Nectin-1: Synaptic processing and functions

描述(申请人提供)：Nectin-1是一种细胞黏附分子，定位于突触中的点状黏附连接。Nectin-1与其他蛋白质结合，共同参与神经元突触的形成。我们假设Nectin-1-以一种活性依赖的方式经历了一系列由几个脱落酶调控的多步骤内切蛋白切割事件，这些事件调节突触发生并有助于突触可塑性。我们的初步数据表明，Nectin-1经历了至少两个脱落酶的胞外结构域脱落，导致两个C-末端片段(CTF)的产生。这些CTF被γ-分泌酶进一步膜内切割，并从质膜上释放出NE-ICD。释放的NEICD移位到细胞核中，我们推测它在那里诱导基因表达。在具体目标1中，我们将通过免疫亲和纯化确定Nectin-1的分泌酶切割位点，然后进行Edman降解测序。我们还将通过CodeLink生物阵列研究NE-ICD在海马神经元中调控哪些基因。然后，利用定量RT-PCR、ICC和Western blotting，我们将确认神经元中差异表达的基因。与NE-ICD相互作用的分子将通过酵母双杂交筛选来鉴定。一旦确定了相互作用因子，就将通过细胞和分子方法来分析它们的生物学功能。在特定目标2中，我们将研究BACE1在Nectin-1加工中的生物学作用。初步实验表明，BACE1与Nectin-1的脱落有关，并参与Nectin-1的脱落。我们将通过ICC、Western blotting和囊泡循环实验来研究Nectin-1脱落的中断是如何通过BACE1功能的丧失影响突触形成和突触可塑性的。我们的初步数据表明，两个Nectin-1点突变T310A和Y311A对BACE1的切割是不敏感的，并且Can/ra“.Y-主要干扰内源性Nectin-1的加工。我们将研究这些点突变是如何通过转导海马神经元和用重组腺相关病毒载体在活体成年海马区影响突触形成和突触功能的。我们将通过ICC、活细胞成像和突触活性来定量测量突触标志物、突触形态和大小的变化。随后，我们检测跨显性Nectin-1突变体的表达是否会影响海马依赖学习

DESCRIPTION (provided by applicant): Nectin-1 is a cell adhesion molecule localized the puncta adherentia junctions in synapses. Nectin-1 associates with other proteins, which collectively participate in the formation of neuronal synapses. We hypothesize that nectin-1-undergoes a regulated multi-step set of endoproteolytic cleavage events by several sheddases in an activity-dependent manner and that these events regulate synaptogenesis and contribute to synaptic plasticity. Our preliminary data indicate that nectin-1 undergoes ectodomain shedding by at least two sheddases that result in the production of two C-terminal fragments (CTFs). These CTFs are further cleaved intramembraneously by y-secretase and liberate the NE-ICD from the plasma membrane. The released NEICD translocates into the nucleus and where we postulate it induces gene expression. In Specific Aim 1, we will determine the secretase cleavage sites of nectin-1 by immunoaffinity purification, followed by Edman degradation sequencing. We will also investigate which genes are regulated by NE-ICD in hippocampal neurons by CodeLink Bioarrays. Then, using quantitative RT-PCR, ICC, and Western blotting we will confirm differentially expressed genes in neurons. The molecules that interact with NE-ICD will be identified by a yeast two-hybrid screen. Once interactors are identified, their biological function will be assayed by cellular and molecular approaches. In Specific Aim 2, we will investigate the biological role of BACE1 in nectin-1 processing. Initial experiments indicate that BACE1 associates with and participates in the shedding of nectin-1. We will investigate how disruption of nectin-1 shedding, through loss of BACE1 function, affects synapse formation and synaptic plasticity by ICC, Western blotting and-vesicle recycling assays. Our preliminary data indicate that two nectin-1 point mutations, T310A and Y311A, are refractory to BACE1 cleavage and can /ra".y-dominantly interfere with processing of endogenous nectin-1. We will examine how these point mutants affect the synapse formation and synaptic function by transduction of hippocampal neurons and in vivo adult hippocampus with recombinant adeno-associated viral vectors. We will quantitatively measure the changes in synaptic markers, synapse morphology, and size by ICC, live cell imaging and synaptic activity. Subsequently, we examine whether expression of trans-dominant nectin-1 mutants will affect hippocampal dependent learning