study, we systematically analyzed the spreading of classical swine fever virus (CSFV) using multiple viral replication analysis in combination with antibody neutralization, transwell assay, and electron microscopy, and identified an extracellular vesicle (EV)-mediated spreading of CSFV in cell cultures. In this approach, intact CSFV virions are enclosed within EVs and transferred into uninfected cells with the movement of EVs, leading to an antibody-resistant infection of the virus. Using fractionation assays, immunostaining, and electron microscopy, we characterized the CSFV-containing EVs and demonstrated that the EVs originated from macroautophagy/autophagy. Taken together, our results showed a new spreading mechanism for CSFV and demonstrated that the EVs in CSFV spreading are closely related to autophagy. These findings shed light on the immune evasion mechanisms of CSFV transmission, as well as new functions of cellular vesicles in virus lifecycles.Abbreviations: 3-MA: 3-methyladenine; CCK-8: Cell Counting Kit-8; CSF: classical swine fever; CQ: chloroquine; CSFV: classical swine fever virus; DAPI, 4-,6-diamidino-2-phenylindole; EVs: extracellular vesicles; hpi: h post infection; IEM: immunoelectron microscopy; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MOI: multiplicity of infection; MVs: microvesicles; ND50: half neutralizing dose; PCR: polymerase chain reaction; PBS: phosphate-buffered saline; SEC: size-exclusion chromatography; siRNA: small interfering RNA; TEM: transmission electron microscopy.
    Keywords:  Classical swine fever virus (CSFV); LC3B; extracellular vesicles (EVs); size-exclusion chromatography (SEC); transmission electron microscopy (TEM)
    DOI:  https://doi.org/10.1080/15548627.2021.1987673
  4. Autophagy. 2021 Oct 31. 1-17
    FAIM regulates autophagy through glutaminolysis in lung adenocarcinoma.
    Tianyu Han, Pengcheng Wang, Yanan Wang, Wenze Xun, Jiapeng Lei, Tao Wang, Zhuo Lu, Mingxi Gan, Wei Zhang, Bentong Yu, Jian-Bin Wang.
      Altered glutamine metabolism is an important aspect of cancer metabolic reprogramming. The GLS isoform GAC (glutaminase C), the rate-limiting enzyme in glutaminolysis, plays a vital role in cancer initiation and progression. Our previous studies demonstrated that phosphorylation of GAC was essential for its high enzymatic activity. However, the molecular mechanisms for GAC in maintaining its high enzymatic activity and protein stability still need to be further clarified. FAIM/FAIM1 (Fas apoptotic inhibitory molecule) is known as an important anti-apoptotic protein, but little is known about its function in tumorigenesis. Here, we found that knocking down FAIM induced macroautophagy/autophagy through suppressing the activation of the MTOR pathway in lung adenocarcinoma. Further studies demonstrated that FAIM could promote the tetramer formation of GAC through increasing PRKCE/PKCε-mediated phosphorylation. What's more, FAIM also stabilized GAC through sequestering GAC from degradation by protease ClpXP. These effects increased the production of α-ketoglutarate, leading to the activation of MTOR. Besides, FAIM also promoted the association of ULK1 and MTOR and this further suppressed autophagy induction. These findings discovered new functions of FAIM and elucidated an important molecular mechanism for GAC in maintaining its high enzymatic activity and protein stability.
    Keywords:  Autophagy; Fas apoptosis inhibitory molecule 1; glutaminase C; protein stability; tetramer formation
    DOI:  https://doi.org/10.1080/15548627.2021.1987672
  5. Nat Commun. 2021 Nov 03. 12(1): 6321
    Competitive binding of E3 ligases TRIM26 and WWP2 controls SOX2 in glioblastoma.
    Tatenda Mahlokozera, Bhuvic Patel, Hao Chen, Patrick Desouza, Xuan Qu, Diane D Mao, Daniel Hafez, Wei Yang, Rukayat Taiwo, Mounica Paturu, Afshin Salehi, Amit D Gujar, Gavin P Dunn, Nima Mosammaparast, Allegra A Petti, Hiroko Yano, Albert H Kim.
      The pluripotency transcription factor SOX2 is essential for the maintenance of glioblastoma stem cells (GSC), which are thought to underlie tumor growth, treatment resistance, and recurrence. To understand how SOX2 is regulated in GSCs, we utilized a proteomic approach and identified the E3 ubiquitin ligase TRIM26 as a direct SOX2-interacting protein. Unexpectedly, we found TRIM26 depletion decreased SOX2 protein levels and increased SOX2 polyubiquitination in patient-derived GSCs, suggesting TRIM26 promotes SOX2 protein stability. Accordingly, TRIM26 knockdown disrupted the SOX2 gene network and inhibited both self-renewal capacity as well as in vivo tumorigenicity in multiple GSC lines. Mechanistically, we found TRIM26, via its C-terminal PRYSPRY domain, but independent of its RING domain, stabilizes SOX2 protein by directly inhibiting the interaction of SOX2 with WWP2, which we identify as a bona fide SOX2 E3 ligase in GSCs. Our work identifies E3 ligase competition as a critical mechanism of SOX2 regulation, with functional consequences for GSC identity and maintenance.
