An autophagy assay reveals the ESCRT-III component CHMP2A as a regulator of phagophore closure

Takahashi, Yoshinori; He, Haiyan; Tang, Zifan; Hattori, Tatsuya; Liu, Ying; Young, Michelle; Serfass, Jacob M.; Chen, Longgui; Gebru, Melat T.; Chen, Chong; Wills, Carson A.; Atkinson, Jennifer M.; Chen, Han; Abraham, Thomas; Wang, Hong Gang

doi:10.1038/s41467-018-05254-w

Cited by 291 publications

(253 citation statements)

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“…While the ESCRT membrane scission machinery was originally discovered for its role in ubiquitinated cargo sorting in the multivesicular body (MVB) pathway (Katzmann et al, 2001), recent studies have revealed its involvement in a number of other cellular processes, including virus budding, cytokinesis, microvesicle generation, plasma membrane wound repair, nuclear envelope reformation, nuclear pore complex quality control, and endolysosomal repair (Hurley, 2015; Christ et al, 2017; Skowyra et al, 2018). Most recently, we have used the HT-LC3 assay and demonstrated the requirement of the ESCRT-III component charged MVB protein 2A (CHMP2A) and VPS4 activation in phagophore closure (Takahashi et al, 2018). In this study, we have combined a genome-wide CRISPR library screen with the HT-LC3 assay and identified the ESCRT-I component vacuolar protein sorting-associated protein 37A (VPS37A) as a novel phagophore closure regulator.…”

Section: Introductionmentioning

confidence: 99%

VPS37A directs ESCRT recruitment for phagophore closure

Takahashi

Liang

Hattori

et al. 2019

Journal of Cell Biology

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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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Section: Introductionmentioning

confidence: 99%

VPS37A directs ESCRT recruitment for phagophore closure

Takahashi

Liang

Hattori

et al. 2019

Journal of Cell Biology

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113

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“…S1E). Finally, we tested whether autophagosome closure or autophagosome-lysosome fusion were required for TRIM5signaling by knocking down the ESCRT component CMHP2A (35), and the autophagosomeassociated (t)-SNARE SNAP29, and the lysosomal (v)-SNARE VAMP8, and found that these proteins were also essential for TRIM5-based signaling ( Fig. 2A,B; S1F).…”

Section: Resultsmentioning

confidence: 99%

A non-canonical role for the autophagy machinery in anti-retroviral signaling mediated by TRIM5α

Saha

Chisholm

Mandell

2020

Preprint

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TRIM5α is a key cross-species barrier to retroviral infection, with certain TRIM5 alleles conferring increased risk of HIV-1 infection in humans. TRIM5α is best known as a speciesspecific restriction factor that directly inhibits the viral life cycle. Additionally, it is also a patternrecognition receptor (PRR) that activates inflammatory signaling. How TRIM5α carries out its multi-faceted actions in antiviral defense remains incompletely understood. Here, we show that proteins required for autophagy, a cellular self-digestion pathway, play an important role in TRIM5α's function as a PRR. Genetic depletion of proteins involved in all stages of the autophagy pathway prevented TRIM5α-driven expression of NF-κB and AP1 responsive genes.One of these genes is the preeminent antiviral cytokine interferon β (IFN-β), whose TRIM5dependent expression was lost in cells lacking the autophagy proteins ATG7, BECN1, and ULK1. Moreover, we found that the ability of TRIM5α to stimulate IFN-β expression in response to recognition of a TRIM5α-restricted HIV-1 capsid mutant (P90A) was abrogated in cells lacking autophagy factors. Stimulation of human macrophage-like cells with the P90A virus protected them against subsequent infection with an otherwise resistant wild type HIV-1 in a manner requiring TRIM5α, BECN1, and ULK1. Mechanistically, TRIM5α was attenuated in its ability to activate the kinase TAK1 in autophagy deficient cells, and both BECN1 and ATG7 contributed to the assembly of TRIM5α-TAK1 complexes. These data demonstrate a non-canonical role for the autophagy machinery in assembling antiviral signaling complexes and demonstrate a role for autophagy in the establishment of a TRIM5α-dependent antiviral state.

