Abstract
Insect hexamerins are actively synthesized by the larval fat body and stored in the hemolymph. During metamorphosis, these proteins are sequestered by the fat body to be used as an amino acid resource for reconstruction of the adult tissues. The hexamerins of some insect species can also function as a source of amino acids for eggs production. There is circumstantial evidence, however, of additional functions in the A. mellifera development. Two sets of data from our Laboratory (Laboratory of Bee Developmental Biology - LBDA) support this hypothesis. The first one refers to the relationship between the levels of hexamerins and juvenile hormone (JH), which has an essential function in metamorphosis and castes differentiation. An in silico analysis of the UTRs (untranslated regions) of the four hexamerin genes revealed a potential binding site for USP (Ultraspiracle protein), which together with other regulatory proteins, integrates the JH nuclear receptor. In support to the functionality of the response element to USP, our in vivo experiments on hormonal manipulation and transcripts quantification by qRT-PCR showed that the hexamerin genes are up-regulated by JH. On the other hand, the contrasting quantitative profiles of both, hexamerin transcripts and JH, suggest that these proteins act as JH-binding proteins, thus controlling JH titer and action. Taken together, these data point to a feedback regulatory loop in which the hexamerins are induced by the increase in JH titer but are also able to bind and inactivate JH, thus regulating its titer. If this is confirmed, the hexamerins have a critical role in JH-regulated developmental processes, including metamorphosis and caste differentiation. The second set of results, obtained by using immunofluorescence and confocal microscopy, showed that in addition to their well-known presence in the fat body cell cytoplasm, the hexamerins are also found in the nuclei of both tissues, fat body and gonads. This unexpected finding suggests that hexamerins have regulatory or structural roles in the cell nuclei. Based on these results and on data from previous experiments (insertion of the four hexamerin genes into bacteria for in vitro production of the respective hexamerins), we are here proposing new experimental approaches, aiming to elucidate the functions of hexamerins. To this end, we intend to use (1) nuclear protein markers to identify the sub-nuclear location of the four hexamerins, (2) calorimetry to verify the molecular interaction between the hexamerin subunits, and the interaction between each subunit and JH; (3) post-transcriptional silencing mediated by RNAi to identify the role of each hexamerin during metamorphosis (pupal and pharate adult developmentin); (4) CHIP-Seq to investigate whether hexamerins function as regulatory proteins and, if so, to characterize their DNA binding sites. These approaches were designed to provide a breakthrough in the functional characterization of hexamerins.
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