Dynein-adaptor interaction mechanisms and malfunction at atomic resolution

项目摘要 神经退行性疾病大多数是导致进行性变性或 神经细胞的死亡。突出的例子，如脊髓性肌萎缩症（SMA）是由以下原因引起的： 细胞质动力蛋白多蛋白复合物中的突变。动力蛋白1参与了多种 细胞活动，从蛋白质、RNA和囊泡的货物运输到核迁移 和细胞分裂。动力蛋白沿着微管的进行性运动需要与微管结合， 必需的共激活动力蛋白和特异性衔接蛋白，其募集货物并促进运动 通过与动力蛋白和动力肌动蛋白形成稳定的三元复合物。我们对这些相互作用的理解是 有限的和许多问题仍然存在：货物适配器如何使动力蛋白和动力肌动蛋白在一起？如何 这是否会导致发动机的激活和运输的启动？哪些因素决定了 适配器特异性？致病突变如何改变这些相互作用？重要的是 越来越明显的是，与动力蛋白1的轻中间链（“LIC 1”）结合是一种常见的 功能上不同的适配器。我们将把调查的重点放在 LIC 1的C-末端尾部与Spindly、BICD 2和Hook 3衔接，它们各自具有重要的，但 在交通运输方面的不同功能。这些研究是复杂的事实，无序性质的C- 终端LIC 1使得难以从X射线获得原子分辨率的全面动态视图 或EM技术。但是，我们将采取创新的方法， (NMR)作为纳米级“放大镜”，以原子水平精确定位蛋白质内的相互作用位点 分辨率与其他方法相比，NMR特别适合研究无序蛋白质， 描述高度动态的相互作用。我们提出了四个目标：（1）结构和动力 游离C-末端动力蛋白LIC 1的表征。(2)动力蛋白LIC 1与 Spindly，BicD 2和Hook 3，以及它们的结合竞争。(3)影响的预测和检验 动力蛋白LIC 1-接头相互作用的突变。(4)相互作用三角形的结构动力学基础 动力蛋白LIC 1、Spindly和ROD/Zw 10/Zwilch复合体之间的关系。因此，我们的实验预计 提供详细的分子洞察人类动力蛋白LIC 1如何与结构多样的 动力蛋白衔接子通过合作，我们将把我们的发现扩展到动物模型中。总体而言，这 研究是重要的，因为动力蛋白和接头蛋白中存在致病性点突变， 蛋白质表明，这些见解也将有助于了解由功能障碍引起的疾病。 动力蛋白驱动的货物运输，并将提供治疗干预目标。

Project Summary Neurodegenerative diseases are mostly incurable conditions that result in progressive degeneration or death of nerve cells. Prominent examples such as Spinal Muscular Atrophy (SMA) are caused by mutations in the cytoplasmic dynein multi-protein complex. Dynein 1 participates in a wide array of cellular activities, ranging from the cargo transport of proteins, RNA, and vesicles to nuclear migration and cell division. Processive movement of dynein along microtubules requires the binding to the essential co-activator dynactin and specific adaptor proteins, which recruit cargo and facilitate movement by forming a stable ternary complex with dynein and dynactin. Our understanding of these interactions is limited and many questions remain: How do cargo adaptors bring dynein and dynactin together? How does this lead to activation of the motor and the initiation of transport? Which elements determine the adaptor specificity? How do the disease-causing mutations modify these interactions? Importantly, it is becoming increasingly evident that binding to light intermediate chain of dynein 1 (`LIC1') is a common feature of functionally distinct adaptors. We will focus our investigations on the interaction between the C-terminal tail of the LIC1 with the adaptors Spindly, BICD2 and Hook3, which each have important but distinct functions in transport. These studies are complicated by the fact that the disordered nature of C- terminal LIC1 makes it difficult to obtain a comprehensive dynamic view at atomic resolution from X-ray or EM techniques. However, we will take the innovative approach of using nuclear magnetic resonance (NMR) as a nano-scale `magnifying glass' that pinpoints interaction sites within the proteins at atomic resolution. Compared to other methods, NMR is uniquely suited to study disordered proteins and to characterize highly dynamic interactions. We propose four aims: (1) Structural and dynamical characterization of free C-terminal dynein LIC1. (2) Characterization of dynein LIC1 interaction with Spindly, BicD2 and Hook3, and their binding competition. (3) Prediction and testing of effects of mutations on dynein LIC1 - adaptor interaction. (4) Structural & dynamic basis of interaction triangle between dynein LIC1, Spindly and ROD/Zw10/Zwilch complex. Our experiments are therefore expected to provide detailed molecular insight into how human dynein LIC1 engages with structurally diverse dynein adaptors. Through collaboration, we will extend our findings into animal models. Overall, this research is significant because the presence of pathogenic point mutations in dynein and adaptor proteins suggests that these insights will also help to understand the diseases caused by malfunction of the dynein-driven cargo transport and will offer therapeutic intervention targets.