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持续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质水解过程或整个细胞的周转从细胞中移除。因此，蛋白质的寿命和维持在有丝分裂后的细胞如神经元中尤为重要。传统上，使用放射性同位素的脉冲追踪技术一直是测量蛋白质表达和周转的一种方法。我们开发了一种体内脉冲追踪方法，使用15N标记的动物来测量蛋白质的寿命，并使用这些方法我们惊讶地观察到内核核孔蛋白是出乎意料的长寿蛋白质。这些研究采用线性速率衰减分析模型，从大鼠3个月开始，一直持续到12个月。在本研究计划中，我们将用15N标记Borchelt阿尔茨海默病小鼠模型，并用14N食物追赶标记的动物。测定14N和15N的比值将决定APP、β 1-42和其他蛋白在聚集体中的寿命。我们的期望是，在聚集体中发现的蛋白质将长期存在，因为去除错误折叠蛋白质的正常过程已经失败。R03提案的第二个目标是开发一种使用l -叠氮多同丙氨酸（AHA）来测量小鼠中新翻译蛋白表达的方法。作为原理证明，我们已经成功地用l -叠氮多同丙氨酸脉冲标记了一只老鼠，以确定安全性和掺入水平。在拟议的研究中，我们将在Borchelt阿尔茨海默氏病小鼠模型中，在设定的时间点对小鼠的食物进行AHA脉冲。我们将在前额皮质和不溶性脑聚集物中富集和测量含有AHA的蛋白质。只有新翻译的蛋白质应该包含AHA，并将通过“点击”化学富集。我们将发现新的蛋白质被翻译和招募到前额皮质和聚集体。我们的假设是，新的蛋白质正在被表达并聚集到聚集体中，以帮助清除它们。

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.