The Role Of ATG3 And ATG8 In Autophagy And Their Interaction In Bombyx Mori

Autophagy is a self-degradation process that widely exists in all organisms. Formation of autophagosome involves in a series of autophagy related proteins, it finally fused with vacuoles or lysosomes to complete the degradation of substrates. Autophagy is closely bound up with insect metamorphosis, on the other hand Bombyx mori is a classic model organism to study the metamorphosis, which is regulated by many signaling pathways related to autophagy, such as nutrient and Ecdysteroids signaling pathways.ATG3 is an E2-like enzyme, which is involved in autophagy-related ubiquitin-like system. The mechanism underlying conjugation of ATG8 and phosphatidylethanolamine (PE) catalyzed by ATG3 have been well studied in the yeast, Saccharomyces cerevisiae. However, the mechanism is unknown in higher animals. This study is based on the titer of ecdysteroids in Bombyx hemolymph, investigated the regulation of ATG8 by 20E, and focus on the temporal expression patterns of Atg3 and Atg8. Combined with dsRNA mediated interference, CRISPR/Cas9 and Baculovirus expression system, we verified the role of Atg3 and Atg8 in autophagy, detected the interaction of ATG3 and ATG8 through cell transfection in vitro; and research the regulation of ATG8 by 20E at last. The details are as below:1. Detection of ecdysteroids in Bombyx mori hemolymphThere are two main ecdysteroids in insect hemolymph, named ecdysone and 20-hydroxyecdysone (20E). Ecdysone is the immediate precursor of 20E, while 20E is the activative form of insect ecdysteroids, called molting hormone. Ecdysone and 20E were separated by reversed-phase high performance liquid chromatographic (RHPLC), which was followed by the Enzyme linked immunosorbent assay (ELISA) to determine their titers respectively. The titer of ecdysone and 20E dominantly increased when the autophagy occurs.2. ATG3 and ATG8 expression pattern in fat body in Bombyx moriThe ATG3 and ATG8 expression pattern were analyzed using western blotting in the larval fat body in Bombyx mori. The results show that the protein level of ATG3 decreased while its mRNA level increased in metamorphosis period, which implied that ATG3 was eliminated during the process of autophagy. Differing from other species, ATG8-PE protein complex maintain low level in feeding stage. Which mean ATG8-PE plays a role in Bombyx development. Meanwhile, ATG8-PE formed puncta when the autophagy accurred.3. The role of ATG3 and ATG8 in autophagy in Bombyx moriBoth dsRNA mediated interference of Atg3 and Atg8 at wondering stage and Knocking Atg3 and Atg8 out by CRISPR-Cas9 resulted in reduction of autophagy. Overexpression of Atg3 and Atg8 by Baculovirus led to upregulation of autophagy. These data indicated that ATG3 and ATG8 are essential for autophagy. Interestingly, Knockout of Atg3 will lead development forward into wondering stage.4. The relationship between ATG3 and ATG8 in Bombyx moriKnockout of Atg3 by CRISPR/Cas9 led to the reduction of ATG8-PE puncta and downregulation of autophagy, which revealed ATG8-PE depend on the catalysis of ATG3. Transfection to overexpress mCherry-ATG3 and EGFP-Atg8 in BmN cells, led to fluorescent dots formation after 20E or starvation treatment. However, ATG3 and ATG8 fluorescent dots are complementary, but not co-localization.5. The regulation of ATG8 by 20E in Bombyx moriThe fat body cells were immunostained by ATG8 antibody at 24h later after the 20E injection treatment in feeding stage. Experimental datas show that ATG8-PE puncta dominantly increased after 20E treatment, and upregulation of autophagy.In conclusion, BmATG3 and BmATG8 play an essential role in autophagy process, and ATG3 is dependent on ATG8 to regulate autophagy.