STRUCTURE/FUNCTION STUDY OF VIAF

VIAF的结构/功能研究

• 批准号: 6413848
• 负责人: DENNIS A TORCHIA
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DENTAL & CRANIOFACIAL RESEARCH
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6413848
• 关键词: apoptosis; cell growth regulation; cofactor; globular protein; molecular cloning; nuclear magnetic resonance spectroscopy; protein sequence; protein structure function; tissue /cell culture

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.

VIAFS是一种保守的蛋白质家族（最初由我们在NCI的Colin Duckett实验室中的合作者确定），该家族与动物IAP（凋亡蛋白抑制剂）相关。 VIAF本身基本上保护细胞免受FAS和BAX诱导的细胞凋亡的影响，而ViaF与次优量的XIAP共表达几乎完全保护了这些诱导剂。 VIAF和XIAP以协同的方式激活JNK。因此，Viar是一种新颖的辅助因子 调节IAP家族的抗凋亡和信号传导特性。全长VIAF包含239个残基，但是由于C末端128个残基足以与杆状病毒的IAP相互作用，因此我们将HVIAF（人Viaf）的158个残基（17.8 kDa）C末端构造克隆到PET-16B载体中，然后将其转化为BL-21细胞。 His-tag蛋白在最小培养基中表达，使用Ni-NTA柱进行纯化，并用Factor-XA处理以删除标签，然后通过Ni-NTA和大小排除柱经过。一升培养可为NMR研究产生足够的蛋白质。 VIAF-C158的15N-HSQCSPECTURM分散得很好，表明它是一种高度结构化的球状蛋白。与VIAF-C158，83ms测得的T2与在298 K时为17.8 kDa蛋白计算出的T2合理一致，105毫秒。我们已经生产了15N标记和15N/13C双标签材料，以确定结构，目前正在优化采集NMR光谱的样本条件。完成后，我们将确定VIAF-C158的三维解决方案结构。 NMR还将研究Viaf-C158与XIAP的相互作用。具体而言，我们将绘制与XIAP相互作用的VIAF-C158的氨基酸残基。