Abstract

   Metamorphosis is an important way for insects to adapt to the
   environment. In this process, larval tissue destruction regulated by
   20-hydroxyecdysone (20E) and adult tissue reconstruction regulated by
   insulin-like peptides (ILPs) occur simultaneously, but the detailed
   mechanism is still unclear. Here, the results of succinylome,
   subcellular localization, and protein interaction analysis show that
   non-succinylated insulin-degrading enzyme (IDE) localizes in the
   cytoplasm, binds to insulin-like growth factor 2 (IGF-2-like), and
   degrades it. When the metamorphosis is initiated, 20E up-regulated
   carnitine palmitoyltransferase 1A (Cpt1a) through transcription factor
   Krüppel-like factor 15 (KLF15), thus increasing the level of IDE
   succinylation on K179. Succinylated IDE translocated from cytoplasm to
   nucleus, combined with ecdysone receptor to promote 20E signaling
   pathway, causing larval tissue destruction, while IGF-2-like was
   released to promote adult tissue proliferation. That is, succinylation
   alters subcellular localization of IDE so that it can bind to different
   target proteins and act as a hub of metamorphosis.
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   Succinylation alters IDE subcellular localization and interaction
   targets to regulate insect metamorphosis.

INTRODUCTION

   Insects are the most diverse group of organisms on earth. One of the
   ways for them to adapt to the environment and maintain the population
   is to show completely different morphologies at different stages of
   development, which is called metamorphosis. Metamorphosis involves not
   only the external morphological changes from larva to adult but also
   the programmed cell death (PCD) of larval tissue and the proliferation
   of adult tissue ([30]1–[31]4). How the destruction and reconstruction
   are carried out at the same time has received considerable critical
   attention in the field of development. Extensive research has shown
   that metamorphosis is synergistically regulated by steroid hormone
   20-hydroxyecdysone (20E), peptide hormone insulin-like peptide (ILP)
   superfamily, and sesquiterpenoid juvenile hormone ([32]5). 20E acts
   through the nuclear receptor complex composed of the ecdysone receptor
   (EcR) and ultraspiracle (USP). Then, the 20E-induced transcriptional
   factors such as hormone receptor 3 (HR3) and metamorphosis initiation
   factor Broad (Br) subsequently mediate the PCD ([33]6, [34]7). In
   addition, ILP superfamily, including insulin, insulin-like growth
   factor (IGF), and relaxin, promotes growth via insulin/IGF signaling
   (IIS) ([35]8). However, the hub that enables both hormone signaling
   pathways to function simultaneously needs to be further identified.

   Organisms can regulate physiological processes according to nutritional
   changes in the environment. One of the important ways is that the
   intermediates of nutrient metabolism act as donors of posttranslational
   modification (PTM) and covalently bind to functional proteins to
   regulate their subcellular localization, activity, stability, and
   protein folding in response to changes in nutrition status
   ([36]9–[37]11). Succinylation is a kind of PTM that has attracted much
   attention in recent years. Succinyl group can bind to ε-amino of lysine
   residues of target proteins in an enzymatic or nonenzymatic way.
   Several enzymes have been reported to be involved in the succinylation
   process, such as histone acetyltransferase 1 (HAT1) and carnitine
   palmitoyltransferase 1A (CPT1A) ([38]12, [39]13). Considering that the
   succinyl group is derived from succinic acid, an intermediate of the
   tricarboxylic acid cycle (TCA), succinylation may be an important
   mechanism for cells to regulate the process of life in response to the
   change of metabolic state. However, its target molecules and regulatory
   mechanisms still need to be further explored. The metabolism and
   physiological activities changed greatly during insect metamorphosis,
   which provides a good model for studying the mechanism of
   succinylation-regulating physiological process.

