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

构成我们身体的细胞是由最简单的脂质膜组成的，脂质膜包含一个由细胞核控制的充满水的空间（DNA储存在这里，细胞核有自己的二级膜来保护）。细胞之间必须相互传递信号，并拥有通信系统，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。所使用的主要信号通路是基于相互结合的蛋白质，以及与DNA和脂质等其他分子结合的蛋白质。这意味着细胞表达的蛋白质种类和水平将决定它的行为以及它与周围环境的相互作用。为了实现细胞信号的有效进化，自然选择依赖于蛋白质“模块”或“结构域”。单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，在每种情况下适应不同的活动。这意味着，不是两个分子之间的相互作用从无到有地反复进化，而是在进化过程中，现有的相互作用界面已经适应了新的角色。这项拨款提案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-膜相互作用有关，但也参与蛋白质-蛋白质界面。当结合膜时，PH结构域经常与特殊的脂质分子相互作用，这些脂质分子被磷酸基团的化学添加修饰，特别是被称为磷脂酰肌醇磷酸（PIPs）的脂质。尽管一些PH结构域已被证明与一种PIP紧密且非常特异性地结合（它们具有高亲和力），但其他结构域似乎具有低亲和力-可能太低而无法与细胞的复杂环境相关-并且对结合伙伴不太挑剔。我们最近发现，一个PH结构域似乎不是与单个PIP结合，而是与聚集在一起的几个PIP结合，这使其结合的紧密程度增加了约300倍。我们提出这种机制，即PH结构域依赖于数量的强度来靶向特定膜，可能与许多其他PH结构域相关，这些PH结构域对其假定的PIP目标的亲和力较低。我们将从三个相关的人类蛋白质的PH结构域开始，分别是kindlin-1、kindlin-2和kindlin-3。在研究了kindin -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，并能识别具有更高亲和力的pip簇。迄今为止我们收集到的所有数据都表明，他们很可能会这样做。Kindlins是一组值得研究的有趣的蛋白质因为它们在细胞的几个不同部位都有非常重要的作用，所以了解它们是如何与细胞的一个部分的细胞膜结合或移动到细胞的另一个部分是非常重要的。例如，众所周知，kindlins可以激活细胞相互粘附的机制，但它们也可以控制生长信号，并移动到细胞核中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“数量上的强度”来加强它们的结合。我们将要研究的蛋白质在疾病中起着重要作用，包括许多不同的癌症，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所起作用的因素——不仅在正常细胞功能中，而且在出现问题时也是如此。

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.