Physiological properties and functions of phosphatidylinositol 5-phosphate 4-kinase gamma

聚磷酸肌醇脂质是构成哺乳动物细胞膜的磷脂的次要组分（约1%），但它们具有与其数量不成比例的生理和病理重要性。它们总共有七种，每种都有至少一种（大多数都有不止一种）生理作用来调节细胞的功能--这些作用几乎发生在细胞内的每一个可以想象的位置。当这些过程没有得到适当的调节时，可能会发生病理学：聚磷酸肌醇脂质与例如癌症、炎症、神经系统疾病和心血管疾病有关。本申请的一个主题是最近发现的聚磷酸肌醇脂质，磷脂酰肌醇5-磷酸盐（PI 5 P），其存在于细胞的许多位置，并且通过大量间接证据，其与细胞响应应激的方式有关（紫外线、热、氧化性自由基），这些反应显然必须作为它们抵御环境引起的疾病的一部分。PI 5 P的水平（以及其功能，因为其作用主要由给定位置存在多少来调节）由一个酶家族调节：PI 5 P 4-激酶（PI 5 P4 Ks）。人类有三个PI 5 P4 K基因（因此有三个不同的PI 5 P4 K），其中一个，PI 5 P4 K γ，到目前为止几乎完全被忽略了（除了我们）。然而，我们发现它具有惊人的表达模式，在肾脏上皮细胞的精确和特定位置以及大脑和脊髓特定部位的神经元中发现了非常高的水平。在表达PI 5 P4 K γ的细胞内，它似乎附着在细胞质内的膜囊泡上，但这些囊泡是什么，它们在做什么还不清楚（澄清是本研究的主要目的之一）。为了进一步改善我们对PI 5 P4 K γ的了解不足，我们计划将分子细胞生物学的许多尖端工具应用于其研究。我们正处于产生缺乏PI 5 P4 K γ基因的小鼠的最后阶段，这种小鼠在肾脏和神经系统中的功能可能会给我们带来新的见解。即使他们没有，这些小鼠的组织和胚胎干细胞的可用性将为我们提供强大的新工具，以了解这种蛋白质的确切功能以及它的作用。我们在这方面的合作者还将研究如何去除斑马鱼胚胎中的PI 5 P4 K γ改变这种生物体中肾脏和神经元的形式和功能。平行地，我们将使用来源于高度表达PI 5 P4 K γ的肾脏相同部分的细胞系，并且我们还将充分利用称为Nalm-6细胞的细胞系，其来源于人B细胞淋巴瘤。Nalm-6细胞的独特性质（非常容易操纵其中的基因）将使我们能够“标记”，删除和改变PI 5 P4 K γ，以找出它与谁交谈，在哪里，以及它说了什么。

Polyphophoinositol lipids are minor components (about 1%) of the phospholipids that make up mammalian cell membranes, but they have a physiological and pathological importance way out of proportion to their quantity. There are seven of them in all, each with at least one (most with more than one) physiological action to regulate how cells function - and these actions take place in almost every conceivable location within a cell. When these processes are not appropriately regulated, pathologies can occur: polyphosphoinositol lipids are implicated in, for example, cancer, inflammation, neurological disorders and cardiovascular disease. One subject of this application is the most recently discovered polyphosphoinositol lipid, phosphatidylinositol 5-phophate (PI5P), which is present in many locations of the cell and which has, by a sigificant body of circumstantial evidence, been implicated in the way in which cells respond to stress (uv light, heat, oxidising radicals), responses which self-evidently must act as part of their defence against environmentally-induced disease. The levels of PI5P (and thus its functions, because its actions are regulated primarily by how much is present at a given location) are regulated by a family of enzymes: the PI5P 4-kinases (PI5P4Ks). Humans have three PI5P4K genes (and thus three different PI5P4Ks), and one of these, PI5P4K gamma, has been ignored almost completely so far (except by us). Yet we have found that it has a striking expression pattern, being found at very high levels in precise and specific locations in epithelial cells in the kidney, and also in neurons within particular parts of the brain and the spinal cord. Inside the cells that express PI5P4K gamma it appears to be attached to membrane vesicles within the cytoplasm, but what these vesicles are, and what they are doing is unclear (and clarification is one of the major aims of this study).To further improve on our paucity of understanding of PI5P4K gamma we plan to apply many of the cutting edge tools of molecular cell biology to its study. We are in the final stages of generating a mouse lacking the PI5P4K gamma gene, and how this mouse functions in its kidneys and nervous system may give us new insights. Even if they do not, the availability of tissues and embryonic stem cells from these mice will give us powerful new tools to understand exactly where this protein is functioning and what it is doing. Our collaborators in this aspect will also investigate how removal of the PI5P4K gamma in zebra fish embryos alters the form and function of kidneys and neurons in this organism. In parallel we will use cell lines derived from the same part of the kidney that expresses PI5P4K gamma highly and also we will make full use of a cell line called Nalm-6 cells, which is derived from a human B cell lymphoma. The unique properties of Nalm-6 cells (it is extremely easy to manipulate genes in them) will enable us to 'tag', remove, and change PI5P4K gamma in order to find out who it talks to, and where, and what it says.