CDK-containing macromolecular assemblies

细胞的行为是由它表达的一组基因以及它对静止或增殖状态的承诺来定义的。细胞周期蛋白依赖性蛋白激酶(CDK)与非催化的细胞周期蛋白亚基结合形成CDK-细胞周期蛋白复合体，在调控基因表达和细胞增殖中发挥重要作用。因此，CDK-细胞周期蛋白在正常细胞和各种疾病情况下都是重要的。我们正在提出一项研究计划，以了解CDK-细胞周期蛋白是如何工作的，它们是如何被调控的，以及它们不适当的活性如何有助于疾病的发展。由于某些CDK-细胞周期蛋白复合体可以互换功能，因此除了一个细胞周期调控CDK外，所有CDK的表达都可以被抑制，而不会影响细胞的分裂能力。CDK1是个例外，它必须具有独特的属性，使其不可或缺。我们对含CDK1的配合物进行了结构解析，发现其具有少量独特的动力学和结构性质。我们建议使用突变技术将这些特性改造成CDK2，CDK1最接近的同源物，并看看产生的CDK2变异体是否能够在功能上补充CDK1。通过这种方式，我们将确定什么是CDK1的属性，解释了它的独特性，解决了CDK生物学核心的一个关键难题。CDK1和CDK2在减数分裂中发挥着重要作用，减数分裂是精子和卵细胞产生的过程。这些CDK可以通过非周期蛋白伴侣的结合和共价修饰来调节，而不是通过磷酸化。我们将探索这些调控机制是如何工作的，以及传统和非传统调控机制如何在减数分裂中发挥作用。不同的CDK-细胞周期蛋白复合体被描述为调节转录(例如CDK9-细胞周期蛋白T)或增殖(例如CDK4-细胞周期蛋白D)。然而，越来越多的证据表明，这种分类过于简单化。特别是Cyclin D似乎与核激素受体(如雄激素和雌激素受体)一起调节转录，我们将研究其背后的机制。由于抑制雄激素受体信号转导是对抗前列腺癌的有效方法，更好地了解细胞周期蛋白D如何参与雄激素受体功能，可能会为治疗对当前治疗产生耐药性的前列腺癌提供方法。一些转录和细胞周期调节CDK依赖于伴侣系统来引导它们形成正确的形状和/或正确的复合体，以执行其细胞角色。CDK的伴侣是由HSP90和CDC37介导的，HSP90是一种混杂的伴侣，而CDC37是一种更特异的适配器。我们在无细胞实验中重新构建了从伴侣到功能性CDK-细胞周期蛋白复合体的“交接”过程。我们现在建议使用生物物理和结构技术相结合的方法来表征切换的结构机制。X射线结晶学为周期蛋白如何激活CDK并在某种程度上将它们导向合适的底物提供了结构上的解释。对于CDK9-Cyclin T，通过两种环境之间的交换提供了更高水平的调节：称为7SK核糖核蛋白(7SKnRNP)的抑制性颗粒和激活环境，其中CDK9-Cyclin T被招募到正在表达的基因中。最近的技术发展使大的复合体，如7SKnRNP，可以通过冷冻电子显微镜进行高分辨率的结构研究，我们将使用这一技术对7SKnRNP进行成像，以了解它保持CDK9-Cyclin T非活性的机制，以及它随后如何切换到激活的设置。

The behaviour of a cell is defined by the set of genes it expresses and by its commitment to either a quiescent or a proliferating state. The cyclin-dependent kinases (CDKs) bind to non-catalytic cyclin subunits to form CDK-cyclin complexes that play central roles in regulating both gene-expression and cell-proliferation. Consequently, CDK-cyclins are important in normal cells and in a variety of disease conditions. We are proposing a programme of research to understand how CDK-cyclins work, how they in turn are regulated, and how their inappropriate activity can contribute to the development of disease.Because certain CDK-cyclin complexes can function interchangeably, expression of all but one cell-cycle regulatory CDK can be inhibited without compromising the cells ability to divide. The exception is CDK1, which must possess unique properties that make it indispensable. We have solved the structure of CDK1-containing complexes, and found that it has a small number of unique kinetic and structural properties. We propose to use mutagenesis to engineer these properties into CDK2, the closest homologue of CDK1, and see if the resulting CDK2-variant can functionally complement CDK1. In this way, we will identify what is the property of CDK1 that explains its uniqueness, resolving a key conundrum at the heart of CDK-biology.CDK1 and CDK2 play important roles in meiosis, the process by which sperm and egg cells are generated. These CDKs can be regulated by binding of non-cyclin partners and by covalent modifications other than phosphorylation. We will explore how these regulatory mechanisms work, and how conventional and unconventional regulatory mechanisms operate in meiosis.Different CDK-cyclin complexes are characterised as regulating either transcription (e.g. CDK9-cyclin T) or proliferation (e.g. CDK4-cyclin D). A growing body of evidence, however, suggests that this classification is over simplified. In particular cyclin D appears to work together with nuclear hormone receptors (such as the androgen and oestrogen receptors) to regulate transcription, and we will investigate the mechanisms that underlie this. Because inhibition of androgen receptor signalling is a well validated way to combat prostate cancer, a better understanding of how cyclin D contributes to androgen receptor function may suggest ways to tackle forms of prostate cancer that have become resistant to current therapies.Several transcriptional and cell-cycle regulatory CDKs depend on a chaperone system to shepherd them into the correct shape and/or correct set of complexes to carry out their cellular roles. The chaperoning of CDKs is mediated by HSP90, a promiscuous chaperone, and by Cdc37, a more specific adaptor. We have reconstituted the process of "hand-off" from chaperone to functional CDK-cyclin complexes in cell-free experiments. We now propose to characterise the structural mechanism of hand off, using a combination of biophysical and structural techniques.X-ray crystallography has provided a structural explanation of how cyclins both activate CDKs and, to some extent, direct them towards appropriate substrates. For CDK9-cyclin T, a further level of regulation is provided by interchange between two settings: an inhibitory particle termed the 7SK ribonucleoprotein (7SKsnRNP) and an activated setting, where CDK9-cyclin T is recruited to genes that are being expressed. Recent technical developments make large complexes such as the 7SKsnRNP accessible to high resolution structural study by cryoelectron microscopy, and we will use this technique to image the 7SKsnRNP to understand the mechanism by which it holds CDK9-cyclin T inactive, and how it subsequently switches into the activated setting.