Dissecting Apoptotic and growth regulatory pathways using structural biology
EMSL Project ID
2431
Abstract
The 3-dimensional structure of proteins and their complexes provides a wealth of information on how proteins interact, how they are regulated and how they can be modulated by therapeutic agents. Furthermore, for cases of newly discovered genes that have not been fully characterized functionally or biochemically, the 3-D structure can often provide clues to, and in some cases identify, important biochemical functions that are not evident from sequence alone. We will use structural biology techniques, in particular NMR spectroscopy, to determine the 3D structures of proteins involved several important apoptotic pathways. The resulting structures and subsequent biochemical analysis will add greatly to our understanding of how these proteins function. For the three projects below (funded by the National Cancer Inst. of Canada) we are able to prepare protein samples that give good NMR HSQC spectra, however, we will require a lot of spectrometer time and in several cases the high sensitivity and spectral dispersion afforded by high field instruments to study samples with limited solubility (PIRH2) or highly dissordered regions of proteins (c-myc and BRCA1). Specific Aim 1. NMR structural studies of p53-regulated genes involved in apoptosis.Our Colleague, Sam Benchimol, has identified several new p53-regulated genes that are specifically involved in p53-mediated apoptosis. Two of these genes, PIHR2 and PIDD, have conserved domains (ring finger and death domains, respectively) that suggest roles in protein-protein interactions which may regulate apoptosis. We will carry out structural studies of these small domains using NMR. Although the 3D structures of a viral ring finger and other death domains are known, it is important to know the high resolution structure of the equivalent domains in PIHR2 and PIDD, as well as additional uncharacterized domains in these proteins. The structures of these subdomains will provide important clues to the function of PIHR2 and PIDD. We also intend to use the same techniques to characterize any newly identified p53-regulated genes arising from work in the Benchimol Lab.Specific Aim 2. Structure-Function studies of c-myc and myc-binding proteins.. We have been investigating the structure of the N-terminal domain of c-myc and its interactions with other proteins. We have shown the N-terminus, including the conserved myc-box I and mycbox II regions is unstructured in the absence of other interacting partners. We are currently studying the myc-binding region of the tumor suppressor, Bin-1, which has been reported to suppress the transformation properties of c-myc, possibly by promoting myc-induced apoptosis. Bin-1 was originally identified in a screen for proteins that bind to the N-terminus of c-myc, and an alternative spliced form of Bin-1, which is incapable of binding to myc, is over-expressed in many melanomas and most cell lines. Specific Aim 3. Structure-Function studies of uncharacterized BRCA1 domains and other novel proteins involved in apoptosis.BRACA1 is a very large protein with large regions that remain uncharacterized, yet contain mutations found in breast cancer patients. We intend to use our domain mapping and NMR techniques to study the structure and function of these uncharacterized regions as well as novel apoptosis genes identified by other team members.
Project Details
Project type
Capability Research
Start Date
2002-04-01
End Date
2002-11-18
Status
Closed
Released Data Link
Team
Principal Investigator