Chemical Biology of Transglutaminases

酶是一种蛋白质，它催化几乎所有维持生命所必需的化学反应。众所周知，酶对其识别和转化的分子（即底物）具有高度的特异性。为了理解一种酶的生物学作用，有必要首先了解它的特异性，以及它所催化的转化的性质。谷氨酰胺转胺酶（TGases）是一个由八种酶组成的家族，它们参与了多种重要的生理过程。然而，我们仍然对这些不同的tgase催化哪些反应，以及与哪些底物催化有有限的了解。例如，它们要么催化不同蛋白质的交联，要么催化生物胺与这些蛋白质的可逆连接，但我们不知道哪种tgase修饰哪种蛋白质，以何种方式，在哪种组织中。凯勒小组是唯一有资格回答这些问题的人。我们有多年研究组织TG2的经验，最近发现了如何制造与TG2特异性反应的小分子，而不是与其他TG2反应。这使得我们在了解TG2如何在癌症转移中发挥作用方面取得了突破。在下一个授权期内，我们将采用相同的方法，制造能够与TGase家族中另外三个成员（Factor XIII， TG1和TG3）特异性反应的分子。TG1和TG3都被认为在皮肤稳定中起作用；然而，其中一种的过度活跃和另一种的缺乏都与皮肤疾病有关。因子XIII在血液中循环并协助凝血过程，但它也参与骨骼发育和凝血障碍。对于这四种tgase，我们将1)确定它们的特异性，2)开发测量其特异性活性的新方法，3)开发阻断其特异性活性的抑制剂，以及4)开发分子探针，以揭示酶及其底物在活细胞中的位置。实现这些目标将使我们能够剖析这些重要酶的相关活性，发现它们独特的生物学作用，并指导未来治疗与它们不受调节的活性相关的生理障碍。实现这些多学科目标所需的详细方法很多，但它们都是我们当前研究计划所固有的。我们经常使用有机合成、分子生物学、蛋白质生物化学、生物分析技术、动力学研究、哺乳动物细胞培养和荧光显微镜。因此，Keillor小组的学员将有机会接受广泛的高度相关技术的培训，而不是只专注于一种技术。这将为他们在现代生物技术或制药行业或学术界担任有影响力的职位做好充分准备。

Enzymes are proteins that catalyse almost all of the chemical reactions necessary to support life. Enzymes are known to show a high degree of specificity with respect to the molecules that they recognize and transform, known as substrates. In order to understand the biological role of an enzyme, it is essential to first understand its specificity, and the nature of the transformation it catalyses. The transglutaminases (TGases) are a family of eight enzymes that have been implicated in a wide variety of important physiological processes. However, we still have a limited understanding of which of these different TGases catalyse which reactions, and with which substrates. For example, they all catalyse either the cross-linking of different proteins, or the reversible attachment of biological amines to those proteins, but we don't know which TGases modify which proteins, in what way, and in which tissues. The Keillor group is uniquely qualified to answer these questions. We have many years' experience studying tissue TGase (TG2), and have recently discovered how to make small molecules that will react specifically with TG2, and not with other TGases. This has led to a breakthrough in understanding how TG2 plays a role in cancer metastasis. Over the next granting period, we will apply the same approach, to make molecules that will react specifically with three additional members of the TGase family: Factor XIII, TG1 and TG3. TG1 and TG3 are both thought to play roles in the stabilization of skin; however, the excess activity of one and the deficiency of the other have both been implicated in skin disorders. Factor XIII circulates in the blood and assists in the coagulation process, but it is also involved in bone development and in clotting disorders. For each of these four TGases, we will 1) determine their specificity, 2) develop novel methods for measuring their specific activity, 3) develop inhibitors for blocking their specific activity, and 4) develop molecular probes that will reveal where the enzymes and their substrates are located, in living cells. Achieving these objectives will allow us to dissect the related activities of these important enzymes, to discover their unique biological roles and to guide the future treatment of physiological disorders related to their unregulated activity. The detailed methods required to achieve these multidisciplinary objectives are numerous, but they are all inherent in our current research program. We routinely employ organic synthesis, molecular biology, protein biochemistry, bioanalytical techniques, kinetic studies, mammalian cell culture and fluorescence microscopy. Trainees in the Keillor group will therefore have the opportunity to be trained across a broad array of highly relevant techniques, rather than specializing in just one. This will prepare them very well for impactful positions in the modern biotech or pharmaceutical industries, or in academia.