DEPLETION OF AUTOPHAGY‐RELATED GENES ATG3 AND ATG5 IN Tenebrio molitor LEADS TO DECREASED SURVIVABILITY AGAINST AN INTRACELLULAR PATHOGEN, Listeria monocytogenes

Abstract

Macroautophagy (autophagy) is an evolutionarily conserved catabolic process involved in physiological and developmental processes including cell survival, death, and innate immunity. Homologues of most of 36 originally discovered autophagy-related (ATG) genes in yeast have been characterized in higher eukaryotes including insects. In this study, the homologues of ATG3 (TmATG3) and ATG5 (TmATG5) were isolated from the coleopteran beetle, Tenebrio molitor by expressed sequence tag and RNAseq approaches. The cDNA of TmATG3 and TmATG5 comprise open-reading frame sizes of 963 and 792 bp encoding polypeptides of 320 and 263 amino acid residues, respectively. TmATG3 and TmATG5 mRNA are expressed in all developmental stages, and mainly in fat body and hemocytes of larvae. TmATG3 and TmATG5 showed an overall sequence identity of 58-95% to other insect Atg proteins. There exist clear one-to-one orthologs of TmATG3 and TmATG5 in Tribolium and that they clustered together in the gene tree. Depletion of TmATG3 and TmATG5 by RNA interference led to a significant reduction in survival ability of T. molitor larvae against an intracellular pathogen, Listeria monocytogenes. Six days post-Listeria challenge, the survival rate in the dsEGFP-injected (where EGFP is enhanced green fluorescent protein) control larvae was significantly higher (55%) compared to 4 and 3% for TmATG3 and TmATG5 double-stranded RNA injected larvae, respectively. These data suggested that TmATG3 and TmATG5 may play putative role in mediating autophagy-based clearance of Listeria in T. molitor model.

MicroRNA-34a Suppresses Autophagy in Tubular Epithelial Cells in Acute Kidney Injury

“…Atg were originally discovered in yeast, and many homologues in higher eukaryotes have been characterized [25]. The process of autophagy is divided into several distinct steps, in which the Atg proteins cooperatively play a part [26].…”

Cloning, Expression Analysis, 20-Hydroxyecdysone Induction, and RNA Interference Study of Autophagy-Related Gene 8 from Heortia vitessoides Moore

“…The developmental expression profile of HvATG8 indicates that HvATG8 is expressed at all stages of development and that its expression is higher at prepupal-adult stages but lower at larval stages (Figure 2). This expression pattern also appears in Aedes albopictus, AaATG8 [41] and Tenebrio molitor, TmATG8 [42]. This may be due to the fact that the prepupal-adult transition requires more material energy than other transitions, and more extensive autophagy occurs during this period than during other periods.…”

“…In T. molitor, TmATG8 is relatively highly expressed in the midgut throughout development [47]. In the process of metamorphosis of Drosophila, larval tissues (midgut, salivary gland, and fat body) undergo autophagic degradation [42]. In G. mellonella, the expressions of GmATG8 are detected in midgut, ovary, malpighian tubules, fat body, and silk gland, with the highest expression level in the midgut of larvae [21].…”

Production of Human ATG Proteins for Lipidation Assays

“…Notably, formation of this E3-like assembly first requires a related conjugation cascade, wherein the UBL ATG12 is covalently conjugated to ATG5 by ATG7 acting as the E1 and ATG10 acting as the E2 (Hanada et al, 2007; Noda, Fujioka, Hanada, Ohsumi, & Inagaki, 2013), and then the ATG12~ATG5 binds ATG16L1 to achieve full E3-like activity toward ATG3~LC3/GABARAP intermediates. Mutations or dysfunction of proteins in the LC3/GABARAP lipidation cascade results in severe diseases and poor prognosis in various eukaryotic organisms including humans (Cadwell, Patel, Komatsu, & Virgin, 2009; Dezelak, Repnik, Koder, Ferkolj, & Potocnik, 2016; Kim et al, 2016; Lenz, Vierstra, Nurnberger, & Gust, 2011; Moloughney et al, 2011; Reed, Morris, Owczarczyk, & Lukacs, 2015; Tindwa et al, 2015; Wu et al, 2014; Xi et al, 2016; Xue, Chiu, & Oleinick, 2010). …”