Structural mechanisms of regulation and assembly in the nephronophthisis INVS-NPHP3-NEK8-ANKS6 module

囊性肾病是一种主要的健康负担，目前几乎没有有效的治疗方法。然而，我们还远未了解这些疾病背后的细胞生物学原理。在这里，我们建议研究在肾病（一种遗传性儿童囊性肾病）中发生突变的蛋白的调控和功能。这些蛋白质协调发育中的肾细胞在细胞表面的天线状结构（称为原纤毛）上接收到的信号。信号传导缺陷导致细胞分裂控制丧失和囊肿形成。我们的目标是为囊性肾病的机制提供新的见解，最终导致新的治疗方法。我们将主要集中精力研究NEK8和INVS两种蛋白。NEK8属于一个叫做蛋白激酶的酶家族。这些修饰其他蛋白质以调节其功能，并且经常在将信息从细胞表面传递到细胞核的信号通路中发现，以便细胞对其环境作出反应。蛋白激酶的活性，也就是它们修饰其他蛋白质的效率，是受到严格控制的。蛋白激酶和其他控制它们的蛋白质在疾病中经常发生突变。突变导致激酶活性失控，从而导致信号通路失灵：它们可能会在不应该传递信号的时候将信号传递给细胞核，或者它们可能会阻断应该传递的信号。我们发现INVS控制着NEK8的活性，我们将INVS负责的部分映射到蛋白质的一个区域，这个区域通常在有INVS突变的患者中被破坏。在许多患者中，疾病似乎是由于缺少INVS的这个区域而引起的。在这个项目的第一部分，我们将研究INVS如何控制NEK8的活性。为了简化问题，我们将只单独研究这两种蛋白质。晶体学将用于产生NEK8的原子分辨率模型，无论是单独的还是与INVS相关的，以显示其活性是如何被最大可能的细节控制的。晶体学产生详细但静态的模型。因此，我们将应用先进的生化方法，使我们能够在许多不同的实验条件下跟踪NEK8随时间的活性。这将使我们详细了解INVS对NEK8的影响，以及因此在INVS突变的患者中缺乏什么。在该项目的第二部分，我们将扩大我们的研究范围，包括与NEK8和INVS相关的另外两种蛋白质，它们也在囊性肾病中发生突变。我们将研究这些蛋白是否和如何结合，以及这些相互作用如何与NEK8的活性相关。最后，我们将研究在囊性肾病患者中发现的这些蛋白质的突变，以发现它们对蛋白质之间相互作用的影响。虽然许多与囊性肾病有关的基因是已知的，但我们对这些基因的突变如何导致疾病的了解甚少。在这个项目中，我们将探索这些突变破坏一组蛋白质之间的相互作用，导致NEK8活性失去控制的想法。这些见解将影响我们思考纤毛信号的方式，以及纤毛信号在一系列相关疾病中是如何出错的。我们将建立一个详细的模型来解释NEK8的活性是如何被INVS控制的，并产生第一个阻断NEK8活性的化合物。我们的工作将为挽救NEK8、INVS和相互作用蛋白突变细胞中的纤毛信号提供策略基础。如果成功，这可能最终会为患者带来新的治疗方法。

Cystic kidney diseases represent a major health burden for which there are few effective treatments. Yet we are far from understanding the cell biology behind these diseases. Here, we propose to study the regulation and function of proteins that are mutated in nephronophthisis, an inherited childhood cystic kidney disease. These proteins coordinate signals received by developing kidney cells at antenna-like structures on the cell surface, called primary cilia. Defective signalling leads to loss of cell division control and cyst formation. Our aim is to provide new insights into cystic kidney disease mechanisms that will ultimately lead to new therapies.We will concentrate our efforts principally on two proteins, NEK8 and INVS. NEK8 belongs to a family of enzymes called protein kinases. These modify other proteins to modulate their functions, and are often found in signalling pathways that pass information from the cell surface to the cell nucleus so that cells may respond to their environment. The activity of protein kinases, that is to say the efficiency with which they modify other proteins, is strictly controlled. Protein kinases and other proteins that control them are very commonly mutated in diseases. The mutations result in uncontrolled kinase activity so that signalling pathways malfunction: they might pass signals to the nucleus when they should not, or they might block signals that ought to be passed on. We discovered that INVS controls the activity of NEK8, and we mapped the part of INVS responsible down to a region of the protein that is usually disrupted in patients that have INVS mutations. In many patients, it seems that disease is caused by missing just this region of INVS. In the first part of this project, we will investigate how INVS controls the activity of NEK8. To simplify matters, we will study just the two proteins in isolation. Crystallography will be used to produce atomic-resolution models of NEK8, both alone and associated with INVS, to show how its activity is controlled in the maximum possible detail. Crystallography produces detailed but static models. So we will apply advanced biochemical methods that enable us to track the activity of NEK8 over time under a number of different experimental conditions. This will provide us with a detailed understanding of what effects INVS has on NEK8, and what is therefore lacking in patients that have mutations in INVS.In the second part of this project, we will broaden our study to encompass two further proteins that are associated with NEK8 and INVS and that are also mutated in cystic kidney disease. We will investigate whether and how these proteins associate and how these interactions relate to the activity of NEK8. Finally, we will examine mutations in these proteins that were found in cystic kidney disease patients, to discover their effects on the interactions between proteins. Although many of the genes involved in cystic kidney disease are known, we have very little insight into how mutations in these genes cause disease. In this project, we will explore the idea that these mutations disrupt the interactions between a group of proteins, leading to loss of control of NEK8 activity. These insights will influence the way we think about signalling in the cilia, and how this goes awry in a range of associated diseases. We will produce a detailed model to explain how NEK8 activity is controlled by INVS and generate the first chemical compounds that block NEK8 activity. Our work will provide the basis for strategies to rescue ciliary signalling in cells with mutations in NEK8, INVS and interacting proteins. If successful, this might eventually lead to new therapies for patients.