Defining the mechanistic and functional details of an evolutionarily conserved non-canonical retromer pathway.

哺乳动物细胞由各种相互连接的膜室组成，每个膜室由蛋白质和脂质的独特组合组成。为了使细胞正常工作，蛋白质和脂质需要被运送到这个错综复杂的细胞膜中的特定隔室。理解细胞如何做到这一点是现代细胞生物学的一个主要挑战。我们的研究重点是两种脂质，PI3P和pi35,5p2，在一种称为内吞网络的特定膜迷宫内的运输调节中的作用。这些脂质水平的改变，以及由此导致的网络缺陷，可导致各种疾病，包括神经退行性疾病，如阿尔茨海默氏症。在目前的提案中，我们概述了一个多学科的研究计划，旨在描述一个pi3p结合蛋白家族-分选连接蛋白-如何调节内吞网络中的特定运输步骤。我们关注的是一种叫做逆转录物的多蛋白复合体。先前的研究已经确定，逆转录酶在进化上是保守的——也就是说，它既存在于相对原始的生物（如酵母）中，也存在于复杂的生物（如人类）中。典型的人类逆转录酶包含以下蛋白质：VPS26、VPS29和VPS35以及分选连接蛋白SNX1、SNX2、SNX5和SNX6。重要的是，研究表明逆转录酶存在于各种疾病状态中。例如，反转录酶功能紊乱可能与阿尔茨海默病有关，各种病原体（如沙门氏菌）的病理也可能需要反转录酶功能。因此，如果我们要充分了解某些人类疾病，表征逆转录功能的细节是很重要的。通过与Rik Korswagen博士实验室（荷兰Hubrecht研究所）的国际合作，我们最近确定了“非经典”逆转录物的存在。虽然它与经典的逆转录蛋白VPS26、VPS29和VPS35相同，但它不包含SNX1、SNX2、SNX5或SNX6，而是包含一个完全不同的分选连接蛋白SNX3。与经典逆转录体一样，非经典逆转录体也是进化保守的。这是一个令人兴奋的发现，因为它已经确定，当研究逆转录物及其在人类疾病中的作用时，还需要考虑非经典逆转录物途径。在当前的项目中，我们建议更详细地定义非经典反转录酶的分子组装，并阐明它如何与经典反转录酶一起通过内吞网络调节特定蛋白质的运输。将进行生物化学、分子细胞生物学和整个生物体遗传学实验，以获得非典型逆转录酶功能的广泛图像，从分子成分和相互作用到整个生物体的生理后果。这项研究的成功完成将产生关于这一新途径的宝贵的基础信息，这将大大增加我们对正常和疾病相关情况下逆转录酶功能的理解。

Mammalian cells are composed of a variety of interconnected membrane compartments each composed of a unique combination of proteins and lipids. For cells to function correctly, proteins and lipids are required to be transported to specific compartments within this maze of membranes. Understanding how cells achieve this is a major challenge in modern cell biology. Our research focuses on the role of two lipids, called PI3P and PI3,5P2 in the regulation of transport within a specific membrane maze called the endocytic network. Altered levels of these lipids, and resulting defects within the network can lead to various diseases including neurodegenerative diseases such as Alzheimer's. In the current proposal, we outline a multidisciplinary programme of research that aims to describe how one family of PI3P-binding proteins - the sorting nexins - regulate a specific transport step within the endocytic network. We focus on a multiprotein complex called the retromer. Previous research has established that retromer is evolutionarily comserved - that is, it is present in relatively primative (e.g. yeast) as well as complex organisms (e.g. humans). Classically the human retromer contains the following proteins: VPS26, VPS29 and VPS35 and the sorting nexins, SNX1, SNX2, SNX5 and SNX6. Importantly, research has implicated retromer in various disease states. For example, perturbed retromer function may be involved in Alzheimer's disease, and various pathogens (e.g. Salmonella) may also require retromer function for their pathology. Characterising the detail of retromer function is therefore important if we are to fully understand certain human diseases. Through an international collaboration with the laboratory of Dr Rik Korswagen (Hubrecht Institute, The Netherlands), we have recently established the presence of a 'non-classical' retromer. While this shares the classic retromer proteins, VPS26, VPS29 and VPS35, it does not contain SNX1, SNX2, SNX5 or SNX6 but instead contains an entirely distinct sorting nexin, called SNX3. Like the classical retromer, the non-classical retromer is also evolutinarily conserved. This is an exciting discovery since it has established that when examining retromer and its role in human diseases, one needs to also consider the non-classical retromer pathway. In the current project we propose to define in more detail the molecular assembly of the non-classic retromer, and elucidate how it functions alongside the classical retromer in regulating transport of specific proteins through the endocytic network. Biochemical, molecular cell biology and experiments in whole organism genetics will be performed in order to obtain a broad picture of the non-canonical retromer function, from the molecular components and interactions through to physiological consequences for the whole organism. Successful completion of the proposed research, will generate invaluable, fundamental information on this new pathway that will add significantly to our understanding of retromer function in normal and disease-related contexts.