This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Chemical cross-linking followed by identification of the cross-linked peptides by mass spectrometry has proved to be especially useful in dynamic and complex systems when crystallization of the protein complex is difficult to achieve. Ffh, the protein subunit of the signal recognition particle (SRP) in bacteria, and the SRP receptor, FtsY, play a critical role in delivering nascent membrane and secretory proteins to the bacterial plasma membrane. At the very heart of this protein targeting reaction is the recognition between Ffh and FtsY. We have investigated the E. Coli Ffh-FtsY complex and the T. Aquaticus Tfh-TftsY complex using chemical cross-linking and mass spectrometry. Geldanamycin-Induced Degradation of p185c-erbB-2: This project involves an important system for the investigation of chaperon complexes and the ubiquitination process. Breast cancer is the leading form of cancer affecting American women and one out of nine women will develop breast cancer during her lifetime. Gene amplification and protein overexpression of c-erbB-2 (also designated as HER-2/ neu) are strongly associated with breast cancer and membrane-associated p185c-erbB-2 exists in a stable complex with 94-kDa glucose-regulated protein (Grp94). Geldanamycin binding to Grp94 disrupts this complex leading to the proteolytic degradation of existing p185c-erbB-2 protein and resulting in growth inhibition in most of the tumor cell lines. In the on-going studies we will further investigate components of this chaperon complex to obtain information on protein-protein interactions; reveal the identification of the degradation signal and of the E3-like or auxiliary proteins that recognize this signal; and characterize nascent p185c-erbB-2 and its corresponding E3 ligase. (Additional effort and instrument time reported under Collaborative projects and other Technical Research and Development projects.)
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