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CG14435 (detour), the Drosophila ZNRF1 and 2 homologue, regulates autophagosome biogenesis critical for healthy aging

ABSTRACT: Autophagy is essential for animal development and homeostasis, and its deregulation is associated with numerous human pathologies. Here we identify a novel regulator of autophagy, the RING domain containing ubiquitin ligase CG14435 (detour). Depletion of CG14435 resulted in increased early-stage autophagic puncta, premature tissue contraction, and an accumulation of autophagy related proteins in young adults. Overexpression of CG14435 or the mammalian homologues, Zinc and RING finger (ZNRF) 1 and ZNRF2, increased late-stage autophagy puncta size. The ablation of ZNRF1 or ZNRF2 in mammalian cells increased basal autophagy. We identified CG14435 interacting proteins including HOPS complex subunits, deep orange (Vps18), Vps16A, and light (Vps41). Furthermore, in the presence of CG14435, dor ubiquitination was increased. The CG14435 mutant adults displayed premature aging, impaired motor function (neurodegeneration), and activation of innate immunity. Our findings reveal a novel role for CG14435 in autophagy, likely through regulation of HOPS complex, required for healthy aging.

INSTRUMENT(S): maXis

ORGANISM(S): Drosophila Melanogaster (fruit Fly)

SUBMITTER: Jarrod Sandow

LAB HEAD: Donna Denton

PROVIDER: PXD036857 | Pride | 2024-05-21

REPOSITORIES: Pride

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The Drosophila ZNRF1/2 homologue, detour, interacts with HOPS complex and regulates autophagy.

Nicolson Shannon S Manning Jantina A JA Lim Yoon Y Jiang Xin X Kolze Erica E Dayan Sonia S Umargamwala Ruchi R Xu Tianqi T Sandow Jarrod J JJ Webb Andrew I AI Kumar Sharad S Denton Donna D

Communications biology 20240215 1

Autophagy, the process of elimination of cellular components by lysosomal degradation, is essential for animal development and homeostasis. Using the autophagy-dependent Drosophila larval midgut degradation model we identified an autophagy regulator, the RING domain ubiquitin ligase CG14435 (detour). Depletion of detour resulted in increased early-stage autophagic vesicles, premature tissue contraction, and overexpression of detour or mammalian homologues, ZNRF1 and ZNRF2, increased autophagic v ...[more]

PMID: 38360932

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Project description:Purpose: To understand the relationship between ciliogenesis and autophagy in the corneal epithelium. Methods: siRNAs for EphA2 or PLD1 were used to inhibit protein expression in vitro. Morpholino-anti-EphA2 was used to knockdown EphA2 in Xenopus skin. An EphA2 knockout mouse was used to conduct loss of function studies. Autophagic vacuoles were visualized by contrast light microscopy. Autophagy flux, was measured by LC3 turnover and p62 protein levels. Immunostaining and confocal microscopy were conducted to visualize cilia in cultured cells and in vivo. Results: Loss of EphA2 (i) increased corneal epithelial thickness by elevating proliferative potential in wing cells, (ii) reduced the number of ciliated cells, (iii) increased large hollow vacuoles, that could be rescued by BafA1; (iv) inhibited autophagy flux and (v) increased GFP-LC3 puncta in the mouse corneal epithelium. This indicated a role for EphA2 in stratified epithelial assembly via regulation of proliferation as well as a positive role in both ciliogenesis and end-stage autophagy. Inhibition of PLD1, an EphA2 interacting protein that is a critical regulator of end-stage autophagy, reversed the accumulation of vacuoles, and the reduction in the number of ciliated cells due to EphA2 depletion, suggesting EphA2 regulation of both end-stage autophagy and ciliogenesis via PLD1. PLD1 mediated rescue of ciliogenesis by EphA2 depletion was blocked by BafA1, placing autophagy between EphA2 signaling and regulation of ciliogenesis. Conclusion: Our findings demonstrate a novel role for EphA2 in regulating both autophagy and ciliogenesis, processes that are essential for proper corneal epithelial homeostasis. Purpose: To understand the relationship between ciliogenesis and autophagy in the corneal epithelium. Methods: siRNAs for EphA2 or PLD1 were used to inhibit protein expression in vitro. Morpholino-anti-EphA2 was used to knockdown EphA2 in Xenopus skin. An EphA2 knockout mouse was used to conduct loss of function studies. Autophagic vacuoles were visualized by contrast light microscopy. Autophagy flux, was measured by LC3 turnover and p62 protein levels. Immunostaining and confocal microscopy were conducted to visualize cilia in cultured cells and in vivo. Results: Loss of EphA2 (i) increased corneal epithelial thickness by elevating proliferative potential in wing cells, (ii) reduced the number of ciliated cells, (iii) increased large hollow vacuoles, that could be rescued by BafA1; (iv) inhibited autophagy flux and (v) increased GFP-LC3 puncta in the mouse corneal epithelium. This indicated a role for EphA2 in stratified epithelial assembly via regulation of proliferation as well as a positive role in both ciliogenesis and end-stage autophagy. Inhibition of PLD1, an EphA2 interacting protein that is a critical regulator of end-stage autophagy, reversed the accumulation of vacuoles, and the reduction in the number of ciliated cells due to EphA2 depletion, suggesting EphA2 regulation of both end-stage autophagy and ciliogenesis via PLD1. PLD1 mediated rescue of ciliogenesis by EphA2 depletion was blocked by BafA1, placing autophagy between EphA2 signaling and regulation of ciliogenesis. Conclusion: Our findings demonstrate a novel role for EphA2 in regulating both autophagy and ciliogenesis, processes that are essential for proper corneal epithelial homeostasis.

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