    DOI:  https://doi.org/10.1038/s41467-021-26653-6
  6. PLoS One. 2021 ;16(11): e0259556
    The SQSTM1/p62 UBA domain regulates Ajuba localisation, degradation and NF-κB signalling function.
    Melanie A Sultana, Carmel Cluning, Wai-Sin Kwong, Nicole Polain, Nathan J Pavlos, Thomas Ratajczak, John P Walsh, Jiake Xu, Sarah L Rea.
      The LIM-domain containing protein Ajuba and the scaffold protein SQSTM1/p62 regulate signalling of NF-κB, a transcription factor involved in osteoclast differentiation and survival. The ubiquitin-associated domain of SQSTM1/p62 is frequently mutated in patients with Paget's disease of bone. Here, we report that Ajuba activates NF-κB activity in HEK293 cells, and that co-expression with SQSTM1/p62 inhibits this activation in an UBA domain-dependent manner. SQSTM1/p62 regulates proteins by targeting them to the ubiquitin-proteasome system or the autophagy-lysosome pathway. We show that Ajuba is degraded by autophagy, however co-expression with SQSTM1/p62 (wild type or UBA-deficient) protects Ajuba levels both in cells undergoing autophagy and those exposed to proteasomal stress. Additionally, in unstressed cells co-expression of SQSTM1/p62 reduces the amount of Ajuba present in the nucleus. SQSTM1/p62 with an intact ubiquitin-associated domain forms holding complexes with Ajuba that are not destined for degradation yet inhibit signalling. Thus, in situations with altered levels and localization of SQSTM1/p62 expression, such as osteoclasts in Paget's disease of bone and various cancers, SQSTM1/p62 may compartmentalize Ajuba and thereby impact its cellular functions and disease pathogenesis. In Paget's, ubiquitin-associated domain mutations may lead to increased or prolonged Ajuba-induced NF-κB signalling leading to increased osteoclastogenesis. In cancer, Ajuba expression promotes cell survival. The increased levels of SQSTM1/p62 observed in cancer may enhance Ajuba-mediated cancer cell survival.
    DOI:  https://doi.org/10.1371/journal.pone.0259556
  7. EMBO Mol Med. 2021 Nov 02. e14824
    Emerging roles of ATG7 in human health and disease.
    Jack J Collier, Fumi Suomi, Monika Oláhová, Thomas G McWilliams, Robert W Taylor.
      The cardinal stages of macroautophagy are driven by core autophagy-related (ATG) proteins, whose ablation largely abolishes intracellular turnover. Disrupting ATG genes is paradigmatic of studying autophagy deficiency, yet emerging data suggest that ATG proteins have extensive biological importance beyond autophagic elimination. An important example is ATG7, an essential autophagy effector enzyme that in concert with other ATG proteins, also regulates immunity, cell death and protein secretion, and independently regulates the cell cycle and apoptosis. Recently, a direct association between ATG7 dysfunction and disease was established in patients with biallelic ATG7 variants and childhood-onset neuropathology. Moreover, a prodigious body of evidence supports a role for ATG7 in protecting against complex disease states in model organisms, although how dysfunctional ATG7 contributes to manifestation of these diseases, including cancer, neurodegeneration and infection, in humans remains unclear. Here, we systematically review the biological functions of ATG7, discussing the impact of its impairment on signalling pathways and human pathology. Future studies illuminating the molecular relationship between ATG7 dysfunction and disease will expedite therapies for disorders involving ATG7 deficiency and/or impaired autophagy.
    Keywords:  ATG7; autophagy; disease; neurodegeneration; therapeutics
    DOI:  https://doi.org/10.15252/emmm.202114824
  8. Science. 2021 Jun 25. 372(6549): eabf6548
    Ubiquitination of G3BP1 mediates stress granule disassembly in a context-specific manner.
    Youngdae Gwon, Brian A Maxwell, Regina-Maria Kolaitis, Peipei Zhang, Hong Joo Kim, J Paul Taylor.
