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The role of the Cdc28 Cbk1 and Tpk1 kinases in the formation of hyphae of the human fungal pathogen Candida albicans

Cdc28 Cbk1 和 Tpk1 激酶在人类真菌病原体白色念珠菌菌丝形成中的作用

基本信息

批准号：
BB/J002305/1
负责人：
Peter Edwin Sudbery
金额：
$ 59.28万
依托单位：
University of Sheffield
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2011
资助国家：
英国
起止时间：
2011 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FJ002305%2F1
关键词：
role Cdc28 Cbk1 Tpk1 kinases

项目摘要

Candida albicans is a fungus that is responsible for causing vaginitis (thrush) in women and oral infections in AIDs patients. In certain groups of vulnerable individuals, C. albicans can enter the blood stream and spread to internal organs. Such bloodstream infections are extremely serious with a mortality rate of up to 50%. Some surveys place Candida infections as the second most common cause of death from infections acquired in hospitals. A particular feature of its biology is its ability to grow as unicellular budding yeasts or in long filaments of tube-like cells called hyphae. This ability to switch growth modes is thought to be important for its pathogenicity. The hyphae allow the fungus to penetrate through mucosal membranes and enter the bloodstream. They also allow the fungus to escape after they have been engulfed by macrophages. Understanding the molecular mechanisms operating when the organisms switches from the yeast to the hyphal growth form is an important first step towards the development of new forms of treatment and prevention. We know the switch from yeast to hyphal growth form involves the production of new types of protein encoded by the activation of genes that are normally silent in yeast cells. One gene that is activated encodes a protein called Hgc1 that partners another protein called Cdc28 which is a type of enzyme called a kinase. Kinases control the activity of other proteins by adding a phosphate group to certain amino acid subunits which make up the proteins and in doing so change their properties in a different ways. We know that kinases play key role orchestrating a wide variety of the complex process in living cells, so it is not surprising that kinases should be important in promoting hyphal growth. Two other kinases, Cbk1 and Tpk1, are known to be required for hyphal growth. The proposed work is designed to identify the proteins that are targeted by Cdc28, Cbk1 and Tpk1. This will tell us which proteins form part of the hyphal growth machinery and allow us to investigate how their properties are altered during hyphal growth. Our existing knowledge of cell growth allows us to guess some of the proteins that might be targeted by the kinases. We have already instigated a program of experiments on this basis and already have some promising results. The first stage of the program aims to finish these studies. Because it would be foolish to suppose that our knowledge is good enough to guess all the proteins involved, the second stage of the investigation will broaden the search for kinase targets based on a procedure to identify which proteins physically associate with each of the kinases. A short stretch of amino acids called an epitope tag will be added to each kinase that is recognized by antibodies attached to magnetic beads which can be collected by a magnet allowing the kinase to isolated from a cell lysate. Any protein to which the kinase physically interacts will also be captured and its identity can be revealed by Mass Spectroscopy. A potential problem is that there may many false positive hits by proteins which attach to the antibody or magnetic beads non-specifically. To recognize these proteins we will mix the lysate culture containing the tagged kinase that recognizes the antibody with an equal amount of lysate from an untagged culture. The tagged culture will be labeled with a heavy isotope. Fragments of protein that bind non-specifically will be present in 1:1 heavy:light ratio because they will be present in equal amounts from the tagged and untagged culture, but proteins that the kinase binds specifically will contain an excess of heavy isotope that can be detected by mass spectroscopy. Despite this modification it will still be necessary to carry out further experiments to verify the hits from the screen. When this has been completed we hope to be able to construct a network showing the proteins modified by the kinases and their role in promoting hyphal growth.

白色念珠菌是一种真菌，它导致女性阴道炎(鹅口疮)和艾滋病患者的口腔感染。在某些易受感染的人群中，白念珠菌可以进入血液并传播到内脏。这种血液感染极其严重，死亡率高达50%。一些调查显示，念珠菌感染是医院感染导致死亡的第二大常见原因。它生物学上的一个特殊特征是它能够以单细胞发芽酵母的形式生长，或者在称为菌丝的管状细胞的长丝中生长。这种转换生长模式的能力被认为是其致病性的重要因素。菌丝使真菌穿透粘膜进入血液。它们还允许真菌在被巨噬细胞吞噬后逃脱。了解当生物体从酵母菌转变为菌丝生长形式时的分子机制是开发新的治疗和预防形式的重要第一步。我们知道，从酵母到菌丝生长形式的转换涉及到通过激活通常在酵母细胞中沉默的基因来编码的新型蛋白质的产生。一个被激活的基因编码一种名为Hgc1的蛋白质，与另一种名为CDc28的蛋白质配对，这是一种称为激酶的酶。激酶通过在组成蛋白质的某些氨基酸亚基上添加一个磷酸基团来控制其他蛋白质的活性，并在这样做的过程中以不同的方式改变其性质。我们知道，在活细胞中，激酶在协调各种复杂的过程中起着关键作用，因此，在促进菌丝生长方面发挥重要作用也就不足为奇了。已知另外两种激酶Cbk1和Tpk1是菌丝生长所必需的。这项拟议的工作旨在识别CDC28、Cbk1和Tpk1靶向的蛋白质。这将告诉我们哪些蛋白质构成了菌丝生长机制的一部分，并使我们能够研究它们的性质在菌丝生长过程中是如何改变的。我们现有的关于细胞生长的知识使我们能够猜测一些可能成为激酶靶标的蛋白质。我们已经在此基础上发起了一项实验计划，并已经取得了一些有希望的结果。该计划的第一阶段旨在完成这些研究。因为假设我们的知识足够好，可以猜测所有涉及的蛋白质是愚蠢的，所以第二阶段的研究将扩大对激酶靶标的搜索，其基础是识别哪些蛋白质与每种激酶物理上相关。一小段称为表位标签的氨基酸将被添加到每个被附着在磁珠上的抗体识别的激酶上，这些抗体可以通过磁铁收集，从而从细胞裂解物中分离出激酶。任何与该激酶物理上相互作用的蛋白质也将被捕获，其身份可以通过质谱学来揭示。一个潜在的问题是，非特异性地附着在抗体或磁珠上的蛋白质可能会产生许多假阳性。为了识别这些蛋白质，我们将含有识别抗体的标记激酶的裂解液培养物与来自非标记培养物的等量裂解物混合。贴上标签的培养物将被贴上重同位素标签。非特异性结合的蛋白质片段将以1：1的重：轻比例存在，因为它们将在标记和未标记的培养物中以等量存在，但激酶特定结合的蛋白质将包含过量的重同位素，可通过质谱仪检测到。尽管做了这样的修改，但仍然需要进行进一步的实验来验证来自屏幕的点击。当这项工作完成后，我们希望能够构建一个网络，展示被激酶修饰的蛋白质及其在促进菌丝生长中的作用。