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author:("Varga, agnes")
1.  Different effects of Atg2 and Atg18 mutations on Atg8a and Atg9 trafficking during starvation in Drosophila☆ 
Febs Letters  2014;588(3):408-413.
Highlights
•Atg9 and Atg18 are required for autophagy upstream of Atg8a, unlike Atg2.•Atg9 accumulates on Ref(2)P aggregates in Atg8a, Atg7 and Atg2 mutants.•Ultrastructurally, Atg9 vesicles cluster around Ref(2)P aggregates in stalled PAS.•Atg9 does not accumulate on Ref(2)P upon loss of Atg18 or Vps34, while FIP200 does.•Atg18 simultaneously interacts with both Atg9 and Ref(2)P.
The Atg2–Atg18 complex acts in parallel to Atg8 and regulates Atg9 recycling from phagophore assembly site (PAS) during autophagy in yeast. Here we show that in Drosophila, both Atg9 and Atg18 are required for Atg8a puncta formation, unlike Atg2. Selective autophagic degradation of ubiquitinated proteins is mediated by Ref(2)P/p62. The transmembrane protein Atg9 accumulates on refractory to Sigma P (Ref(2)P) aggregates in Atg7, Atg8a and Atg2 mutants. No accumulation of Atg9 is seen on Ref(2)P in cells lacking Atg18 or Vps34 lipid kinase function, while the Atg1 complex subunit FIP200 is recruited. The simultaneous interaction of Atg18 with both Atg9 and Ref(2)P raises the possibility that Atg18 may facilitate selective degradation of ubiquitinated protein aggregates by autophagy.
Structured summary of protein interactions
Ref(2)Pphysically interacts with Atg18 by anti tag coimmunoprecipitation (View interaction) Atg18physically interacts with Atg2 by anti tag coimmunoprecipitation (View interaction) CG8678physically interacts with Atg2 by anti tag coimmunoprecipitation (View interaction) Atg18physically interacts with atg9 by anti tag coimmunoprecipitation (View interaction)
doi:10.1016/j.febslet.2013.12.012
PMCID: PMC3928829  PMID: 24374083
Atg, autophagy-related; PAS, phagophore assembly site; PI3P, phosphatidylinositol 3-phosphate; Ref(2)P, refractory to Sigma P; ULK, uncoordinated-51 like autophagy kinase; Vps, vacuolar protein sorting; WIPI, WD40 repeat domain phosphoinositide-interacting protein; Atg2; Atg7; Atg8a; Atg9; Atg18; Ref(2)P/p62
2.  Autophagosomal Syntaxin17-dependent lysosomal degradation maintains neuronal function in Drosophila 
The Journal of Cell Biology  2013;201(4):531-539.
Lysosomal degradation and recycling of sequestered autophagosome content is crucial to maintain proper functioning of the fly nervous system.
During autophagy, phagophores capture portions of cytoplasm and form double-membrane autophagosomes to deliver cargo for lysosomal degradation. How autophagosomes gain competence to fuse with late endosomes and lysosomes is not known. In this paper, we show that Syntaxin17 is recruited to the outer membrane of autophagosomes to mediate fusion through its interactions with ubisnap (SNAP-29) and VAMP7 in Drosophila melanogaster. Loss of these genes results in accumulation of autophagosomes and a block of autolysosomal degradation during basal, starvation-induced, and developmental autophagy. Viable Syntaxin17 mutant adults show large-scale accumulation of autophagosomes in neurons, severe locomotion defects, and premature death. These mutant phenotypes cannot be rescued by neuron-specific inhibition of caspases, suggesting that caspase activation and cell death do not play a major role in brain dysfunction. Our findings reveal the molecular mechanism underlying autophagosomal fusion events and show that lysosomal degradation and recycling of sequestered autophagosome content is crucial to maintain proper functioning of the nervous system.
doi:10.1083/jcb.201211160
PMCID: PMC3653357  PMID: 23671310
3.  Myc-Driven Overgrowth Requires Unfolded Protein Response-Mediated Induction of Autophagy and Antioxidant Responses in Drosophila melanogaster 
PLoS Genetics  2013;9(8):e1003664.