      Tailoring stress responsesWhen faced with environmental stress, cells respond by shutting down cellular processes such as translation and nucleocytoplasmic transport. At the same time, cells preserve cytoplasmic messenger RNAs in structures known as stress granules, and many cellular proteins are modified by the covalent addition of ubiquitin, which has long been presumed to reflect degradation of stress-damaged proteins (see the Perspective by Dormann). Maxwell et al. show that cells generate distinct patterns of ubiquitination in response to different stressors. Rather than reflecting the degradation of stress-damaged proteins, this ubiquitination primes cells to dismantle stress granules and reinitiate normal cellular activities once the stress is removed. Gwon et al. show that persistent stress granules are degraded by autophagy, whereas short-lived granules undergo a process of disassembly that is autophagy independent. The mechanism of this disassembly depends on the initiating stress.Science, abc3593 and abf6548, this issue p. eabc3593 and p. eabf6548; see also abj2400, p. 1393.
    DOI:  https://doi.org/10.1126/science.abf6548
  9. Science. 2021 Jun 25. 372(6549): eabc3593
    Ubiquitination is essential for recovery of cellular activities after heat shock.
    Brian A Maxwell, Youngdae Gwon, Ashutosh Mishra, Junmin Peng, Haruko Nakamura, Ke Zhang, Hong Joo Kim, J Paul Taylor.
      Tailoring stress responsesWhen faced with environmental stress, cells respond by shutting down cellular processes such as translation and nucleocytoplasmic transport. At the same time, cells preserve cytoplasmic messenger RNAs in structures known as stress granules, and many cellular proteins are modified by the covalent addition of ubiquitin, which has long been presumed to reflect degradation of stress-damaged proteins (see the Perspective by Dormann). Maxwell et al. show that cells generate distinct patterns of ubiquitination in response to different stressors. Rather than reflecting the degradation of stress-damaged proteins, this ubiquitination primes cells to dismantle stress granules and reinitiate normal cellular activities once the stress is removed. Gwon et al. show that persistent stress granules are degraded by autophagy, whereas short-lived granules undergo a process of disassembly that is autophagy independent. The mechanism of this disassembly depends on the initiating stress.Science, abc3593 and abf6548, this issue p. eabc3593 and p. eabf6548; see also abj2400, p. 1393.
    DOI:  https://doi.org/10.1126/science.abc3593
  10. Cell Commun Signal. 2021 Nov 03. 19(1): 107
    Beclin1 controls caspase-4 inflammsome activation and pyroptosis in mouse myocardial reperfusion-induced microvascular injury.
    Wenjing Sun, Hongquan Lu, Shujuan Dong, Rui Li, Yingjie Chu, Nan Wang, Yu Zhao, Yabin Zhang, Limeiting Wang, Lin Sun, Di Lu.
      BACKGROUND: Myocardial reperfusion injury is often accompanied by cell death and inflammatory reactions. Recently, pyroptosis is gradually recognized as pivotal role in cardiovascular disease. However, little is known about the regulatory role of beclin1 in the control of caspase-4 activation and pyroptosis. The present study confirmed whether beclin1 regulates caspase-4 mediated pyroptosis and thereby protects Human Cardiac microvascular endothelial cells (HCMECs) against injury.METHODS: TTC and Evan's blue dye, western blot, immunofluorescence and immunohistochemistry staining were performed in wild mice and transgenic mice with overexpression of beclin 1(BECN1-Tg). CMECs were transfected with a beclin1 lentivirus. The cell cytotoxicity was analyzed by LDH-Cytotoxicity Assay Kit. The protein levels of autophagy protein (Beclin1, p62 and LC3II/LC3I) and caspase-4/GSDMD pathway were determined by western blot. Autophagic vacuoles in cells were monitored with RFP-GFP-LC3 using fluorescence microscope.
    RESULTS: I/R caused caspase-4 activity and gasdermin D expression increase in vivo and in vitro. Overexpression of beclin1 in heart tissue and CMECs suppressed the caspase-4 activity and decreased the levels of gasdermin D; meanwhile beclin1 overexpression also reduced IL-1β levels, promoted autophagy (p62 expression was inhibited while LC3II expression was increased) in the heart and CMECs. Interestingly, beclin1 overexpression increased animal survival and attenuated myocardial infarct size (45 ± 6.13 vs 22 ± 4.37), no-reflow area (39 ± 5.22 vs 16 ± 2.54) post-myocardial ischemia reperfusion.
    CONCLUSIONS: Induction of beclin-1 signaling can be a potential therapeutic target in myocardial reperfusion-induced microvascular injury. Video Abstract.
    Keywords:  Beclin1; Caspase-4 inflammasome; Ischemia/reperfusion; Pyroptosis
    DOI:  https://doi.org/10.1186/s12964-021-00786-z
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