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

聚磷脂是构成哺乳动物细胞膜的磷脂中的次要成分(约1%)，但它们具有与其数量不成比例的生理和病理重要性。总共有七个细胞，每个细胞至少有一个(大多数有不止一个)生理动作来调节细胞的功能-这些动作发生在细胞内几乎所有可以想象到的位置。当这些过程没有得到适当的调控时，可能会发生病理：多磷肌醇脂质与癌症、炎症、神经疾病和心血管疾病等有关。这一应用的一个主题是最近发现的多磷肌醇脂质，磷脂酰肌醇5-磷酸(PI5P)，它存在于细胞的许多位置，通过大量的间接证据，它与细胞对压力(紫外线、热、氧化自由基)的反应方式有关，不言而喻，这些反应必须作为它们对环境诱导的疾病的防御的一部分。PI5P的水平(以及它的功能，因为它的作用主要由给定位置存在的量来调节)受一系列酶的调节：PI5P4-激酶(PI5P4Ks)。人类有三个PI5P4K基因(因此有三个不同的PI5P4K)，其中一个，PI5P4K Gamma，到目前为止几乎完全被忽视(除了我们)。然而，我们发现它有一个惊人的表达模式，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分的神经元中，都发现了非常高的水平。在表达PI5P4K伽马的细胞内，它似乎附着在细胞质内的膜小泡上，但这些小泡是什么，它们在做什么尚不清楚(澄清是本研究的主要目的之一)。为了进一步改善我们对PI5P4K伽马的了解，我们计划将许多分子细胞生物学的尖端工具应用到它的研究中。我们正处于产生一只缺乏PI5P4K伽马基因的小鼠的最后阶段，这只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。即使没有，这些小鼠的组织和胚胎干细胞的可获得性也将给我们提供强大的新工具，让我们确切地了解这种蛋白质在哪里发挥作用，它在做什么。我们在这方面的合作者还将调查斑马鱼胚胎中PI5P4K伽马的移除如何改变这种有机体中肾脏和神经元的形态和功能。与此同时，我们将使用来自肾脏高表达PI5P4K伽马的同一部分的细胞系，我们还将充分利用一种名为NALM-6细胞的细胞系，它来自人类B细胞淋巴瘤。NALM-6细胞的独特性质(操纵其中的基因非常容易)将使我们能够对PI5P4K伽马进行标记、移除和更改，以便找出它与谁交谈，在哪里交谈，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.