Molecular and Cellular Analysis of Brain Enriched PTP's

描述（由申请方提供）：拟定研究的目的是研究酪氨酸磷酸酶STEP的调节。先前的研究确定STEP使ERK 1/2、p38和酪氨酸激酶Fyn失活。在上一个周期中，我们发现STEP还诱导NR 1/NR 2B和GluR 1/GluR 2内化。 STEP与脆性X综合征（FXS）、阿尔茨海默病（AD）和精神分裂症（SZ）的病理生理学有关。在这些疾病中，STEP水平增加。目前的模型表明，STEP的增加导致NR 1/NR 2B和GluR 1/GluR 2受体的不适当内化。然而，STEP水平增加的机制在这些疾病中不同。在AD和SZ中，存在泛素化和降解STEP的中断。在FXS中，由于缺乏FMRP，STEP mRNA的翻译增加。 STEP在特定形式的突触活动后迅速降解。突触刺激导致STEP的泛素化，然后通过蛋白酶体系统降解。我们假设STEP在突触部位被降解以促进突触强化。突触外NMDAR刺激导致钙蛋白酶介导的蛋白水解STEP。我们认为这种机制抑制p38信号传导并启动细胞死亡途径。 调节这些过程的分子机制尚不清楚。Aim 1专注于STEP泛素化。我们包括突变STEP的功能实验，使其不再被泛素化和降解。我们预测，这种结构将破坏LTP，以及注入杏仁核后巩固的恐惧条件反射。我们将确定促进或阻止STEP泛素化的激酶。目的2着重于STEP蛋白水解。我们包括破坏STEP蛋白水解的功能研究，并将确定这对细胞死亡的影响，使用体内兴奋性毒性模型。我们预测阻断STEP蛋白水解将显著减少细胞死亡。目标3侧重于STEP翻译。两个细胞质聚腺苷酸化元件（CPE）存在于STEP mRNA，我们将确定这些元件是否调节STEP mRNA的翻译。我们包括功能实验，将突变这些CPE。我们预测这将破坏STEP在调节其底物（包括谷氨酸受体运输）方面的正常功能。这些研究将推进我们对STEP如何参与正常认知过程的理解，以及STEP在存在显著认知缺陷的几种疾病中被破坏的可能机制。 公共卫生相关性：上一个周期的一个重大发现是富含酪氨酸的酪氨酸磷酸酶（STEP）调节谷氨酸受体的运输，并在特定形式的突触刺激后从突触膜上清除NR 1/NR 2B和GluR 1/GluR 2受体。这些后者的研究结果牵连STEP在脆性X综合征，精神分裂症和阿尔茨海默病的病理生理学。拟议的研究检查了调节STEP活性的分子机制，并将阐明这一重要的调节蛋白家族如何参与突触可塑性，细胞死亡和人类疾病。

DESCRIPTION (provided by applicant): The goals of the proposed investigation are to study the regulation of the tyrosine phosphatase STEP. Previous studies established that STEP inactivates ERK1/2, p38 and the tyrosine kinase Fyn. Over the last cycle, we have discovered that STEP also induces NR1/NR2B and GluR1/GluR2 internalization. STEP has been implicated in the pathophysiology of Fragile X syndrome (FXS), Alzheimer's disease (AD), and schizophrenia (SZ). In these disorders, there is an increase in STEP levels. The current model suggests that the increase in STEP leads to the inappropriate internalization of NR1/NR2B and GluR1/GluR2 receptors. However, the mechanisms by which STEP levels are increased differ in these disorders. In AD and SZ, there is a disruption in the ubiquitination and degradation STEP. In FXS, there is an increase in the translation of STEP mRNA due to the absence of FMRP. STEP is rapidly degraded after specific forms of synaptic activity. Synaptic stimulation results in the ubiquitination of STEP, which is then degraded through the proteasome system. We hypothesize that STEP is degraded at synaptic sites to promote synaptic strengthening. Extrasynaptic NMDAR stimulation results in a calpain-mediated proteolysis STEP. We propose that this mechanism prolongs p38 signaling and initiates cell death pathways. The molecular mechanisms that regulate these processes are unknown. Aim 1 focuses on STEP ubiquitination. We include functional experiments that mutate STEP so that it can no longer be ubiquitinated and degraded. We predict that this construct will disrupt both LTP, as well as the consolidation of fear conditioning after infusion into the amygdala. We will determine the kinases that promote or prevent STEP ubiquitination. Aim 2 focuses on STEP proteolysis. We include functional studies that disrupt proteolysis of STEP, and will determine the effects this has on cell death using in vivo excitotoxic models. We predict that blocking STEP proteolysis will significantly decrease cell death. Aim 3 focuses on STEP translation. Two cytoplasmic polyadenylation elements (CPEs) are present in STEP mRNA and we will determine whether these elements regulate the translation of STEP mRNA. We include functional experiments that will mutate these CPEs. We predict this will disrupt the normal function of STEP in the regulation of its substrates, including glutamate receptor trafficking. These studies will advance our understanding of how STEP is involved in normal cognitive processes and possible mechanisms by which it is disrupted in several diseases in which there are significant cognitive deficits. PUBLIC HEALTH RELEVANCE: A major discovery over the last cycle is that STriatal-Enriched tyrosine Phosphatase (STEP) regulates glutamate receptor trafficking, and removes both NR1/NR2B and GluR1/GluR2 receptors from synaptic membranes after specific forms of synaptic stimulation. These latter findings have implicated STEP in the pathophysiology of fragile X syndrome, schizophrenia, and Alzheimer's disease. The proposed studies examine the molecular mechanisms that regulate STEP activity and will clarify how this important family of regulatory proteins are involved in synaptic plasticity, cell death, and human disease.