Identification and functional characterization of a novel ovarian-specific Kelch domain-containing gene involved in the ecdysteroid signaling pathway of Penaeus monodon

Abstract

In crustaceans, vitellogenin (Vg) synthesis is regulated by complex hormonal and molecular networks. This study investigates the role of the ecdysone receptor (PmEcR) in regulating Vg expression and identifies downstream effectors in Penaeus monodon. Silencing of PmEcR using RNA interference resulted in a two-fold increase in Vg expression and a significant rise in the gonadosomatic index (GSI), suggesting PmEcR acts as a suppressor of vitellogenesis. Through a suppression subtractive hybridization (SSH) technique, an uncharacterized LOC 113805388 gene, was identified as being downregulated upon PmEcR silencing. Functional and structural characterization revealed this transcript as an ovarian-specific protein with a β-propeller structure (Kelch-like domain-containing protein; PmKel), suggesting that it belongs to a Kelch-like protein family generally involved in protein-protein interactions in cellular regulation. LC-MS/MS analysis of the ovarian proteome confirmed the presence of the PmKel protein, verifying its expression in the ovary. PmKel expression displayed an inverse pattern to that of Vg during ovarian development, and its knockdown was associated with increased Vg transcript levels, suggesting a possible role in the regulation of Vg gene expression. In addition, a recombinant PmKel protein was produced and used as a bait to identify its partners in ovarian nuclear extract by affinity purification mass spectrometry (AP-MS). The isolated set of interacting proteins highlighted functional enrichment in mRNA and protein metabolic processes involved in developmental pathways. Also, these proteins could interact with a Kelch-like domain predicted on PmKel 3D protein structure by protein network analysis and protein-protein docking. These findings suggest that PmKel is a novel ovarian specific Kelch-containing protein that plays a key role in the vitellogenesis regulatory network via the EcR pathway. Collectively, these results add to the current knowledge of shrimp reproduction and may suggest future direction for improving reproductive per formance in aquaculture.