UGT PROTEIN NETWORK AND THE CONTROL OF CELL METABOLISM.

由udp -葡萄糖醛基转移酶（UGTs）催化的葡萄糖醛酸化作用在高等真核生物中至关重要，可以保护生物免受内源性（如激素、神经递质）和有害外源性（如污染物、药物）的失衡。在人类中，九种功能性UGTs酶由一个名为UGT1A的基因编码。作为该领域的重大突破，我们发现了一种新的细胞糖醛酸化转录后调控机制，涉及通过选择性剪接产生的另外9种较短的UGT1A蛋白（命名为i2s）。这些i2s酶无活性，但通过蛋白质-蛋白质相互作用抑制内质网（ER）中的i1酶，从而作为细胞代谢的负调节因子。此外，i2s定位于内质网外，表明其可能具有其他作用并与新的伴侣蛋白相互作用。我们假设ugt1a包含一个高度组织的蛋白质网络，该网络由可能参与调节ugt1a介导的活性的蛋白质塑造（如最初在i2s中观察到的），并可能通过与新伙伴的相互作用揭示该蛋白家族的其他细胞功能。目的1：研究内质网i1酶和i2调节因子之间的蛋白-蛋白相互作用，并确定所涉及的相互作用域。目的2：确定新的相互作用伙伴（ER-resident和non-resident），为ugt1a相关蛋白参与糖醛酸化活性调节提供新的见解，并探索ugt可能的新功能。我们将使用互补的方法，包括质谱法和在上一个资助期建立的模型和工具。这些发现可能为调节葡萄糖醛酸化途径提供新的途径。识别和理解蛋白质-蛋白质相互作用的作用是在系统水平上理解葡萄糖醛酸化和开发新的治疗方法的基础。本课题组首次发现UGT1A剪接变异体，揭示其调控功能；因此，我们拥有独特的专门知识和工具来实现这项建议中提出的目标。高素质的学生将在整个研究项目中继续接受培训。

Glucuronidation, catalyzed by UDP-glucuronosyltransferases (UGTs), is crucial in higher eukaryotes for protection against imbalances in endobiotics (e.g., hormones, neurotransmitters) and harmful xenobiotics (e.g., pollutants, drugs). In humans, nine functional UGTs enzymes are encoded by a single gene, UGT1A. As a major breakthrough in the field, we discovered a novel post-transcriptional regulatory mechanism of cellular glucuronidation involving nine other shorter UGT1A proteins (named i2s) produced by alternative splicing. These i2s are enzymatically inactive but act as negative modulators of cellular metabolism by inhibiting the i1 enzymes in the endoplasmic reticulum (ER) through protein-protein interaction. In addition, i2s localize outside the ER, suggesting potential additional roles and interactions with new partner proteins. We hypothesized that UGT1As comprise a highly organized protein network that is shaped by proteins likely involved in modulating UGT1A-mediated activities (as initially observed for i2s) and possibly through interaction with novel partners that would reveal additional cellular functions for this family of proteins. Aim 1: To study protein-protein interactions between i1 enzymes and i2 regulators in the ER and define the interaction domains involved. Aim 2: To identify novel interacting partners (ER-resident and non-resident) to provide new insights into UGT1A-associated proteins involved in modulating glucuronidation activity and explore putative new functions for UGTs. We will use complementary approaches including mass spectrometry and the models and tools established in the last grant period. The findings may provide novel approaches to regulate or modulate the glucuronidation pathway. Identifying and understanding the role of protein-protein interactions is fundamental to understanding glucuronidation at the systems level and developing novel therapeutics. Our group has discover UGT1A splice variants and reveal their regulatory function for the first time; we thus have the unique expertise and tools to achieve the goals set forth in this proposal. Highly qualified students will continue to be trained throughout this research program.