Atg8 proteins play a major role in autophagy. Atg8 is involved in the formation of autophagosome and also, serves to recruit selective autophagy receptors, autophagy regulators and autophagy substrates. These receptors, regulators and substrates are characterized by the presence of the LIR motif that mediates Atg8 interaction. Despite the discovery of several Atg8 interactors in human and yeast, knowledge of these interactors in Drosophila is limited. The known Atg8a interactors (Drosophila homolog of Atg8) in Drosophila include Ref(2)P, functions in selective autophagy of ubiquitinated substrates whereas Atg1, functions in autophagy initiation. We searched for novel Atg8a interactors in Drosophila and their role in selective autophagy of ubiquitinated protein aggregates and autophagy regulation. Using a bioinformatics approach, we identified novel putative LIR-containing proteins in Drosophila proteome and characterized three of the promising candidates in vivo using various biochemical and molecular biology techniques. One of the candidates was UbcD4, a ubiquitin ligase containing putative LIR and UBA (involved in ubiquitin binding) domains. Despite this, we could not detect UbcD4 interaction with Atg8a, suggesting that the putative LIR is not functional. Localization and western blot analysis showed that UbcD4 accumulates as aggregates under Atg8a-deficienct conditions and colocalizes with various markers of protein aggregation. Knockdown of UbcD4 indicated that UbcD4 mediates aggregate formation in old flies but not in young flies. Thus, we identified a novel component of ubiquitinated protein aggregates that mediate aggregate formation in Drosophila brains under autophagy-deficient condition. The second candidate was PAR1, a serine/tyrosine kinase with putative LIR and UBA domains. Interaction studies demonstrated that PAR1 interacts with Atg8a. Further, PAR1 is not a major component of protein aggregates formed in response to Atg8-deficiency, demonstrating that PAR1 is not found with ubiquitinated protein aggregates and thus does not participate in the removal of ubiquitinated proteins through ubiquitin-dependent selective autophagy. Further, we showed that overexpression of kinase-dead PAR1 induces mCherry-Atg8a puncta (a marker of autophagy) under fed condition, suggesting a novel role of PAR1 in autophagy regulation. The third candidate was Sequoia, a putative LIR, and ZnF-C2H2 domains (involved in DNA-binding) protein. Sequoia interacts with Atg8a, and it does not accumulate under Atg8a-deficient condition. Knockdown of Sequoia and overexpression of LIR-mutated Sequoia induces autophagy under fed conditions in the larval fat body. Additionally, expression analysis indicated that Sequoia acts as a repressor of Atg7. Thus, we identified a novel Atg8-ineracting protein that negatively regulates autophagy under fed condition. To further the understanding of selective autophagy in Drosophila, we characterized the role of Ref(2)P in mediating the cytotoxicity associated with the expression of mutant huntingtin (Htt), a toxic protein that accumulates in Huntington’s disease (HD) brains. We examined whether overexpression of Ref(2)P ameliorates HD-associated phenotypes in a Drosophila HD model. This HD model faithfully recapitulates HD-associated phenotypes such as Htt aggregation, motor dysfunction, and short lifespan. It was observed that overexpression of Ref(2)P does not rescue the above-mentioned HD-associated phenotypes. These analyses suggest that selective autophagy receptor Ref(2)P does not mediate Htt-induced toxicity in Drosophila. Finally, a label-free shotgun proteomics was employed to identify proteins with increased accumulation in Atg8a-deficient versus wild-type conditions to further the knowledge of autophagy mechanisms. An increase in the accumulation of 69 and 57 proteins was identified in Triton-soluble and Triton-insoluble fraction respectively. These proteins may rely on autophagy for their degradation. These proteins were further searched computationally for the occurrence of the putative LIR motif. The results presented here open the pathway for the discovery of novel Atg8a interactors and autophagy substrates and thus provide insights into novel mechanisms of autophagy in Drosophila.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:720436 |
| Date | January 2016 |
| Creators | Mulakkal, Nitha C. |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/89801/ |
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