Functional analysis of a tissue context-dependent role for beta-TRCP in lipid metabolism and tumorigenesis

适当的细胞周期转变是由APC/C和SCF E3泛素连接酶复合物对关键细胞周期调节因子的泛素依赖性降解的协调波驱动的。其中，SCFβ-TRCP是基于Cullin 1的E3泛素连接酶之一，通过促进-catenin、Emi1和Ib等关键调节蛋白的降解，参与许多重要的细胞过程。在上一个资助周期中，我们的团队和其他人通过分别将DEPTOR、VEGFR、Mdm2和Cdh1定义为SCFβ-TRCP的下游底物，为进一步了解β-TRCP在自噬、细胞迁移、DNA损伤反应和细胞周期调节等各种生理功能中的关键作用做出了重大贡献。然而，在体内，上游信号通路如何控制β-TRCP的稳定性和生理功能仍是未知的。为此，我们的初步结果表明，与Fbw7一样，β-TRCP通过自身泛素化来负向控制其自身的稳定性，而OTUD3与β-TRCP特异性相互作用并去泛素化以控制其稳定性，而不是其他OTU家族的DUBs。因此，OTUD3的缺失显著降低了β-TRCP的丰度。在Aim #1中，我们打算从机制上探索β-TRCP信号通路如何受动态自泛素化和去泛素化过程的控制，从而影响体内β-TRCP的生物学功能。此外，除了β-TRCP在肿瘤发生中的组织环境依赖性作用外，β-TRCP在代谢（如脂质稳态）中的生理作用尚未被描述。我们推断，鉴定额外的β-TRCP泛素底物将进一步确定其生理功能。为了克服在大多数E3连接酶底物筛选中使用异位过表达条件的担忧，我们开发了一种新的筛选系统，使用β-TRCP磷酸化-degron特异性抗体介导的质谱方法来鉴定内源性水平的β-TRCP底物。我们确定了许多已知的β-TRCP靶点，验证了这种筛选方法，并将Lipin1和Lyric表征为新的β-TRCP底物。这一发现提供了β-TRCP与β-TRCP1-/-小鼠中观察到的组织特异性代谢表型之间的新联系。因此，另一个主要焦点是探索β-TRCP如何通过调节Lipin1蛋白稳定性和随后抑制SREBP1转录活性来控制肝细胞脂质代谢的机制（Aim # 2）。最后，我们还打算揭示β-TRCP在通过调节抒情蛋白稳定性来控制肠细胞脂质吸收途径和肿瘤发生中的关键生理作用（目的3）。我们相信这些建议的研究将显著扩展我们对β-TRCP如何发挥组织环境依赖作用来控制脂质代谢途径等重要过程的理解，并进一步暗示除了肿瘤发生外，-TRCP信号通路的异常调节可能导致其他人类疾病，包括脂质稳态紊乱，这将最终为开发更好的治疗方法提供理论依据。

Proper cell cycle transitions are driven by coordinated waves of ubiquitin-dependent degradation of key cell cycle regulators by APC/C and SCF E3 ubiquitin ligase complexes. Among them, SCFβ-TRCP is one of the well-characterized Cullin 1-based E3 ubiquitin ligases involved in numerous important cellular processes through promoting the degradation of critical regulatory proteins including -catenin, Emi1 and IBs. During the last funding cycle, our group and others have made significant contributions to further our understanding of the critical role of β-TRCP in various physiological functions such as autophagy, cell migration, DNA damage response and cell cycle regulation by defining DEPTOR, VEGFR, Mdm2 and Cdh1, respectively, as downstream substrates of SCFβ-TRCP. However, it remains largely unknown how upstream signaling pathways control β-TRCP stability and physiological functions in vivo. To this end, our preliminary results reveal that like Fbw7, β-TRCP undergoes auto-ubiquitination to negatively control its own stability, while OTUD3, but not other OTU family of DUBs, specifically interacts with, and deubiquitinates, β-TRCP to control its stability. As such, depletion of OTUD3 significantly reduced β-TRCP abundance. In Aim #1, we intend to explore mechanistically how the β-TRCP signaling pathway is governed by the dynamic auto-uibiquitination and deubiquitination processes to influence biological functions of β-TRCP in vivo. Furthermore, other than tissue context-dependent roles for β-TRCP in tumorigenesis, the physiological role of β-TRCP in metabolism such as lipid homeostasis has not been described. We reasoned that identification of additional β-TRCP ubiquitin substrate(s) would further define its physiological functions. To overcome the concern of using ectopic overexpression conditions in most E3 ligase-substrate screenings, we developed a novel screening system to identify β-TRCP substrates at endogenous levels using a β-TRCP phospho-degron specific antibody- mediated mass spectrometry approach. We identified many known β-TRCP targets, validating this screening method, and characterized Lipin1 and Lyric as novel β-TRCP substrates. This finding provides a novel link between β-TRCP and tissue-specific metabolic phenotypes observed in β-TRCP1-/- mice. Therefore, another major focus is to explore mechanistically how β-TRCP controls hepatocyte lipid metabolism through regulating Lipin1 protein stability and subsequent inhibition of SREBP1 transcriptional activity (Aim # 2). Lastly, we also intend to reveal a critical physiological role for β-TRCP in controlling enterocyte lipid absorption pathways and tumorigenesis by governing Lyric protein stability (Aim #3). We believe that these proposed studies will significantly extend our understanding of how β-TRCP exerts tissue context-dependent roles to control important process such as lipid metabolic pathways, and further implicate that in addition to tumorigenesis, aberrant regulation of -TRCP signaling pathway may lead to other human diseases including lipid homeostasis disorders, which will ultimately provide the rationale to develop better therapies.