Protein-protein interactions mediated by Cys2His2 zinc-fingers

摘要: The C2H2 zinc finger motif is a compact ~ 30 amino acid molecular recognition domain that comprises beta-hairpin followed by an alpha-helix. These domains are typically found as tandem arrays mediate specific interactions with various macromolecules including DNA, RNA and other proteins. Although very well characterized DNA-binding domain, relatively little currently understood about the details of protein-protein mediated ZFs. Ikaros Hunchback transcription factor family provides ideal model system for studying ZF interactions. Ikaros, founding member this defined classical protein composed cluster four ZFs at N-terminus two additional C-terminus. While N-terminal involved in DNA recognition, C-terminal (termed Dimerization Zinc Finger or DZF domain) has been shown to homo- hetero-typic In thesis, have examined using combination genetic, biochemical functional assays. To test if protein-interacting can be used create novel interaction specificities, libraries synthetic DZFs were constructed shuffling individual from human related factors. Using bacterial-based selection system, we identified heterodimeric activation reporter gene bacterial cells. also reconstitute bi-partite activator nucleus cell, which results transcriptional endogenous VEGF-A gene. In addition, these two-finger linked together more extended interfaces. Analysis specificities led discovery anti-parallel mode domain. The homo-typic different was greater detail mutational analysis. studies narrowed down residues likely important dimerization obtain further information physical chemical surface attempted purify active peptides consisting X-ray crystallography. highly purified obtained, attempts refold into resulted formation aggregates DZFs. Based on findings cell culture systems, started exploring dimerizes Drosophila melanogaster its essential function protein. Constructs encoding full-length harboring natural modified generated expressed transgenic flies. transgenics perform vivo Neuroblast specification during development. We confirmed previous maintaining specifying early-born temporal identity neural stem lineages. Importantly, our indicate functionally replaced heterologous (i.e., non fly) suggesting importance due ability dimerization.