Unravelling the structural biology of a hypoxia scaffold protein using novel NMR approaches

氧气平衡受到严格调节，以确保细胞和组织获得足够的氧气。快速反应和适应低氧浓度（“缺氧”）可以减少损伤，使细胞保持活力。这个过程被称为“缺氧反应”。在低氧条件下，缺氧诱导转录因子（hif）控制促进缺氧反应的因子的表达。然而，在常氧条件下，HIF亚基（HIFa）中的特定脯氨酸残基被脯氨酸羟化酶（PHD）羟基化。这些羟脯氨酸修饰导致HIF被VHL E3泛素连接酶复合物泛素化并最终降解。一种名为LIMD1的支架蛋白对缺氧反应信号通路至关重要。LIMD1与PHD和VHL相互作用并进行配位。iCASE博士项目的目标是表征LIMD1在缺氧信号传导中介导的蛋白/蛋白相互作用的结构生物学。该博士生将使用结构、生物物理和生化方法来探索LIMD1的结构/功能关系，包括研究超分子蛋白质复合物的尖端核磁共振方法。LIMD1具有结构化结构域和内在无序区域，两者都包含重要的蛋白质识别位点。我们已经与NMR- bio合作，这是一家总部位于格勒诺布尔的公司，该公司开发了新型试剂，有助于对大型蛋白质复合物进行核磁共振分析。该学生将在格勒诺布尔度过三个月，在那里他们将接受核磁共振研究中细菌和真核蛋白生产技术的培训。在约克进行的LIMD1的结构和生物物理分析结果将推动与伦敦巴茨癌症研究所泰森夏普博士小组合作进行的分子细胞生物学研究。缺氧反应的功能障碍与许多人类疾病有关，包括癌症的生长和扩散。在iCASE博士项目中进行的结构/功能分析将阐明LIMD1在这些过程中的作用。

Oxygen homeostasis is tightly regulated to ensure that cells and tissues are supplied with sufficient oxygen. Rapid reaction and adaptation to low oxygen concentrations ('hypoxia') can reduce damage and enable cells to remain viable. This process is known as the 'hypoxic response'. In low oxygen conditions, hypoxia-inducible transcription factors (HIFs) control expression of factors that facilitate the hypoxic response. However, under normoxic conditions, specific proline residues in one of the HIF subunits (HIFa) are hydroxylated by a prolyl hydroxylase (PHD). These hydroxyproline modifications lead to ubiquitination of HIF by the VHL E3 ubiquitin ligase complex and its eventual degradation.A scaffold protein called LIMD1 is critical to the hypoxic response signalling pathway. LIMD1 interacts with and collocates PHD and VHL. The objective of this iCASE PhD project is to characterize the structural biology of the protein/protein interactions mediated by LIMD1 in hypoxic signalling. The PhD student will use structural, biophysical and biochemical approaches to explore the structure/function relationship of LIMD1, including cutting-edge NMR methods for studying supramolecular protein complexes. LIMD1 has structured domains and intrinsically disordered regions, both of which contain important protein recognition sites. We have teamed up with NMR-Bio, a Grenoble-based company that develops novel reagents that facilitate NMR analyses of large protein complexes. The student will spend three months in Grenoble where they will receive training in bacterial and eukaryotic protein production techniques for NMR studies. The results of the structural and biophysical analyses of LIMD1 conducted in York will drive molecular cell biology studies performed in collaboration with Dr Tyson Sharp's group at the Barts Cancer Institute, London.The dysfunction of the hypoxic response is implicated in many human diseases, including the growth and spread of cancers. The structure/function analyses conducted in this iCASE PhD project will illuminate the role of LIMD1 in these processes.