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

许多重要的生物学过程，我们的细胞和组织的正常功能的基础依赖于细胞内结构或细胞器的运动。这种运动是由马达蛋白调节的，马达蛋白沿着沿着细胞内称为细胞骨架的轨道运输细胞器。一类称为肌球蛋白的马达蛋白的活性和对特定细胞内细胞器的靶向由称为Rabs的细胞器特异性蛋白调节。在皮肤和眼睛中的色素颗粒的情况下，一种称为Rab 27 a的Rab蛋白通过分别与称为Mlph或MyRIP的第三类蛋白质相互作用，间接地将称为MyoVa或MyoVIIa的肌球蛋白招募到颗粒中。在皮肤的色素产生细胞（黑素细胞）中色素的正常细胞内运输在形成皮肤颜色和保护免受来自太阳的紫外线辐射方面是至关重要的，而在眼睛中，重要的是维持位于视网膜色素上皮（RPE）附近的感光细胞的功能。这项工作的目的是确定Mlph/MyRIP蛋白与MyoVa/VIIa相互作用并激活MyoVa/VIIa的机制。特别地，Mlph仅与MyoVa相互作用并激活MyoVa，而MyRIP与MyoVa和MyoVIIa相互作用并激活两者。Mlph和MyRIP的氨基酸序列的比较表明，这两种蛋白质含有共同的部分，而MyRIP含有Mlph中不存在的额外部分。我们将测试这样的想法，即共同部分或蛋白质结构域参与与MyoVa的相互作用/激活，而MyRIP特异性结构域参与与MyoVIIa的相互作用/激活。为了做到这一点，我们将生产和纯化的共同和MyRIP特定的结构域，并测试其相互作用的强度与MyoVa和MyoVIIa蛋白在试管中。这将为我们提供有关与MyoVa和MyoVIIa相互作用所需的Mlph和MyRIP关键组件的信息。然后，我们将进行细胞生物学实验，以确认我们的相互作用研究的结果，并测量这些组分对活细胞中MyoVa和MyoVIIa激活的影响。如上所述，色素颗粒在皮肤中的色素产生细胞（称为黑素细胞）中的运动取决于MyoVa的活性，特别是其通过Mlph向色素颗粒的募集。同时，在眼睛中，色素颗粒的运动由MyoVIIa调节，随后MyRIP将其募集到颗粒表面。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由这些蛋白质丢失引起的色素转运缺陷的能力。这将使我们能够了解Mlph/MyRIP与肌球蛋白家族蛋白相互作用的基础，Mlph/MyRIP允许激活马达的机制，并为未来研究Mlph/MyRIP与肌球蛋白马达复合物的三维结构奠定基础。

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.