Mechanism and design of a pH sensor at the organelle-cytoskeleton interface

细胞拥有许多特殊的部件，这些部件必须在正确的时间出现在正确的位置，才能完成它们的功能。使用后，这些部件必须被运走以进行回收或降解。此外，细胞必须能够调整其组织，以满足功能需求或对环境的变化做出反应。这些运输过程的错误调节或中断可能会导致从阿尔茨海默病等神经退行性疾病到癌症的各种人类疾病。此外，在HIV-1病毒感染或沙门氏菌等细菌感染期间，细胞的自然运输系统可能会被“劫持”。因此，询问这些运输系统和过程是了解细胞、疾病和感染的自然运作的关键。为了移动组件，细胞使用由被称为微管的电缆网络组成的运输系统。就像铁路网一样，这些电缆将细胞的各个区域连接在一起。细胞还拥有沿这个被称为马达蛋白的网络行进的“交通工具”。在这些最重要的马达中，Kinesin-1是我们建议研究的主题。马达蛋白可以选择性地附着在细胞成分上，并在微管网络上移动它们。他们还可以通过相互滑动电缆来控制网络本身的形状和组织。尽管马达蛋白在细胞生物学的许多领域都很重要，但我们对像Kinesin-1这样的复杂机器是如何控制的缺乏正确的理解。这项提议完全是关于理解和利用细胞内的这种控制。我们已经做了初步的观察，表明在控制机制中有一个新的和未知的因素。这是细胞的酸碱度(其pH值)之间的平衡，它似乎控制着Kinesin-1马达的活动，因此控制着细胞的组织。这一点很重要，因为当细胞获得或缺乏营养，细胞成分受到破坏，以及癌症中肿瘤产生局部酸性环境时，pH值就会发生变化。我们还将探索这种“pH依赖”是否可以被利用来开发在这些环境中扰乱细胞的类药物分子。我们的方法是独一无二的。这项联合提案源于生物化学学院的Dodding小组和布里斯托尔大学化学学院的Woolfson小组之间的一项新的、成功的合作。它结合了细胞生物学和蛋白质设计两个学科，两者相互作用。我们卓有成效的合作工作突出了Kinesin-1如何附着于其携带的细胞组件以及如何利用蛋白质设计和工程来获得对自然运输系统的新见解的重要方面。通过这一提议，我们询问了Kinesin-1如何感知pH，如何将其转化为运输活动，以及是否可以使用类药物分子来操纵这一过程。我们寻求应用我们从这个自然系统中产生的知识来开发新的蛋白质设计或合成生物学方法，这些方法将测试我们的理解，并导致在活细胞中发挥作用的合成运输机器的开发。该提案的结果将导致对蛋白质马达及其协调的细胞过程的更深层次的理解。反过来，这可能会导致合成生物学、生物技术和医学等更多应用领域的进步。

Cells possess many specialised components that must be in the right place at the right time to fulfil their functions. After their use, these components must be transported away for recycling or degradation. In addition, cells must be able to adapt their organisation to meet functional demands or respond to changes in their environment. Mis-regulation or disruption of these transport processes can contribute to human diseases ranging from neurodegenerative conditions such as Alzheimer's disease to cancer. Also, the natural transport systems of the cell can be 'hijacked' during viral infections by HIV-1 or bacterial infections such as Salmonella. Therefore, interrogating these transport systems and processes is key to understanding the natural workings of cells, diseases and infections. To move components around, cells use a transport system composed of a network of cables known as microtubules. Much like a railway network, these cables link together regions of the cell. Cells also possess 'vehicles' that travel along this network known as motor proteins. On of the most important of these motors, kinesin-1, is the subject of our proposed study. Motor proteins can selectively attach to cellular components and move them on the microtubule network. They can also control the shape and organisation of the network itself by sliding the cables against one another. Despite the importance of motor proteins across many areas of cell biology, we lack a proper understanding of how complex machines like kinesin-1 are controlled. This proposal is all about understanding and exploiting this control within cells. We have made preliminary observations that suggest a new and unexplored factor in the control mechanism. This is the balance between acidity and alkalinity of the cell (its pH), which appears to control activity of the kinesin-1 motor, and so controls cellular organisation. This is important because changes in pH occur when cells gain or lack nutrients, cellular components are damaged, and, in cancer, where tumours generate a local acidic environment. We will also explore whether this 'pH-dependence' can be exploited to develop drug-like molecules that disrupt cells in these contexts. Our approach is unique. This joint proposal stems from a new and successful collaboration between the Dodding group in the School of Biochemistry and the Woolfson Group in the School of Chemistry of the University of Bristol. It combines two disciplines of cell biology and protein design, with each informing the other. Our fruitful collaborative work has highlighted important aspects of how kinesin-1 attaches to the cellular components it carries and how protein design and engineering can be used to obtain new insights into natural transport systems. Through this proposal, we ask how kinesin-1 senses pH, how this is translated into transport activities, and whether this can be manipulated using drug-like molecules. We seek to apply our knowledge emerging from this natural system to develop new protein-design or synthetic-biology approaches that will test our understanding and lead to the development of synthetic transport machines that function in living cells. The outcomes of the proposal will lead to a deeper understanding of protein motors and the cellular processes that they orchestrate. In turn, this may lead to advances in more-applied fields such as synthetic biology, biotechnology and medicine.