Structure/function analysis of Rab- and Myosin-interacting proteins in organelle motility

细胞器运动中 Rab 和肌球蛋白相互作用蛋白的结构/功能分析

• 批准号: BB/E021689/1
• 负责人: Miguel Seabra
• 金额: $ 45.69万
• 依托单位: Imperial College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2007
• 资助国家: 英国
• 起止时间: 2007 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FE021689%2F1
• 关键词: Structure; function; analysis; Rab; Myosin

Many important biological processes that underlie the normal function of our cells and tissues rely upon the movement of intracellular structures or organelles. This movement is regulated by motor proteins that transport organelles along intracellular tracks known as cytoskeleton. The activity and targeting of one class of motor protein known as Myosins to specific intracellular organelles is regulated by organelle specific proteins known as Rabs. In the case of pigment granules in the skin and eye one Rab protein called Rab27a recruit Myosins known as MyoVa or MyoVIIa to the granule indirectly via interaction with a third class of protein known as Mlph or MyRIP, respectively. Normal intracellular transport of pigment in the pigment producing cells of the skin (melanocytes) is critical in forming skin colour and protection from ultraviolet radiation from the sun, while in the eye it is important to maintain the function of the light sensing photoreceptor cells that lie adjacent to the retinal pigment epithelium (RPE). The aim of the proposed work is to determine the mechanism by which Mlph/MyRIP proteins interact with and activate MyoVa/VIIa. In particular Mlph interacts with and activates only MyoVa while MyRIP interacts with and activates both MyoVa and MyoVIIa. Comparison of the amino acid sequence of Mlph and MyRIP indicate that both proteins contain common parts and that MyRIP contains extra parts that are not present in Mlph. We will test the idea that common parts or protein domains are involved in interaction with/activation of MyoVa while MyRIP-specific domains are involved in interaction with/activation of MyoVIIa. To do this we will produce and purify the common and MyRIP specific domains and test the strength of their interaction with MyoVa and MyoVIIa proteins in a test-tube. This should provide us with information about the key components of Mlph and MyRIP required for interaction with MyoVa and MyoVIIa. We will then conduct cell biological experiments to confirm the results of our interaction studies and also to measure the effects of these components on the activation of MyoVa and MyoVIIa in living cells. As indicated above movement of pigment granules in pigment producing cells, known as melanocytes, in the skin is dependent upon the activity of MyoVa and in particular its recruitment to the pigment granule by Mlph. Meanwhile in the eye movement of pigment granules is regulated by MyoVIIa following its recruitment to the granule surface by MyRIP. In parallel studies we will introduce common domains and MyRIP specific domains to skin melanocytes and RPE cells that lack Mlph and MyRIP, respectively, and test the ability of introduced proteins to restore the pigment transport defects resulting from loss of these proteins. This will allow us to understand the basis of the interaction of Mlph/MyRIP with Myosin family proteins, the mechanism by which Mlph/MyRIP allow activation of the motors and lay the groundwork for future studies of the three-dimensional structure of Mlph/MyRIP in complex with Myosin motors.

许多重要的生物过程是我们细胞和组织正常功能的基础，它们依赖于细胞内结构或细胞器的运动。这种运动是由运动蛋白调节的，运动蛋白沿着细胞内轨道运输细胞器，称为细胞骨架。一类被称为肌球蛋白的运动蛋白对特定胞内细胞器的活性和靶向是由细胞器特异性蛋白Rabs调节的。在皮肤和眼睛中的色素颗粒中，一种称为Rab27a的rabb蛋白通过与第三类称为Mlph或MyRIP的蛋白质相互作用，间接地将称为MyoVa或MyoVIIa的肌球蛋白招募到颗粒中。在皮肤色素生成细胞（黑色素细胞）中，正常的细胞内色素运输对于形成皮肤颜色和保护皮肤免受太阳紫外线辐射至关重要，而在眼睛中，维持视网膜色素上皮（RPE）附近的感光细胞的功能非常重要。这项工作的目的是确定Mlph/MyRIP蛋白与MyoVa/VIIa相互作用和激活的机制。特别是Mlph只与MyoVa相互作用并激活，而MyRIP与MyoVa和MyoVIIa相互作用并激活。Mlph和MyRIP的氨基酸序列比较表明，两者都含有共同的部分，而MyRIP含有Mlph中不存在的额外部分。我们将测试共同部分或蛋白质结构域参与MyoVa的相互作用/激活，而myrip特异性结构域参与MyoVIIa的相互作用/激活。为了做到这一点，我们将产生和纯化共同和MyRIP特异性结构域，并在试管中测试它们与MyoVa和MyoVIIa蛋白相互作用的强度。这将为我们提供与MyoVa和MyoVIIa相互作用所需的Mlph和MyRIP关键成分的信息。然后，我们将进行细胞生物学实验，以确认我们相互作用研究的结果，并测量这些成分对活细胞中MyoVa和MyoVIIa活化的影响。如上所述，皮肤中黑色素细胞中色素颗粒的运动依赖于MyoVa的活性，特别是MyoVa通过Mlph向色素颗粒募集。同时在眼球运动中，MyoVIIa调节色素颗粒的运动，MyRIP将其招募到颗粒表面。在平行研究中，我们将分别向缺乏Mlph和MyRIP的皮肤黑素细胞和RPE细胞引入共同结构域和MyRIP特异性结构域，并测试引入的蛋白质恢复因这些蛋白质缺失而导致的色素运输缺陷的能力。这将使我们了解Mlph/MyRIP与Myosin家族蛋白相互作用的基础，Mlph/MyRIP激活马达的机制，并为Mlph/MyRIP与Myosin马达复合物的三维结构的未来研究奠定基础。