Autophagy, a lysosomal self-degradation and recycling pathway, plays dual roles in tumorigenesis. Autophagy deficiency predisposes to cancer, at least in part, through accumulation of the selective autophagy cargo p62, leading to activation of antioxidant responses and tumor formation. While cell growth and autophagy are inversely regulated in most cells, elevated levels of autophagy are observed in many established tumors, presumably mediating survival of cancer cells. Still, the relationship of autophagy and oncogenic signaling is poorly characterized. Here we show that the evolutionarily conserved transcription factor Myc (dm), a proto-oncogene involved in cell growth and proliferation, is also a physiological regulator of autophagy in Drosophila melanogaster. Loss of Myc activity in null mutants or in somatic clones of cells inhibits autophagy. Forced expression of Myc results in cell-autonomous increases in cell growth, autophagy induction, and p62 (Ref2P)-mediated activation of Nrf2 (cnc), a transcription factor promoting antioxidant responses. Mechanistically, Myc overexpression increases unfolded protein response (UPR), which leads to PERK-dependent autophagy induction and may be responsible for p62 accumulation. Genetic or pharmacological inhibition of UPR, autophagy or p62/Nrf2 signaling prevents Myc-induced overgrowth, while these pathways are dispensable for proper growth of control cells. In addition, we show that the autophagy and antioxidant pathways are required in parallel for excess cell growth driven by Myc. Deregulated expression of Myc drives tumor progression in most human cancers, and UPR and autophagy have been implicated in the survival of Myc-dependent cancer cells. Our data obtained in a complete animal show that UPR, autophagy and p62/Nrf2 signaling are required for Myc-dependent cell growth. These novel results give additional support for finding future approaches to specifically inhibit the growth of cancer cells addicted to oncogenic Myc.
Author Summary
The evolutionarily conserved transcription factor Myc promotes protein synthesis, cell growth and cancer progression through incompletely understood mechanisms. In this work, we show that forced expression of Myc induces the accumulation of abnormal proteins leading to unfolded protein responses (UPR), presumably by overloading the protein synthetic capacity of cells in Drosophila. UPR then results in autophagy-mediated breakdown and recycling of cytoplasmic material, and at the same time, to activation of antioxidant responses in these cells. Blocking the UPR stress signaling, autophagy and antioxidant pathways genetically, or by feeding larvae an autophagy-inhibiting drug, prevents overgrowth of Myc-expressing cells, but these treatments do not affect the growth of control cells in the same tissues. These results, together with recent reports in mammalian cancer models, suggest that drugs targeting UPR, autophagy and antioxidant responses may specifically inhibit cancer cell proliferation driven by oncogenic Myc.
doi:10.1371/journal.pgen.1003664
PMCID: PMC3738540  PMID: 23950728
4.  Impaired proteasomal degradation enhances autophagy via hypoxia signaling in Drosophila 
BMC Cell Biology  2013;14:29.
Background
Two pathways are responsible for the majority of regulated protein catabolism in eukaryotic cells: the ubiquitin-proteasome system (UPS) and lysosomal self-degradation through autophagy. Both processes are necessary for cellular homeostasis by ensuring continuous turnover and quality control of most intracellular proteins. Recent studies established that both UPS and autophagy are capable of selectively eliminating ubiquitinated proteins and that autophagy may partially compensate for the lack of proteasomal degradation, but the molecular links between these pathways are poorly characterized.
Results
Here we show that autophagy is enhanced by the silencing of genes encoding various proteasome subunits (α, β or regulatory) in larval fat body cells. Proteasome inactivation induces canonical autophagy, as it depends on core autophagy genes Atg1, Vps34, Atg9, Atg4 and Atg12. Large-scale accumulation of aggregates containing p62 and ubiquitinated proteins is observed in proteasome RNAi cells. Importantly, overexpressed Atg8a reporters are captured into the cytoplasmic aggregates, but these do not represent autophagosomes. Loss of p62 does not block autophagy upregulation upon proteasome impairment, suggesting that compensatory autophagy is not simply due to the buildup of excess cargo. One of the best characterized substrates of UPS is the α subunit of hypoxia-inducible transcription factor 1 (HIF-1α), which is continuously degraded by the proteasome during normoxic conditions. Hypoxia is a known trigger of autophagy in mammalian cells, and we show that genetic activation of hypoxia signaling also induces autophagy in Drosophila. Moreover, we find that proteasome inactivation-induced autophagy requires sima, the Drosophila ortholog of HIF-1α.
