Pro-Apoptotic Function Analysis of the Reaper Homologue IBM1 in Spodoptera frugiperda

Abstract

As an important type of programmed cell death, apoptosis plays a critical role in lepidopteran insects in response to various internal and external stresses. It is controlled by a network of genes such as those encoding the inhibitor of apoptosis proteins. However, there are few studies on apoptosis-related genes in Spodoptera frugiperda. In this study, an orthologue to the Drosophila reaper gene, named Sf-IBM1, was identified from S. frugiperda, and a full-length sequence was obtained by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends PCR (RACE-PCR). The expression pattern of Sf-IBM1 was determined in different developmental stages and various tissues. Apoptotic stimuli including azadirachtin, camptothecin, and ultraviolet radiation (UV) induced the expression of Sf-IBM1 at both transcript and protein levels. Overexpression of Sf-IBM1 induced apoptosis in Sf9 cells, and the Sf-IBM1 protein was localized in mitochondria. The apoptosis induced by Sf-IBM1 could be blocked by the caspase universal inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK) and Sf-IAP1. Our results provide valuable information that should contribute to a better understanding of the molecular events that lead to apoptosis in lepidopterans.Apoptosis is inhibited by the inhibitor of apoptosis proteins (IAPs), which are endogenous caspase inhibitors and were originally identified in the genomes of lepidopteran baculoviruses [8]. Numerous IAPs have been identified from various insects and have been demonstrated to be the negative regulators of apoptosis [9]. For example, the Drosophila IAP1 (DIAP1) can enhance ubiquitylation and inhibit the activity of Dronc both in vitro and in vivo [10]. Co-expression of Bm-IAP1 in Bombyx mori suppresses apoptosis induced by overexpression of Bm-Dronc in BM-N cells [9]. IAPs contain two functional domains, the baculoviral IAP repeat (BIR) domain and the Really Interesting New Gene (RING) domain. The IBR domain may be involved in direct inhibition of apoptosis, whereas the RING domain may take part in protein-protein interactions. IAPs can bind to both initiator and effector caspases directly and degrade activated caspases through the E3 ubiquitin ligase activity, resulting in inhibition of apoptosis [11].The anti-apoptotic function of IAPs can be neutralized by IAP antagonists. IAP antagonists are the proteins containing the evolutionarily conserved IAP binding motif (IBM), which consists of several amino acids at the N-terminal [12]. In Drosophila, five RHG (Rpr, Hid, Grim) family proteins including Reaper, Hid, Grim, Sickle, and Jafrac2 have been identified, each with an IBM motif in the N-terminus and identified as the IAP antagonists [13]. Similarly, two IAP antagonists, Smac/Diablo and HtrA2/Omi have also been identified in mammals [14]. IAP antagonists compete with caspases by binding to the BIR domains of IAPs with different affinities via IBM directly [15]. Besides, the IAP antagonists can also function as positive regulato...

Deep Conservation of Hid-Like RHG Gene Family Homologs in Winged Insects Revealed by “Taxon Hopping” BLAST

“…This is further supported by the fact that both IMB and MTS are essential for hid's cell death-inducing capacity [20]. Interestingly, also, the lepidopteran S-isoform is mitochondrially localized, based on immunohistochemical detection in the armyworm moth S. frugiperda [26]. At first glance, this suggests a higher level of functional conservation between the derived S-isoforms and the ancestrally organized L-isoforms in the Lepidoptera compared to that between hid vs. grim, rpr, and skl in Drosophila.…”

“…In Diptera, RHG homologs have been analyzed in the blowfly species Lucilia cuprina and L. sericata [21,22], the Caribbean fruit fly species Anastrepha suspensa (Tephritidae) [23], and, most recently, the scuttle fly Megaselia scalaris [20]. In the Lepidoptera, RHG homologs have been studied in the silkmoth Bombyx mori and the fall armyworm Spodoptera frugiperda [24][25][26]. The existence of RHG homologs outside the Lepidoptera and Diptera, however, has remained elusive.…”

Deep conservation ofHid-like RHG gene family homologs in winged insects

“…This is further supported by the fact that both the IMB and MTS domain are essential for Hid's cell death inducing capacity (Yoo et al 2017) . Interestingly, also the lepidopteran S-isoform is mitochondrially localized based on immunohistochemical detection in the armyworm moth S. frugiperda (Shu et al 2020) , suggesting at a higher level of functional conservation between the derived S-and ancestrally organized L-isoforms in the Lepidoptera compared to that between hid vs grim , rpr , and skl in Drosophila .…”

“…The first RHG homologs identified outside dipterans via bioinformatic search in a new genome sequence was Ibm1 of B. mori (Bryant et al 2009) , which is now identified as the derived S-isoform of the B. mori RHG homolog locus. Paralleling the situation in mosquitoes, it is the ancestrally organized L-RHG isoform that has to be studied (Bryant et al 2009;Shu et al 2020) . Future analyses of both lepidopteran isoforms have the potential to inform about the subfunctionalization trajectories of newly emerging RHG homologs.…”

“…In the Lepidoptera, RHG homologs have been studied in the silkmoth Bombyx mori and the fall armyworm Spodoptera frugiperda (Bryant et al 2009;Wu et al 2013;Shu et al 2020) .…”