Peptide aptamers as tools to study protein function in Drosophila.

摘要

Two-hybrid technology provides a simple way to isolate small peptides that specifically recognize and strongly bind to a protein of interest. Such peptide aptamers have the potential to dominantly interfere with specific activities of their target proteins, and therefore could be used as in vivo inhibitors. We developed an approach in which peptide aptamers are used to inhibit protein function in Drosophila. In our approach peptides are isolated with an advanced version of the two-hybrid system. Using a specially designed two-hybrid assay, aptamers that disrupt protein-protein interactions can then be identified. These disruptive aptamers can be delivered into living Drosophila to disrupt specific protein interactions in vivo. We tested the aptamer approach by demonstrating that peptides directed against cyclin-dependent kinases (Cdks) could be used to specifically target the Cdks during Drosophila development.; We also used peptide aptamers to explore the function of cyclin J, a previously uncharacterized Drosophila protein. Our cytological and biochemical analyses have shown that cyclin J is present in early embryos, where it forms active kinase complexes with cyclin dependent kinase 2 (Cdk2). This suggested a possible role for cyclin J in the control of the DNA synthesis (S) phases during the syncytial nuclear division cycles. These early cycles have a number of features that distinguish them from later cell cycles, including very rapid S phases, the lack of certain checkpoints, and the absence of gap phases. The molecular mechanisms that account for these unique features are largely unknown. Here we show that antibodies and peptide aptamers that inhibit cyclin J-mediated Cdk activity cause phenotypes consistent with a requirement of cyclin J for DNA replication during the syncytial cycles. Thus, the results obtained with peptide aptamers confirmed that cyclin J is a unique embryonic cell cycle regulator. Combined, the results of this study demonstrate that peptide aptamers can serve as inhibitory reagents to target specific proteins and protein interactions in vivo. Potentially, the aptamer approach based on the use of inhibitory peptides may become useful for systematic analysis of protein function.