Grants and Contracts Details
Nebulette is a modular protein that functions in the organization of the cardiac sarcomere. The 108 KDa protein was identified by its antigenic similarity to the giant skeletal muscle protein nebulin. Nebulette is 70% homologous to the C-terminal region of nebulin. There are four functional domains of nebulette, the N-terminal domain, the repeat region, a linker domain and the C-terminal SH3 domain. The SH3 domain has been shown to bind a-actinin, a major component of the Z-line and along with the linker domain is proposed to target nebulette to the Z-line. The repeat region is comprised of 35-residue nebulin repeats, which mediate the interaction of nebulette with actin. A binding partner has not been identified for the N-terminal domain, however there is 71 % identity or conserved sequence between the chicken and human N-termini. Nebulette has been identified as a binding partner for myopalladin and zyxin, however the interactions were not characterized. The goal of the proposed research is to identify and characterize binding partners for nebulette in the cardiac sarcomere. The proposed research has two specific aims. (1) The yeast two-hybrid system will be used to find candidate binding partners for nebulette. Binding partners will be characterized genetically and biochemically. The screen will employ a cDNA library constructed from embryonic chicken heart mRNA. The b-galactosidase reporter assay will be used to study the relative binding strength between interacting proteins and deletion constructs. Microtiter plate binding assays will be performed using bacterially expressed individual domains of nebulette probed with serial dilutions of biotinylated interacting proteins. (2) Confirmation of the sub-cellular localization of nebulette binding partners to the cardiac sacrcomere will be demonstrated by expressing GFP fusions in chicken embryo cardiomyocytes and colocalization with immunofluorescent labeled myofibrillar proteins. The GFP fusions will be visualized using epifluorescence microscopy. Expression of GFP fusions with sections deleted will allow the identification of those domains important for localization. This research will provide detailed information on molecular interactions in the cardiac thin filament and Z-line. Mutations in nebulette and other sarcomeric proteins have been associated with inherited cardiomyopathy.
|Effective start/end date||7/1/02 → 6/30/04|
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