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“…6D; 1-0-10 ). During autophagy, CHMP2A translocates to the phagophore to regulate separation of the inner and outer autophagosomal membranes to form double-membrane autophagosomes [171]. CHMP2B is a member of the ESCRT-III complex required for efficient autophagy and has reduced expression in melanoma [172,173], raising the hypothesis that gemcitabine could have efficacy in that context.…”

Section: Resultsmentioning

confidence: 99%

A humanized yeast phenomic model of deoxycytidine kinase to predict genetic buffering of nucleoside analog cytotoxicity

Santos

Icyuz

Pound

et al. 2019

Preprint

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10Knowledge about synthetic lethality can be applied to enhance the efficacy of 11 anti-cancer therapies in individual patients harboring genetic alterations in their cancer 12 that specifically render it vulnerable. We investigated the potential for high-resolution 13 phenomic analysis in yeast to predict such genetic vulnerabilities by systematic, 14 comprehensive, and quantitative assessment of drug-gene interaction for gemcitabine 15 and cytarabine, substrates of deoxycytidine kinase that have similar molecular structures 16 yet distinct anti-tumor efficacy. Human deoxycytidine kinase (dCK) was conditionally 17 expressed in the S. cerevisiae genomic library of knockout and knockdown (YKO/KD) 18 strains, to globally and quantitatively characterize differential drug-gene interaction for 19 gemcitabine and cytarabine. Pathway enrichment analysis revealed that autophagy, 20 histone modification, chromatin remodeling, and apoptosis-related processes influence 21 gemcitabine specifically, while drug-gene interaction specific to cytarabine was less 22 enriched in Gene Ontology. Processes having influence over both drugs were DNA 23 repair and integrity checkpoints and vesicle transport and fusion. Non-GO-enriched 24 genes were also informative. Yeast phenomic and cancer cell line pharmacogenomics 25 data were integrated to identify yeast-human homologs with correlated differential gene 26 2 expression and drug-efficacy, thus providing a unique resource to predict whether 27 differential gene expression observed in cancer genetic profiles are causal in tumor-28 specific responses to cytotoxic agents. 29 30 Keywords: 31 yeast phenomics, gene-drug interaction, genetic buffering, quantitative high 32 throughput cell array phenotyping (Q-HTCP), cell proliferation parameters (CPPs), 33 gemcitabine, cytarabine, recursive expectation-maximization clustering (REMc), 34 pharmacogenomics 35 36 Introduction: 37Genomics has enabled targeted therapy aimed at cancer driver genes and 38 oncogenic addiction [1], yet traditional cytotoxic chemotherapeutic agents remain among 39 the most widely used and efficacious anti-cancer therapies [2]. Changes in the genetic 40 network underlying cancer can produce vulnerabilities to cytotoxic chemotherapy that 41 further influence the therapeutic window and provide additional insight into their 42 mechanisms of action [3,4]. A potential advantage of so-called synthetic lethality-based 43 treatment strategies is that they could have efficacy against passenger gene mutation or 44 compensatory gene expression, while classic targeted therapies are directed primarily at 45 driver genes ( Fig. 1A). Quantitative high throughput cell array phenotyping of the yeast 46 knockout and knockdown libraries provides a phenomic means for systems level, high- 47resolution modeling of gene interaction [5][6][7][8][9], which is applied here to predict cancer-48 relevant drug-gene interaction through integration with cancer pharmacogenomics 49 resources ( Fig. 1B). 50Nucleoside analogs include a diverse group of co...

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An autophagy assay reveals the ESCRT-III component CHMP2A as a regulator of phagophore closure

Cited by 291 publications

References 46 publications

VPS37A directs ESCRT recruitment for phagophore closure

VPS37A directs ESCRT recruitment for phagophore closure

A non-canonical role for the autophagy machinery in anti-retroviral signaling mediated by TRIM5α

A humanized yeast phenomic model of deoxycytidine kinase to predict genetic buffering of nucleoside analog cytotoxicity

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