Role of kinesin light chain 1 in binding to specific cargoes.

驱动蛋白轻链 1 在与特定货物结合中的作用。

• 批准号: BB/V008307/1
• 负责人: Viki Allan
• 金额: $ 72.51万
• 依托单位: University of Manchester
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2021
• 资助国家: 英国
• 起止时间: 2021 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FV008307%2F1
• 关键词: Role; kinesin; light; chain; binding

Cells of all organisms except bacteria contain filaments, called microtubules, that act as tracks for the transport of material from one region to another. This vital traffic is carried by proteins that 'walk' along microtubules, acting as minute motors that move many different cargoes. There are two families of microtubule motor proteins, the kinesins and dyneins, with most kinesins carrying cargo away from the cell centre towards the cell periphery. These motors and the cargoes they carry are absolutely vital for the health of nerve cells and for brain function, since mutations in them can cause or contribute to diseases such as muscular dystrophy, Alzheimer's disease, Huntington's disease, spastic paraplegia and schizophrenia. Their function is vital in all cells of the body, not just neurons.Kinesin-1 transports many different kinds of cargo, ranging from membrane organelles through to cytoskeletal proteins. It is made up of two types of protein subunits: the KIF5 subunits provide the motor activity, while the kinesin light chains (KLCs) bind to cargo proteins and also control kinesin's activity. The KLCs come in several different types, so one possibility is that each cargo uses kinesin-1 containing a particular KLC. There are four KLC genes, with the KLC1 gene being alternatively spliced to generate at least 17 different proteins (isoforms) that vary in amino acid sequence only at one end, their C-terminal. Our functional studies showed that two isoforms, KLC1B and KLC1D, each control the movement of different membrane types, supporting the idea that KLC variation is crucial for cargo selection. KLC1 splicing is important physiologically, as changes in the levels of certain splice forms have been linked to Alzheimer's disease and schizophrenia. Altogether, it is clear that KLCs are central to kinesin-1 cargo binding and subsequent activation. However, we do not fully understand the importance of different KLCs-and particularly KLC1 isoforms-in kinesin-1 function. As a first step, we must identify the cellular cargoes to which they bind, and the specific proteins that recruit them to those cargoes. To begin to do this, we have used a technique called BioID that adds a biotin molecule to any protein in the near neighbourhood of a specific KLC isoform. Biotinylated proteins are then isolated and identified using mass spectrometry. We found that KLC1D, but not KLC2 or 3, was in close proximity to three proteins involved in endocytosis. This pathway is the route by which cells take up material (nutrients and growth factors, for example) from outside the cell. Two of the KLC1D near-neighbours, SNX1 and CCDC22, are involved in sorting material that should be recycled from that to be degraded. The third, BIRC6, is needed for cells to divide into two after cell division in a process called cytokinesis. A major goal of this project is to test how kinesin-1 containing the KLC1D isoform contributes to protein sorting at the early endosome, focussing on the two pathways that require SNX1 and CCDC22. We will also investigate kinesin's role in cytokinesis. To do this, we will make a new kinesin tool-box that will allow us to remove KLC1 rapidly from cells, or replace the cell's kinesin on cargo with an inactive version that lacks the motor domains. We will use light microscopy and biochemical assays to monitor what happens to endocytosis after disrupting kinesin function. We will also determine if SNX1, CCDC22 and BIRC6 can bind directly to KLC1D, and if so, dissect the regions of each protein needed for this binding. Finally, we will use BioID to test if KLC1 isoform do indeed target kinesin-1 to specific organelles, using three additional KLC1 splice forms. Overall, this project will provide important insight into how KLC1 splicing affects kinesin function and association with specific cargoes.

除细菌以外的所有生物的细胞中含有细丝，称为微管，这些细胞是从一个区域传输到另一个区域的轨道。这种重要的交通由沿着微管“行走”的蛋白质携带，充当了移动许多不同货物的微小电动机。有两个微管运动蛋白的家族：驱动蛋白和动力蛋白，大多数驱动蛋白将货物从细胞中心延伸到细胞外围。这些电动机及其携带的货物对于神经细胞的健康和脑功能绝对至关重要，因为其中的突变会导致或导致诸如肌肉营养不良，阿尔茨海默氏病，亨廷顿氏病，刺激性瘫痪和精神分裂症等疾病。它们的功能在人体的所有细胞中都至关重要，而不仅仅是神经元。Kinesin-1运输许多不同种类的货物，从膜细胞器到细胞骨架蛋白。它由两种类型的蛋白质亚基组成：KIF5亚基提供运动活性，而驱动蛋白轻链（KLCS）与货物蛋白结合并控制驱动蛋白的活性。 KLC有几种不同类型的类型，因此，每辆货物使用含有特定KLC的驱动蛋白-1。有四个KLC基因，KLC1基因被剪接以产生至少17种不同的蛋白质（同工型），仅在其C末端（其C末端）在氨基酸序列中变化。我们的功能研究表明，两个同工型KLC1B和KLC1D都控制着不同膜类型的运动，这支持KLC变异对于货物选择至关重要的想法。 KLC1​​剪接在生理上很重要，因为某些剪接形式水平的变化与阿尔茨海默氏病和精神分裂症有关。总的来说，很明显，KLCS是驱动蛋白-1货物结合和随后的激活的核心。但是，我们不完全理解不同KLC的重要性，尤其是KLC1同工型IN驱动蛋白-1功能。作为第一步，我们必须确定其结合的细胞货物以及将其募集到这些货物的特定蛋白质。为了开始做到这一点，我们使用了一种称为生物剂的技术，该技术将生物素分子添加到特定KLC同工型的近附近的任何蛋白质中。然后使用质谱法分离并鉴定生物素化蛋白。我们发现KLC1D（而不是KLC2或3）与参与内吞作用的三种蛋白质紧邻。该途径是细胞从细胞外吸收材料（例如营养和生长因子）的途径。 KLC1​​D接近邻居的两个SNX1和CCDC22参与了分类材料，这些材料应从中降低。第三个BIRC6在一个称为细胞力学的过程中需要细胞分成两个细胞分裂的两个。该项目的一个主要目标是测试含有KLC1D同工型的驱动蛋白-1如何在早期内体上有助于蛋白质排序，重点是需要SNX1和CCDC22的两种途径。我们还将研究动力蛋白在细胞因子中的作用。为此，我们将制作一个新的动力蛋白工具箱，该箱将使我们能够从单元格中快速删除KLC1，或用缺少电动机域的不活动版本替换货物上的单元格的动力蛋白。我们将使用光学显微镜和生化测定法来监测破坏驱动蛋白功能后内吞作用的情况。我们还将确定SNX1，CCDC22和BIRC6是否可以直接与KLC1D结合，如果是这样，请剖析这种结合所需的每种蛋白质的区域。最后，我们将使用生物剂来测试KLC1同工型是否确实使用三种额外的KLC1剪接形式靶向运动蛋白-1。总体而言，该项目将为KLC1剪接如何影响运动蛋白功能以及与特定货物的关联提供重要的见解。