How does the clustering of phosphatidylinositol phosphates assist in pleckstrin homology domain binding of membranes?

组成我们身体的细胞在最简单的水平上由脂膜组成，脂膜包含一个充满水的空间，由细胞核从中心控制（DNA储存在那里，并且有自己的次级膜用于保护）。细胞必须相互发出信号，并拥有通讯系统，以调节生命的基本过程：DNA复制、细胞分裂、细胞生长，以及通过细胞间相互作用形成组织和器官。所采用的主要信号传导途径是基于蛋白质，这些蛋白质彼此结合并与其他分子如DNA和脂质结合。这意味着细胞表达哪些蛋白质以及表达水平将决定细胞的行为以及与周围环境的相互作用。为了使细胞信号转导的有效进化成为可能，自然选择依赖于蛋白质“模块”或“结构域”。各个模块以多种形式出现，每种模块都有一个共同的基本结构，在每种情况下都适用于不同的活动。这意味着，而不是两个分子之间的相互作用已经从零开始反复进化，在进化过程中，现有的相互作用界面已经适应了新的roles.This拨款建议涉及一个重要的模块类型，pleckstrin同源（或PH）域。PH结构域与蛋白质-膜相互作用相关，但也参与蛋白质-蛋白质界面。当结合膜时，PH结构域通常与通过化学添加磷酸基团修饰的特殊脂质分子相互作用，特别是称为磷脂酰肌醇磷酸（PIP）的脂质。虽然一些PH结构域已经被证明与一种PIP紧密且非常特异性地结合（它们具有高亲和力），但其他PH结构域似乎具有低亲和力-可能太低而与细胞的复杂环境无关-并且对结合伴侣不太挑剔。我们最近发现，一个PH结构域似乎不与单个PIP结合，而是与聚集在一起的几个PIP结合，这使其结合的紧密性增加了约300倍。我们提出，这种机制，即一个PH结构域依赖于强度的数字，以目标特定的膜可能是相关的一些其他PH结构域，其测量的亲和力为他们假定的PIP目标是low. We的出发点将着眼于PH结构域的三个相关的人类蛋白质，称为kindlin-1，kindlin-2和kindlin-3。在研究了kindlin-3之后，我们想看看这个小蛋白家族的其他两个成员是否具有相似的特性，并以更高的亲和力识别PIP簇。迄今为止我们收集的所有数据都表明，它们很可能会这样做。Kindlin是一组值得研究的有趣蛋白质，因为它们在细胞中的几个不同点上都起着非常重要的作用，因此了解它们如何结合到细胞某个部分的一个膜上或移动到细胞的另一个部分是非常重要的。例如，众所周知kindlin激活细胞相互粘附的机制，但它们也控制生长信号等，并移动到细胞核以进行完全不同的活动，控制基因表达。我们将比较PIP聚类增强kindlin PH结构域结合的能力，以及我们已经知道与单个PIP分子紧密结合的其他蛋白质。然后，我们将测试是否有更多被认为具有低PIP亲和力的蛋白质利用“数量优势”来加强它们的结合。我们将要研究的蛋白质已知在疾病中很重要，包括大量不同的癌症，以及心脏病和中风。这意味着研究一种新的机制的可能性，这种机制是它们生物学效应的基础，可以帮助我们更好地理解影响它们发挥作用的因素-不仅在正常细胞功能中，而且在出现问题时。

The cells which make up our bodies consist at the simplest level of a lipid membrane containing a water-filled space controlled from the centre by the cell nucleus (where DNA is stored and which has its own secondary membrane for protection). Cells have to signal to one another and possess systems of communication in order to regulate the basic processes of life: DNA replication, cell division, cell growth and, by cell-cell interaction, the formation of tissues and organs. The major signalling routes employed are based on proteins which bind to one another and to other molecules like DNA and lipids. This means that which proteins a cell expresses and at what levels will determine how it behaves and the interactions it enjoys with its surroundings.To enable the efficient evolution of cell signalling natural selection relies on protein "modules" or "domains". Individual modules crop up in multiple forms, and each kind shares a common basic structure adapted in each case to a different activity. This means that rather than interactions between two molecules having evolved repeatedly from scratch, over the course of evolution existing interaction interfaces have been adapted for new roles.This grant proposal concerns one important module type, the pleckstrin homology (or PH) domain. PH domains are associated with protein-membrane interactions but also get involved in protein-protein interfaces. When binding membranes, PH domains often interact with special lipid molecules which have been modified by the chemical addition of phosphate groups, in particular lipids called phosphatidyl inositol phosphates (PIPs). Although some PH domains have been shown to bind tightly and very specifically to one kind of PIP (they have high affinity), others seem to have a low affinity - maybe too low to be relevant in the complex environment of a cell - and not to be very choosy about binding partners. We recently found that one PH domain seems to bind not to a single PIP but to several PIPs clustered together and that this increases the tightness of its binding about 300x. We are proposing that this mechanism whereby a PH domain relies on strength in numbers to target specific membranes could be relevant to a number of other PH domains whose measured affinity for their putative PIP target is low.Our starting point will be to look at the PH domains of three related human proteins, called kindlin-1, kindlin-2 and kindlin-3. Having looked at kindlin-3 already, we want to see if the other two members of this small protein family have similar properties and recognise clusters of PIPs with increased affinity. All the data we have collected to date suggest that they probably will.Kindlins are an interesting group of proteins to study because they have very imporant roles at several different points in the cell, and so understanding how it is that they bind to one membrane in one part of the cell or move to another part of the cell is something that matters very much. For example, the kindlins are well known to activate mechanisms by which cells stick to one another, but they also control things like growth signals and move to the cell nucleus to take up completely different activities, controlling gene expression.We will compare the power of PIP clustering to enhance kindlin PH domain binding, to other proteins which we already know bind tightly to a single PIP molecule. Then we will test whether more proteins considered to have low PIP affinity make use of "strength in numbers" to tighten their binding. The proteins we are going to study are known to be important in disease, including a large number of different cancers, and also heart disease and stroke. This means that investigating the possibility of a new kind of mechanism which underlies their biological effect is something that can help us understand better factors influencing the roles they play - not only in normal cell function but also when things go wrong.