CALSENILIN INTERACTIONS WITH PRESENILIN 2

早老素（PS1和PS2）最近才被分离并鉴定为主要的家族性阿尔茨海默病（FAD）基因，因此对其正常生物学作用或早老素突变如何导致AD相关的淀粉样蛋白β（ABeta）水平增加知之甚少。 为了更多地了解PS1和PS2的功能，以及它们共同的细胞途径，例如涉及致病性ABeta产生增加的途径，我们专注于与两种蛋白质中同源结构域相互作用的蛋白质的表征。 C-末端是这样一个同源结构域，最近的合作努力已经导致鉴定出一种新的蛋白质，该蛋白质与PS1和PS2的C-末端相互作用。 该蛋白与神经元钙结合蛋白家族具有重要的同源性，因此我们将其命名为钙结合早老素相互作用蛋白calsenilin。我们已经发现，calsenilin是一种真正的钙结合蛋白，在大脑中特异性表达。 亲水性分析表明钙生蛋白缺乏跨膜结构域，这得到了我们的免疫荧光研究的支持，该研究表明钙生蛋白定位于转染细胞的细胞质中。 有趣的是，当PS2和钙生素在细胞中共表达时，钙生素的亚细胞定位似乎与PS2重新分布并共定位于ER和高尔基体。 这一发现是兼容与当前模型的早老素拓扑结构，预测的C-末端结构域位于细胞质面对的ER/高尔基体膜，并表明这两种蛋白质可以在体内相互作用。 最后，我们的初步数据表明，在PS2的正常切割模式的改变的钙生素的表达结果。在这个建议中，我们概述了旨在提供有关钙生素的基本生物学特性的信息的实验。此外，我们将证实和扩展我们的初步研究结果，这表明，钙生素的表达影响PS2的加工，我们将确定是否钙生素有能力介导的影响，早老素突变对APP代谢和ABeta的产生。 我们还将评估是否这些属性，或calsenilin-PS2的相互作用，被改变的存在下，FAD相关的突变早老素。本申请中描述的实验应提供有关钙生素的基本细胞生物学及其与PS2相互作用的基本但关键的信息。 这些信息将是一个宝贵的资产，了解早老蛋白生物学，以及了解早老蛋白相关的钙在大脑中的信号通路。 由于钙生素与两种早老素相互作用，其作用的阐明应提供有关这两种蛋白质共享的途径的信息，例如参与AD相关的ABeta增加的途径。 有趣的是，最近的数据表明，FAD相关的增加依赖于C-末端结构域（与calsenilin相互作用的结构域）的存在，因此，该提议可能提供一个平台，最终基于有效的研究和治疗策略以及未来的研究。

The presenilins (PS1 and PS2) have only recently been isolated and identified as major familial Alzheimer's disease (FAD) genes, consequently little is known about their normal biological role or about how the presenilin mutations lead to the AD-associated increase in the levels of amyloid beta (ABeta). To learn more about the function of PS1 and PS2, and about cellular pathways that they have in common, such as those involving the pathogenic increased production of ABeta, we have focused on the characterization of protein(s) that interact with domains that are homologous in the two proteins. The C-terminus is such a homologous domain and a recent collaborative effort has resulted in the identification of a novel protein that interacts with the C-terminal of both PS1 and PS2. This protein shares significant homology with a family of neuronal Ca2+-binding proteins, therefore we have named this novel Ca2+-binding, presenilin-interacting protein calsenilin. We have found that calsenilin is a bone fide Ca2+-binding protein that is expressed specifically in the brain. Hydropathy analyses indicating that calsenilin lacks a membrane spanning domain(s) are supported by our immunofluorescence studies which indicate that calsenilin is localized in the cytoplasm of transfected cells. Interestingly, when PS2 and calsenilin are co-expressed in cells, the subcellular localization of calsenilin appears to redistribute and co-localize with PS2 to the ER and Golgi. This finding is compatible with current models of presenilin topology, which predict that the C-terminal domain is located at the cytoplasmic face of the ER/Golgi membranes, and indicates that the two proteins can interact in vivo. Finally, our preliminary data indicate that the expression of calsenilin results in alterations of the normal cleavage pattern of PS2. In this proposal we have outlined experiments designed to provide information about the basic biological properties of calsenilin. In addition, we will confirm and extend our preliminary findings, which indicate that the expression of calsenilin effects the processing of PS2, and we will determine if calsenilin has the ability to mediate the effect that the presenilin mutations have on APP metabolism and ABeta generation. We will also assess whether these properties, or the calsenilin-PS2 interaction, are altered by the presence of FAD-associated mutations in the presenilins. The experiments described in this application should supply fundamental but critical information about the basic cell biology of calsenilin and its interaction with PS2. This information will be a valuable asset for understanding presenilin biology, as well understanding presenilin-associated Ca2+-signaling pathways in the brain. Because calsenilin interacts with both of the presenilins, the elucidation of its role should provide information about pathways shared by the two proteins, such as those involved in the AD-associated increases in ABeta. Interestingly, recent data indicates that FAD-associated increases are dependent of the presence of the C-terminal domain (the domain that interacts with calsenilin) therefore, this proposal may provide a platform on which to ultimately base effective investigational and therapeutic strategies as well as future studies.