Atg2A/B deficiency switches cytoprotective autophagy to non-canonical caspase-8 activation and apoptosis

Tang, Zifan; Takahashi, Yoshinori; Chen, Chong; Liu, Ying; He, Haiyan; Tsotakos, Nikolaos; Serfass, Jacob M.; Gebru, Melat T.; Chen, Han; Young, Michelle; Wang, Hong Gang

doi:10.1038/cdd.2017.133

Cited by 65 publications

(62 citation statements)

References 61 publications

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“…This is consistent with the observation in D. melanogaster Atg2 mutant cells that p62 extensively colocalizes with Atg9 but isolation membranes (phagophores) were only rarely observed around p62 structures [11,12]. Recently, another group also established ATG2A/B DKO THP-1 cells [9]. In contrast to our observation, these authors reported that immature autophagosomelike structures were generated in these cells.…”

Section: Discussionsupporting

confidence: 93%

Differential requirement for ATG2A domains for localization to autophagic membranes and lipid droplets

et al. 2017

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ATG2 is one of the autophagy-related (ATG) proteins essential for autophagosome formation and localizes to isolation membranes and lipid droplets in mammalian cells. Here, we investigated the requirement of regions in ATG2A for its organellar localization and function. The N-terminal amino acids 1-198 and the C-terminal amino acids 1830-1938 are required for the localization to isolation membranes and lipid droplets, respectively. The C-terminal region is not required for the localization to isolation membranes and for autophagy. We also identified an amphipathic helix in ATG2A that is required for both its localization to organelles and autophagosome formation. These data suggest that the dual localization of ATG2A is regulated by different regions.

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Section: Discussionsupporting

confidence: 93%

Differential requirement for ATG2A domains for localization to autophagic membranes and lipid droplets

et al. 2017

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“…The activation of apoptosis-related caspase can degrade autophagy-related proteins, thus inhibiting autophagy in tumor cells (Man et al 2017, Tang et al 2017. However, whether the apoptosis could regulate autophagy in germ cells in hypoxia remains to be elucidated.…”

Section: Discussionmentioning

confidence: 99%

Elevation of autophagy rescues spermatogenesis by inhibiting apoptosis of mouse spermatocytes

Yin

Yang

et al. 2018

Reproduction

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Autophagy and apoptosis are interlocked in an extensive crosstalk. Our previous study demonstrated that hypotonic hypoxia-induced marked apoptosis of a spermatocyte-derived cell line (GC-2). However, whether hypoxia-induced apoptosis is mediated by inhibition of autophagy under hypoxic conditions remains unclear. In this study, GC-2 cells were cultured in 1% O 2 and harvested at different time points. Autophagy was determined by acridine orange staining, cyto-ID staining, mCherry-GFP-LC3B adenovirus transfection and Western blotting for various autophagy markers. Apoptosis was detected by TUNEL staining, flow cytometry, JC-1 staining and Western blotting of apoptosis-related proteins. We found that hypoxia-induced apoptosis of GC-2 cells through mitochondrial and death receptor pathways and inhibited autophagic flux in GC-2 cells in a time-dependent manner. However, while marked autolysosome formation was observed in GC-2 cells before 24-h culture in hypoxic conditions, apparent apoptosis was observed only after 24-h culture in hypoxic conditions. Caspase-8 siRNA treatment induced cell survival, accompanied by induction of the mature autophagosome, acidic vesicular organelle formation and autophagic flux. Furthermore, Beclin-1 overexpression markedly attenuated the impairment of spermatogenesis in mice by inhibiting apoptosis of spermatocytes. The results of this study demonstrate that hypoxia inhibits autophagy, which further enhances hypoxia-induced apoptosis of mouse spermatocytes by promoting caspase-8 activation in a time-dependent manner, suggesting that combined application of apoptosis inhibition and autophagy activation might be a therapeutic strategy for treating hypoxia-induced male infertility.Reproduction (2018) 156 545-558 546 Reproduction (2018) 156 545-558 https://rep.bioscientifica.com The activation of apoptosis can also inhibit autophagy under hypoxia. Caspases that are activated during apoptosis, mainly caspase-8 and caspase-3, can cleave autophagy-related proteins, such as ATG3, ATG4D and Beclin1, suppressing autophagy and resulting in cell damage (Li et al. 2011, Oral et al. 2012, Lamy et al. 2013). In addition, autophagy can block the induction of apoptosis and attenuate cellular injury under hypoxia. The activation of autophagy inhibits apoptosis by clearing damaged mitochondria, inhibiting caspase-3 activation and clearing reactive oxygen species (ROS)-damaged proteins. Thus, autophagy plays a protective role under hypoxia (Zhang et al. 2013, Li et al. 2014). However, in specific cases, autophagy or autophagy-relevant proteins may help in inducing apoptosis, which can aggravate cellular injury under hypoxia. The underlying mechanisms include the activation of caspases and clearance of the Bruce protein (an anti-apoptotic protein, a member of the inhibitors of apoptosis proteins family) (Nezis et al. , Young et al. 2012. Taking all the above into consideration, the interaction between autophagy and apoptosis in spermatocytes under hypoxia treatment remains unclear.In our previous study, we...