Conclusions
We have characterized proteasome inactivation- and hypoxia signaling-induced autophagy in the commonly used larval Drosophila fat body model. Activation of both autophagy and hypoxia signaling was implicated in various cancers, and mutations affecting genes encoding UPS enzymes have recently been suggested to cause renal cancer. Our studies identify a novel genetic link that may play an important role in that context, as HIF-1α/sima may contribute to upregulation of autophagy by impaired proteasomal activity.
doi:10.1186/1471-2121-14-29
PMCID: PMC3700814  PMID: 23800266
Autophagy; Drosophila; HIF-1α/sima; Hypoxia; p62/Ref2P; Proteasome
5.  Analysis of CPD Ultraviolet Lesion Bypass in Chicken DT40 Cells: Polymerase η and PCNA Ubiquitylation Play Identical Roles 
PLoS ONE  2012;7(12):e52472.
Translesion synthesis (TLS) provides a mechanism of copying damaged templates during DNA replication. This potentially mutagenic process may operate either at the replication fork or at post-replicative gaps. We used the example of T-T cyclobutane pyrimidine dimer (CPD) bypass to determine the influence of polymerase recruitment via PCNA ubiquitylation versus the REV1 protein on the efficiency and mutagenic outcome of TLS. Using mutant chicken DT40 cell lines we show that, on this numerically most important UV lesion, defects in polymerase η or in PCNA ubiquitylation similarly result in the long-term failure of lesion bypass with persistent strand gaps opposite the lesion, and the elevation of mutations amongst successful TLS events. Our data suggest that PCNA ubiquitylation promotes CPD bypass mainly by recruiting polymerase η, resulting in the majority of CPD lesions bypassed in an error-free manner. In contrast, we find that polymerase ζ is responsible for the majority of CPD-dependent mutations, but has no essential function in the completion of bypass. These findings point to a hierarchy of access of the different TLS polymerases to the lesion, suggesting a temporal order of their recruitment. The similarity of REV1 and REV3 mutant phenotypes confirms that the involvement of polymerase ζ in TLS is largely determined by its recruitment to DNA by REV1. Our data demonstrate the influence of the TLS polymerase recruitment mechanism on the success and accuracy of bypass.
doi:10.1371/journal.pone.0052472
PMCID: PMC3525536  PMID: 23272247
6.  Loss of the starvation-induced gene Rack1 leads to glycogen deficiency and impaired autophagic responses in Drosophila 
Autophagy  2012;8(7):1124-1135.
Autophagy delivers cytoplasmic material for lysosomal degradation in eukaryotic cells. Starvation induces high levels of autophagy to promote survival in the lack of nutrients. We compared genome-wide transcriptional profiles of fed and starved control, autophagy-deficient Atg7 and Atg1 null mutant Drosophila larvae to search for novel regulators of autophagy. Genes involved in catabolic processes including autophagy were transcriptionally upregulated in all cases. We also detected repression of genes involved in DNA replication in autophagy mutants compared with control animals. The expression of Rack1 (receptor of activated protein kinase C 1) increased 4.1- to 5.5-fold during nutrient deprivation in all three genotypes. The scaffold protein Rack1 plays a role in a wide range of processes including translation, cell adhesion and migration, cell survival and cancer. Loss of Rack1 led to attenuated autophagic response to starvation, and glycogen stores were decreased 11.8-fold in Rack1 mutant cells. Endogenous Rack1 partially colocalized with GFP-Atg8a and early autophagic structures on the ultrastructural level, suggesting its involvement in autophagosome formation. Endogenous Rack1 also showed a high degree of colocalization with glycogen particles in the larval fat body, and with Shaggy, the Drosophila homolog of glycogen synthase kinase 3B (GSK-3B). Our results, for the first time, demonstrated the fundamental role of Rack1 in autophagy and glycogen synthesis.
doi:10.4161/auto.20069
PMCID: PMC3429548  PMID: 22562043
antimicrobial peptides; Atg8; autophagy; Drosophila; fat body; glycogen; GSK-3B; microarray; Rack1; starvation

Results 1-6 (6)