MRI: Acquisition of a Phosphorimager to Investigate Phosphorylation and Regulation of TACE

在摩尔博士的监督下，向卡拉马祖学院提供了一笔赠款，用于购买一台磷光成像仪，这将使教师能够研究TNF-α转化酶（TACE）的磷酸化和调节。 TACE是一种蛋白质，其作为酶起作用，催化将大的全长前体（称为TACE的底物）切割成较小的片段。 了解TACE酶的活性是如何调节的，将提供关于这些片段是如何机械产生的见解。 计划进行特定实验以确定TACE上被PKC磷酸化的位点（特定氨基酸），并确定TACE的磷酸化状态是否直接影响其活性。 前者涉及使用缺乏关键磷酸化位点的突变形式的TACE。 该实验将确定在TACE上这些磷酸基团被添加的位置。 后者还涉及在切割测定中使用上述突变形式的TACE，以确定TACE底物（TACE作用并切割的其他蛋白质）的切割是否受到磷酸化状态的影响。除了提供有关TACE酶调节的基本科学信息外，上述实验将产生更广泛的影响。 例如，我们对TACE酶的了解可以推广到与TACE相同的蛋白质“家族”中的类似蛋白酶。 此外，了解TACE被“打开”的机制将阐明细胞“打开”蛋白酶的一般策略。 这项工作的更广泛影响在于揭示细胞功能的复杂性：科学家对细胞如何正常工作的了解越多，科学家就能更好地确定疾病或损伤状态下功能失常的细胞中出现的问题。 此外，由于这些实验将与本科生合作者一起完成，因此将提供重要的教育经验。 此外，学院的其他几位教职员工也急于学习这项技术的应用，他们将从校园里的这台设备中受益。来自生物和化学系的教师和学生都将参与利用这些设备的研究。

A grant has been awarded to Kalamazoo College under the supervision of Dr. Moore,to acquire a phosphoimager, which will allow faculty to investigate phosphorylation and regulation of the TNF-alpha converting enzyme (TACE). TACE is a protein that works as an enzyme, catalyzing the cleavage of large, full-length precursors (known as substrates of TACE) into smaller fragments. Understanding how the activity of the TACE enzyme is regulated will provide insights as to how, mechanistically, these fragments are generated. Specific experiments are planned to determine the sites (specific amino acids) on TACE that are phosphorylated by PKC, and to determine whether the phosphorylation state of TACE directly influences its activity. The former involves the use of mutated versions of TACE that lack critical proposed phosphorylation sites. This experiment will determine where on TACE these phosphate groups are added. The latter also involves the use of the aforementioned mutant versions of TACE in cleavage assays to determine whether the cleavage of TACE substrates (other proteins that TACE acts on and cleaves) is affected by phosphorylation state. In addition to providing basic science information about the regulation of the TACE enzyme, the experiments described above will have a much broader impact. For example, much of what we learn about the TACE enzyme may be generalized to similar proteases that are in the same protein "family" as TACE. Furthermore, understanding the mechanisms whereby TACE is "turned on" will illuminate general strategies used by cells to "turn on" protein enzymes. The broader impact of this work lies in uncovering the complexities of cellular function: the more scientist know about how cells work normally, the better prepared scientists are to determine what goes wrong in malfunctioning cells in disease or injury states. Moreover, because these experiments will be done in conjunction with undergraduate student collaborators, an important educational experience will be provided. In addition several other faculty members in the college are anxious to learn application of the technique, and they will benefit from the presence of this piece of equipment on the campus. Both faculty and students from the biology and chemistry departments will be involved in research utilizing the equipment.