   This study seeks to obtain data in Helicoverpa armigera (Lepidoptera:
   Noctuidae), a holometabolous agricultural pest, toexplore the role of
   succinylation in insect metamorphosis coordinated by 20E and ILPs. We
   took advantage of liquid chromatography–mass spectrometry/mass
   spectrometry (LC-MS/MS) to screen for the different succinylated
   proteins in the feeding stage and the metamorphic molting stage of the
   last-instar larvae. Among the differentially succinylated proteins
   involved in hormone regulation, we noticed a protein with important
   functions in both ILP and steroid hormone signaling pathway,
   insulin-degrading enzyme (IDE). IDE is a multifunctional
   zinc-metallo-endopeptidase that regulates development ([40]14), which
   can exert protease activity to degrade insulin ([41]15), glucagon
   ([42]16), and other small molecular peptides ([43]17), thereby
   regulating nutrient metabolism, cell proliferation, and apoptosis. It
   can also participate in various physiological processes through
   interaction with other proteins, such as regulating hormone signal
   pathways by binding to steroid hormone receptors ([44]18). Dysfunction
   of IDE can lead to diabetes ([45]19), Alzheimer’s disease ([46]17), and
   other diseases. IDE has different subcellular localization in different
   cells, and it can be localized in the peroxisome, endosome,
   mitochondria, rough endoplasmic reticulum, cytoplasm, and other
   subcellular compartments and secreted into the extracellular space
   ([47]20, [48]21). IDE action targets are numerous and distributed in
   different locations of cells. However, the exact mechanism of how IDE
   alters subcellular localization to combine with different targets to
   perform different functions remains unclear. Here, we found that
   non-succinylated IDE localizes in the cytoplasm, where it binds to
   IGF-2-like and degrades it. After entering metamorphosis, 20E
   up-regulates the expression of carnitine palmitoyltransferase 1A
   (Cpt1a) through Krüppel-like factor 15 (KLF15) and then induces the
   succinylation of IDE-K179. Succinylated IDE can translocate from the
   cytoplasm into the nucleus and bind to the EcR-USP1 complex, promoting
   the 20E signaling pathway and thus promoting the PCD of larval tissue
   of H. armigera. Meanwhile, IGF-2-like was released to promote adult
   tissue proliferation. The results of this study revealed that
   succinylation regulates the subcellular localization of IDE to bind to
   different target proteins, participates in different signaling
   pathways, and actsas a hub mediating the coordination of 20E and ILPs
   in insect metamorphosis.

RESULTS

Succinylome profiling analysis identified 192 differentially succinylated
proteins during metamorphosis

   The life cycle of the cotton worm consists of four stages: egg, larva,
   pupa, and adult. The larva stage was segmented into six instars by
   molts. After eating for more than 48 hours, sixth-instar larvae got
   into the metamorphic commitment stage at 72 hours and then pupation at
   about 144 hours. The whole bodies of bleeding larvae at the
   sixth-instar feeding (6F, 12 to 60 hours after ecdysis into sixth
   instar) and metamorphic commitment stages (6M, 72 to 120 hours after
   ecdysis into sixth instar) were collected to provide detailed
   illustrations of the variation of lysine succinylation in the process
   of metamorphosis. The sampling time point and the morphology of the
   larvae are shown in [49]Fig. 1A.

Fig. 1. Succinylome profiling analysis identified differentially succinylated
proteins during metamorphosis.

   [50]Fig. 1.
   [51]Open in a new tab

   (A) Representative images of cotton worm (H. armigera) during feeding
   and metamorphosis stages. 6F, sixth-instar feeding (12 to 60 hours
   after ecdysis into sixth instar) stage; 6M, metamorphic commitment
   stages (72 to 120 hours after ecdysis into sixth instar). Scale bar, 1
   cm. (B) Overlap of the identified, quantified, and differentially
   modified succinylated proteins (fold change > 1.3). (C) Up-regulated
   and down-regulated succinylated proteins or sites during 6M compared
   with 6F. (D) Percentage of subcellular localization of differentially
   succinylated proteins during metamorphosis. (E) Motif analyses of
   flanking sequence preferences for succinylation sites with 1.3-fold