Using viruses to study kinesin-1 recruitment, regulation and function

细胞需要将货物分类并运输到细胞的不同部位，这是细胞存活的基础，也是决定细胞结构和功能的基础。为此，细胞拥有一个由微管组成的骨架（细胞骨架）。微管与马达有关，马达以高度特定和受管制的方式捆绑和运输货物。一种被称为运动蛋白-1的马达负责将货物运送到细胞内的特定位置。运动蛋白-1运输缺陷导致人类疾病，从与年龄相关的黄斑变性和白内障，到侵袭性癌症和神经系统疾病。运动蛋白-1的运输对于沿着神经轴突长距离移动货物是非常重要的，运动蛋白-1的缺陷会导致几种神经退行性疾病，包括帕金森病、亨廷顿病、阿尔茨海默病和遗传性痉挛性截瘫。运动蛋白-1如何与微管结合并沿着微管移动已经得到了很好的研究。相比之下，激酶-1如何招募特定的货物以及激酶-1的活性和目标目的地如何被调节仍然知之甚少。病毒在细胞内复制，并进化出劫持细胞机制以增强其复制和传播的机制。一些病毒在其复制周期中利用驱动蛋白-1，例如帮助将新形成的病毒颗粒输出到细胞外。牛痘病毒（VACV）就是一个例子，它是痘病毒的原型，也是用于根除天花的疫苗。我们的实验室以VACV对激酶-1的招募为模型，研究控制货物与激酶-1结合和马达激活的机制。驱动蛋白-1复合体由两条驱动蛋白重链（KHC）和两条驱动蛋白轻链（KLC）组成。其活性受自身抑制调节。激活涉及货物蛋白的多个结合事件，这些结合事件诱导与微管结合的复合物发生结构变化。哺乳动物细胞表达多种驱动蛋白-1类型（同种异构体），每种类型都具有略微不同的生化特性，可以结合不同的货物或移动到不同的目的地。三种疫苗病毒蛋白，A36和F12/E2复合物，参与招募运动蛋白-1将病毒颗粒运输到细胞表面。为了了解这些蛋白质的作用，它们之间的相互作用以及与酪蛋白-1复合物组分的相互作用将通过生物化学和结构生物学方法进行表征。我们已经证明F12/E2和A36与KLC的不同结构域结合，并优先结合不同的KLC异构体。使用RNA干扰的基因敲除和使用CRISPR/ cas9介导的基因编辑的基因敲除将用于确定不同的KLC亚型对病毒运输和正常细胞功能的重要性。各种KLC异构体在细胞功能中的作用在很大程度上仍然未知。单个KLC或KLC组合被敲除的细胞将通过显微镜进行表征。此外，单个KLCs的细胞功能将通过鉴定和比较与它们相关的蛋白质来确定。还将研究与病毒转运复合体相关的蛋白质，以确定它们是否是病毒输出所必需的。病毒蛋白、个体KLC异构体和细胞蛋白对激酶1激活的贡献，以及它们在其调节中的可能作用，将通过体外和细胞试验的结合来测量。拟议的研究项目旨在通过表征病毒和所涉及的细胞蛋白来研究VACV招募和激活酪蛋白-1的机制。这项研究也将有助于我们理解正常的细胞过程，以及如果这些功能障碍，疾病是如何随之而来的。有关病毒与驱动蛋白-1相互作用的结构信息将有助于开发抑制这一过程的化合物，并预防由痘病毒引起的疾病。

Cells need to sort and transport cargo to distinct parts of the cell and this is fundamental for cell survival and for determining cell structure and function. To do this cells possess a skeleton (cytoskeleton) that is composed of microtubules. Microtubules are associated with motors that bind and transport cargo in a highly specific and regulated manner. One type of motor, called kinesin-1, is responsible for transporting cargos to specific locations within the cell. Defects in kinesin-1 transport cause human diseases, ranging from age-related macular degeneration and cataracts, to invasive cancers and neurological disorders. Kinesin-1 transport is very important for moving cargos longer distances along nerve axons and defects in kinesin-1 cause several neurodegenerative disorders, including Parkinson's, Huntington's, Alzheimer's and inherited spastic paraplegia. How kinesin-1 binds to and moves along microtubules is quite well studied. In contrast, how kinesin-1 recruits specific cargos and how kinesin-1 activity and target destination are regulated remain poorly understood.Viruses replicate inside cells and have evolved mechanisms to hijack cellular machinery to enhance their replication and spread. Several viruses exploit kinesin-1 during their replication cycle, for instance to help export newly formed virus particles out of the cell. An example is vaccinia virus (VACV), the prototype poxvirus and the vaccine used to eradicate smallpox. Our lab uses the recruitment of kinesin-1 by VACV as a model to study the mechanisms that control the binding of cargo to kinesin-1 and activation of the motor.The kinesin-1 complex is made up of two kinesin heavy chains (KHC) and two kinesin light chains (KLC). Its activity is regulated by autoinhibition. Activation involves multiple binding events by cargo proteins that induce a structural change in the complex enabling association with microtubules. Mammalian cells express multiple kinesin-1 types (isoforms), each with slightly different biochemical properties that may bind different cargos or move to different destinations.Three VACV proteins, A36 and the F12/E2 complex, are involved in recruiting kinesin-1 to transport virus particles to the cell surface. To understand the role of each of these proteins, their interaction with each other and with components of the kinesin-1 complex will be characterised biochemically and using structural biology approaches. We have shown that F12/E2 and A36 associate with different domains of KLC and preferentially bind to different KLC isoforms. Gene knock-down using RNA interference and gene knock-out using CRISPR/Cas9-mediated gene editing will be used to determine the importance of different KLC isoforms for virus transport and for normal cellular function.The roles of various KLC isoforms in cell function remain largely unknown. Cells in which individual KLCs or combinations of KLC have been knocked-out will be characterised by microscopy. Additionally, the cellular functions of individual KLCs will be determined by identifying and comparing the proteins that associate with them. Proteins that are also found to associate with the viral transport complex will be studied to determine if they are required for virus export. The contribution of viral proteins, individual KLC isoforms and cellular proteins to the activation of kinesin-1, and their possible role in its modulation will be measured using a combination of in vitro and in cell assays.The proposed research project aims to investigate the mechanisms used by VACV to recruit and activate kinesin-1 by characterising the virus and cellular proteins involved. This research will also help our understanding of the normal cellular processes and how disease ensues if these dysfunction. Structural information about the interaction of the virus with kinesin-1 will aid the development of compounds that inhibit this process and prevent disease caused by poxviruses.