CALSENILIN INTERACTIONS WITH PRESENILIN 2

早老素(PS1和PS2)是最近被分离和鉴定为家族性阿尔茨海默病(FAD)的主要基因，因此对它们的正常生物学作用或早老素突变如何导致AD相关的淀粉样β蛋白(ABeta)水平的升高知之甚少。为了更多地了解PS1和PS2的功能，以及它们共同的细胞通路，例如那些涉及ABETA致病活性增加的通路，我们重点研究了与这两种蛋白质中同源结构域相互作用的蛋白质(S)的特征。C-末端就是这样一个同源结构域，最近的一项合作努力导致了一种新的蛋白质的鉴定，它与PS1和PS2的C-末端都相互作用。该蛋白与一个神经元钙结合蛋白家族有显著的同源性，因此我们将其命名为钙结合、早老素相互作用蛋白Calsenlin。我们已经发现，钙生蛋白是一种骨骼钙结合蛋白，在大脑中特异表达。亲水性分析表明钙生蛋白缺乏跨膜结构域(S)，免疫荧光研究表明钙生蛋白定位于细胞质中。有趣的是，当PS2和钙生蛋白在细胞中共表达时，钙生蛋白的亚细胞定位似乎重新分布并与PS2共同定位到内质网和高尔基体。这一发现与现有的早老素拓扑学模型相一致，该模型预测C-末端结构域位于ER/Golgi膜的细胞质表面，表明这两种蛋白在体内可以相互作用。最后，我们的初步数据表明，钙化蛋白的表达导致了PS2的正常切割模式的改变。在这项提案中，我们概述了旨在提供关于钙钙素基本生物学特性的信息的实验。此外，我们还将证实和推广我们的初步发现，这些发现表明钙化蛋白的表达影响PS2的加工，并将确定钙化蛋白是否具有介导早老素突变对APP代谢和ABETA生成的影响的能力。我们还将评估这些属性或钙化蛋白-PS2相互作用是否会因早老素中FAD相关突变的存在而改变。本申请中描述的实验应该提供关于钙化蛋白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。这些信息将是了解早老素生物学以及了解大脑中与早老素相关的钙信号通路的宝贵财富。由于Calsenin与这两种早老素相互作用，对其作用的阐明应该提供有关这两种蛋白共有的途径的信息，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(与钙钙素相互作用的结构域)的存在，因此，这一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.