VIAFs are a conserved protein family (initially identified by our collaborators in Colin Duckett's lab in NCI), that associate with animal IAPs (inhibitor of apoptosis proteins). VIAF itself substantially protects cells from Fas- and Bax-induced apoptosis, while coexpression of VIAF with suboptimal quantities of XIAP conferred almost complete protection from these inducers. VIAF and XIAP activated JNK in a synergistic manner. Hence, VIAR is a novel cofactor which modulates the anti-apoptotic and signaling properties of the IAP family. Full length VIAF contains 239 residues, but because the C-terminal 128 residues are sufficient (and necessary) for interaction with IAPs from baculovirus, we have cloned a 158 residue (17.8 kDa) C-terminal construct of hVIAF (human VIAF) into a pET-16b vector which was then transformed into BL-21 cells. The His-tag protein was expressed in minimal media, purified using Ni-NTA column, treated with factor-Xa to remove the tag and subsequently passed over Ni-NTA and size exclusion columns. A one liter culture yields sufficient protein for NMR studies. The 15N-HSQCspecturm of VIAF-C158 is well dispersed, indicating that it is a highly structured globular protein. The the T2 measured for VIAF-C158, 83ms is in reasonable agreement with that calculated for a 17.8 kDa protein at 298 K,105 ms. We have produced both 15N labeled and 15N/13C double-labeled material for a structure determination and are currently optimizing sample conditions for acquiring NMR spectra. When this is completed, we will determine the three dimensional solution structure of VIAF-C158. The interaction of VIAF-C158 with XIAP will also be studied by NMR; specifically, we will to map the amino acid residues of VIAF-C158 that interact with XIAP.

National Institute of Health (NIH)
National Institute of Dental & Craniofacial Research (NIDCR)
Intramural Research (Z01)
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Dental & Craniofacial Research
United States
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