Pulse-Chase Labeling with 15N and AHA in an Alzheimer's Mouse Model

描述（由申请人提供）：蛋白质在细胞或细胞外基质中存在的时间长度可能是蛋白质在疾病中作用的重要决定因素。随着时间的推移，蛋白质可能被破坏或修饰，然后必须通过涉及细胞内蛋白酶体或自噬系统的蛋白水解过程或通过整个细胞的周转从细胞中除去。因此，蛋白质寿命和维持在有丝分裂后细胞如神经元中特别重要。传统上，使用放射性同位素的脉冲追踪技术是测量蛋白质表达和周转的方法。我们已经开发了一种体内脉冲追踪方法，使用15 N标记的动物来测量蛋白质的寿命，并且使用这些方法，我们令人惊讶地观察到内核核孔蛋白是出乎意料的长寿命蛋白质。这些研究在大鼠中使用了线性速率衰减分析模型，从3个月开始，一直持续到12个月。在本研究计划中，我们将用15 N标记Borchelt阿尔茨海默病小鼠模型，并用14 N食物追踪标记的动物。14 N与15 N比率的测量将确定APP、abeta 1 -42和聚集体中存在的其他蛋白质的寿命。我们的期望是，在聚集体中发现的蛋白质将是长寿的，因为去除错误折叠蛋白质的正常过程已经失败。该R 03提案的第二个目标是开发一种使用L-叠氮基高丙氨酸（AHA）来测量小鼠中新翻译的蛋白质的表达的方法。作为原理的证明，我们已经成功地用L-叠氮基高丙氨酸脉冲标记小鼠以确定安全性和掺入水平。在拟议的研究中，我们将在Borchelt阿尔茨海默氏症小鼠模型的设定时间点在小鼠食物中脉冲AHA。我们将丰富和测量蛋白质结合AHA在前额叶皮层和不溶性脑聚集体。只有新翻译的蛋白质应该已经掺入AHA，并将使用“点击”化学富集。我们将识别新的蛋白质被翻译和招募到前额叶皮层和聚集。我们的假设是，新的蛋白质被表达并聚集在一起，以帮助去除它们。

DESCRIPTION (provided by applicant): The length of time a protein persists in a cell or the extracellular matrix can be an important determinant of the protein's role in disease. Over time proteins can be damaged or modified and then must be removed from the cell by proteolytic processes involving the proteasome or autophagic systems within the cell or by turnover of the whole cell. Accordingly, protein lifetimes and maintenance are particularly important in post-mitotic cells such as neurons. Traditionally, pulse-chase techniques using radioactive isotopes have been a method to measure the expression and turnover of proteins. We have developed an in vivo pulse-chase method using 15N labeled animals to measure the lifetimes of proteins and using these methods we observed surprisingly that inner core nuclear pore proteins are unexpectedly long- lived proteins. These studies used a linear rate decay analysis model in rats starting at 3 months and extending through 12 months. In this research proposal, we will label the Borchelt Alzheimer's disease mouse model with 15N and chase the labeled animal with 14N food. Measurement of the 14N to 15N ratios will determine the lifetime of APP, abeta1-42 and other proteins present in aggregates. Our expectation is that protein found in the aggregates will be long lived because the normal processes that remove misfolded proteins have failed. A second goal of this R03 proposal is to develop a method using L-azidohomoalanine (AHA) to measure the expression of newly translated proteins in mice. As proof of principle we have successfully pulse labeled a mouse with L-azidohomoalanine to determine safety and incorporation levels. In the proposed research we will pulse AHA in the mouse's food at set timepoints in a Borchelt Alzheimer's mouse model of Alzheimer's disease. We will enrich and measure proteins incorporating AHA in the prefrontal cortex and in insoluble brain aggregates. Only newly translated proteins should have incorporated AHA and will be enriched using "click" chemistry. We will identify new proteins being translated and recruited to the prefrontal cortex and to aggregates. Our hypothesis is that new proteins are being expressed and recruited to aggregates to help remove them.