Abstract
Autophagy requires the conjugation of autophagy-related protein 12 (ATG12) to autophagy-related protein 5 (ATG5) through covalent attachment. However, the signals regulating ATG12–ATG5 conjugation are unclear. The larval midgut of lepidopteran insects performs autophagy and apoptosis sequentially during the transition of larvae to pupae under regulation by the steroid hormone 20-hydroxyecdysone (20E), thus representing a model to study steroid hormone regulation of ATG12–ATG5 conjugation. In the present study, using the lepidopteran insect Helicoverpa armigera as a model, we report that 20E regulates the conjugation of ATG12–ATG5 in a concentration and time-dependent manner. The ATG12–ATG5 conjugate was abundant in the epidermis, midgut, and fat body during metamorphosis from the larvae to the pupae; however, the ATG12–ATG5 conjugate level decreased at the time of pupation. At low concentrations (2–5 µM) over a short time course (1–48 h), 20E promoted the conjugation of ATG12–ATG5; however, at 10 µM and 72 h, 20E repressed the conjugation of ATG12–ATG5. ATG12 was localized in the larval midgut during metamorphosis. Knockdown of ATG12 in larvae caused death with delayed pupation, postponed the process of midgut programmed cell death (PCD), and repressed ATG8 (also called LC3-I) transformation to LC3-II and the cleavage of caspase-3; therefore, knockdown of ATG12 in larvae blocked both autophagy and apoptosis. Knockdown of ATG12 in H. armigera epidermis cell line cells also repressed 20E-induced autophagosome formation and caspase-3 activation. The results suggested that 20E plays key role in the regulation of ATG12–ATG5 conjugation in a concentration and time-dependent manner for autophagy or apoptosis, and that ATG12 is necessary by both autophagy and apoptosis during insect midgut PCD.
Highlights
Programed cell death (PCD) includes apoptosis with features of nuclear condensation and fragmentation, formation of apoptotic bodies, and caspase activation; autophagic cell death, with the accumulation of autophagosomes and autolysosomes, along with cell death; necrotic cell death, which involves cell swelling and membrane rupture [1]; and ferroptosis, a non-apoptotic forms of cell death induced by the small molecule erastin [2]
We report that low concentrations, a short period of 20E promote autophagy-related protein 12 (ATG12)–autophagy-related protein 5 (ATG5) conjugation and high concentrations, a long period repress ATG12–ATG5 conjugation during midgut PCD in the lepidopteran insect H. armigera
We tested whether ATG12 and ATG5 formed a conjugate in an H. armigera Epidermis Cell Line (HaEpi) and midgut
Summary
Programed cell death (PCD) includes apoptosis (type I PCD) with features of nuclear condensation and fragmentation, formation of apoptotic bodies, and caspase activation; autophagic cell death (type II PCD), with the accumulation of autophagosomes and autolysosomes, along with cell death; necrotic cell death (type III PCD), which involves cell swelling and membrane rupture [1]; and ferroptosis, a non-apoptotic forms of cell death induced by the small molecule erastin [2]. ATG12 is an ubiquitin-like protein that conjugates with ATG5 to play vital roles during autophagy [4]. ATG12 forms an isopeptide bond with ATG5 through activation of ATG7 and under the mediation of ATG10, forming the ATG12–ATG5 conjugate [5], which associates with ATG16 to form a larger ATG12–ATG5–ATG16 complex [6]. The complex functions as an ubiquitin–protein ligase (E3)-like enzyme to transfer microtubule-associated protein 1A/1B-light chain 3 (LC3 in mammals, known as ATG8 in yeast) from ATG3 to phosphatidylethanolamine (PE) by stimulating the E2 activity of ATG3 [7]. The ATG12–ATG5–ATG16 complex and ATG8/LC3 drive autophagosome membrane expansion and fusion [8]. It is unclear how upstream factors, especially steroid hormones, regulate ATG12–ATG5 conjugation
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