Biophysical and biological characterization of FIH-catalysed post-translational asparaginyl hydroxylation.

我们提出要做的工作是基于我们最近对细胞感知氧气的方式所获得的见解。调节向组织输送氧气对所有生物来说都是一个问题，尤其是由数十亿细胞组成的大型动物（如人类）。许多人类疾病，如心脏病、中风、癌症和贫血，都涉及低氧水平（缺氧）对细胞功能的损害。在之前的工作中（其中的重要组成部分得到了BBSRC的支持），我们已经确定了一组加氧酶（催化大气氧气进入其底物的酶），它们作为细胞的“氧传感器”，催化HIF（缺氧诱导因子）蛋白质中特定氨基酸残基的羟基化（涉及氧原子的添加）。羟基化破坏并使HIF失活，但由于它需要氧气，该反应在缺氧条件下被抑制，从而使HIF在缺氧细胞中被激活（因此得名）。HIF是一种转录因子（基因表达的一种调节因子），当它被打开时，它会调节许多基因，这些基因参与改变细胞代谢，生长新血管，增加血液产生和其他帮助身体在缺氧中生存的行为。关于HIF的工作提出了很多问题，如这种类型的修饰（羟基化）是否发生在细胞内其他类型的蛋白质上，以及可能的影响是什么。最近，我们发现hif羟化酶中的一种酶也羟化蛋白质中一个非常常见的结构域，锚蛋白重复结构域（ARD）。ARDs发生在许多具有不同功能的蛋白质中，如转录、细胞信号传导、细胞结构、离子通道、染色体完整性和衰老、炎症和分化。这些发现开辟了一个新的研究领域，研究这种蛋白质修饰的作用，它是如何被缺氧调节的，以及它是如何影响细胞对缺氧的反应的。它还可能提供有关调节HIF的“氧感应”过程类型如何进化的基本线索。这项工作是目标导向的，正在寻求解决基础科学中的一个问题，这个问题对许多不同的生物和生物医学领域可能很重要。这两位申请人来自不同的背景（生物学和化学），他们正在合作，将他们在几种生物物理学、生物化学和生物学方法方面的专业知识结合起来，找出这种类型的蛋白质修饰有多普遍，它如何影响蛋白质的物理性质，以及它如何改变细胞功能。

The work we are proposing to do is based on recent insights we have obtained into the way cells sense oxygen. Regulating the delivery of oxygen to tissues is a problem for all organisms - particularly large animals (such as humans) that are composed of many billions of cells. Many human diseases such as heart attacks, strokes, cancer, and anaemia involve compromise of cell function by low oxygen levels (hypoxia). In previous work (important components of which were supported by the BBSRC) we have identified a group of oxygenases (enzymes that catalyse the incorporation of atmospheric oxygen into their substrates) that act as cellular 'oxygen sensors', catalysing the hydroxylation (involving addition of an oxygen atom) of specific amino acid residues in a protein called HIF (hypoxia inducible factor). Hydroxylation destroys and inactivates HIF, but since it requires oxygen this reaction is suppressed in hypoxia, allowing HIF to become activate in hypoxic cells (hence its name). HIF is a transcription factor (a type of regulator of gene expression) and when it is switched on, it regulates a lot of genes that are involved in altering cell metabolism, growing new blood vessels, increasing blood production and other actions that help the body survive hypoxia. The work on HIF has raised a lot of questions as to whether this type of modification (hydroxylation) occurs for other types of protein within cells and what the effects might be. Recently we have found that one of the HIF-hydroxylases enzymes also hydroxylates a very common structural domain in proteins, the ankyrin repeat domain (ARD). ARDs occur in many types of protein with many different functions, such as in transcription, cell signalling, cell structure, ion channels, chromosome integrity and aging, inflammation and differentiation. These findings have opened up a new field of research on this type of protein modification, what it does, how it is regulated by hypoxia, and how it affects the cell's responses to hypoxia. It might also give fundamental clues about how the type of 'oxygen sensing' process that regulates HIF evolved. The work is goal directed and is seeking to address a problem in basic science that is potentially important for a number of different biological and biomedical fields. The two applicants are from different backgrounds (Biology and Chemistry) and are working in a partnership to combine their expertise across several biophysical, biochemical, and biological approaches to find out how common this type of protein modification is, how is affects the physical properties of the protein, and how this alters cell function.