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“…To generate a lentiviral vector encoding human VPS28, the VPS37A cDNA at the XhoI–NotI site in pCDH1-CMV-GFP-FL was replaced to the human VPS28 cDNA amplified from the plasmid pQTEV-VPS28 using the primer 5′-TATACTCGAGGATTTCATGGGATCCCAGCC-3′ and 5′-TCGAGCGGCCGCTCAGGCATG-3′. For the tandem fluorescent-tagged VPS37A (RFP-GFP-VPS37A) expression constructs, the EGFP-LC3 cassette in tfLC3 (pCDH-RFP-EGFP-LC3; Tang et al, 2017) was replaced to GFP-FL or GFP-ΔPUEV at the Age1–Bamh1 site. All other reagents were obtained from the following sources: autoMACS Rinsing Solution (130–091-222), Miltenyi Biotec; BafA1 (B-1080), LC Laboratories; BSA (126575), EMD Millipore; MIL (AF488-conjugated, G1001; AF660-conjugated, G8471), Promega; MPL (TMR-conjugated, G8251), Promega; normal goat serum (G9023), Sigma-Aldrich; Nucleofector Kit V (VCA-1003), Lonza; PFA (15710), Electron Microscopy Sciences; recombinant human EGF (PHG0311), Invitrogen; and XF Plasma Membrane Permeabilizer/recombinant perfringolysin O mutant (102504–100), Seahorse Bioscience.…”

Section: Methodsmentioning

confidence: 99%

VPS37A directs ESCRT recruitment for phagophore closure

Takahashi

Liang

Hattori

et al. 2019

Journal of Cell Biology

Self Cite

113

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The process of phagophore closure requires the endosomal sorting complex required for transport III (ESCRT-III) subunit CHMP2A and the AAA ATPase VPS4, but their regulatory mechanisms remain unknown. Here, we establish a FACS-based HaloTag-LC3 autophagosome completion assay to screen a genome-wide CRISPR library and identify the ESCRT-I subunit VPS37A as a critical component for phagophore closure. VPS37A localizes on the phagophore through the N-terminal putative ubiquitin E2 variant domain, which is found to be required for autophagosome completion but dispensable for ESCRT-I complex formation and the degradation of epidermal growth factor receptor in the multivesicular body pathway. Notably, loss of VPS37A abrogates the phagophore recruitment of the ESCRT-I subunit VPS28 and CHMP2A, whereas inhibition of membrane closure by CHMP2A depletion or VPS4 inhibition accumulates VPS37A on the phagophore. These observations suggest that VPS37A coordinates the recruitment of a unique set of ESCRT machinery components for phagophore closure in mammalian cells.

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Atg2A/B deficiency switches cytoprotective autophagy to non-canonical caspase-8 activation and apoptosis

Cited by 65 publications

References 61 publications

Differential requirement for ATG2A domains for localization to autophagic membranes and lipid droplets

Differential requirement for ATG2A domains for localization to autophagic membranes and lipid droplets

Elevation of autophagy rescues spermatogenesis by inhibiting apoptosis of mouse spermatocytes

VPS37A directs ESCRT recruitment for phagophore closure

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