UGT PROTEIN NETWORK AND THE CONTROL OF CELL METABOLISM.

在高等真核生物中，由UDP-葡萄糖醛酸基转移酶(UGTS)催化的葡萄糖醛酸化反应对于抵御内生生物(如激素、神经递质)和有害外源生物(如污染物、药物)的不平衡是至关重要的。在人类中，9种具有功能的UGTS酶由单个基因UGT1A编码。作为该领域的一项重大突破，我们发现了一种新的细胞葡萄糖醛酸化反应的转录后调控机制，涉及通过选择性剪接产生的其他9种较短的UGT1A蛋白(命名为I2S)。这些I2s在酶活性上是不活跃的，但通过蛋白质-蛋白质相互作用抑制内质网(ER)中的I1酶，从而起到细胞代谢的负调节作用。此外，I2S定位于内质网外，暗示了潜在的额外作用和与新伙伴蛋白的相互作用。我们假设UGT1A包括一个高度组织化的蛋白质网络，该网络由可能参与调节UGT1A介导的活动的蛋白质(如最初观察到的I2S)组成，并可能通过与新伙伴的相互作用而形成，这将揭示该蛋白质家族的额外细胞功能。目的1：研究内质网中i1酶和i2调节因子之间的蛋白质-蛋白质相互作用，并确定相互作用区域。目的2：寻找新的相互作用伙伴(内质网驻留和非驻留)，为UGT1a相关蛋白参与调节葡萄糖醛酸化活性提供新的见解，并探索UGT可能的新功能。我们将使用补充方法，包括质谱学和在上一次赠款期间建立的模型和工具。这一发现可能为调节或调节葡萄糖醛酸化途径提供新的方法。识别和了解蛋白质-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作用和开发新的治疗方法的基础。我们的团队已经发现了UGT1A剪接变异体，并首次揭示了它们的调控功能；因此，我们拥有独特的专业知识和工具来实现本提案中提出的目标。在整个研究项目中，高素质的学生将继续接受培训。

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.