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

我们计划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。对所有生物体--尤其是由数十亿个细胞组成的大型动物(如人类)--来说，调节氧气向组织的输送是一个问题。许多人类疾病，如心脏病发作、中风、癌症和贫血，都涉及低氧水平(缺氧)对细胞功能的损害。在以前的工作中(其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(催化将大气中的氧气结合到其底物中的酶)，它们作为细胞的“氧传感器”，催化称为HIF(缺氧诱导因子)的蛋白质中特定氨基酸残基的羟化(涉及氧原子的添加)。羟化作用破坏和失活HIF，但由于它需要氧气，这一反应在低氧条件下被抑制，允许HIF在低氧细胞中激活(因此而得名)。HIF是一种转录因子(一种基因表达调节因子)，当它被打开时，它调节许多基因，这些基因涉及改变细胞代谢、生长新血管、增加血液产量和其他帮助身体在缺氧中生存的行为。对HIF的研究提出了许多问题，即这种类型的修饰(羟化)是否发生在细胞内的其他类型的蛋白质上，以及可能产生的影响。最近我们发现，HIF-羟基酶中的一种酶还可以羟化蛋白质中一个非常常见的结构域，即锚蛋白重复结构域(ARD)。ARDS存在于多种蛋白质中，具有多种不同的功能，如转录、细胞信号、细胞结构、离子通道、染色体完整性和衰老、炎症和分化。这些发现为这种蛋白质修饰开辟了一个新的研究领域，包括它的作用，它如何受到低氧的调节，以及它如何影响细胞对低氧的反应。这也可能给出调节HIF的“氧气感应”过程如何进化的基本线索。这项工作是以目标为导向的，正在寻求解决基础科学中的一个问题，该问题对许多不同的生物和生物医学领域具有潜在的重要意义。这两位申请者来自不同的背景(生物学和化学)，他们正在合作，将他们在几种生物物理、生化和生物学方法方面的专业知识结合起来，以了解这种类型的蛋白质修饰有多常见，它如何影响蛋白质的物理性质，以及这种修饰如何改变细胞功能。

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.