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, SCF?-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 ?-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 SCF?-TRCP. However, it remains largely unknown how upstream signaling pathways control ?-TRCP stability and physiological functions in vivo. To this end, our preliminary results reveal that like Fbw7, ?-TRCP undergoes auto-ubiquitination to negatively control its own stability, while OTUD3, but not other OTU family of DUBs, specifically interacts with, and deubiquitinates, ?-TRCP to control its stability. As such, depletion of OTUD3 significantly reduced ?-TRCP abundance.
In Aim #1, we intend to explore mechanistically how the ?-TRCP signaling pathway is governed by the dynamic auto-uibiquitination and deubiquitination processes to influence biological functions of ?-TRCP in vivo. Furthermore, other than tissue context-dependent roles for ?-TRCP in tumorigenesis, the physiological role of ?-TRCP in metabolism such as lipid homeostasis has not been described. We reasoned that identification of additional ?-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 ?-TRCP substrates at endogenous levels using a ?-TRCP phospho-degron specific antibody- mediated mass spectrometry approach. We identified many known ?-TRCP targets, validating this screening method, and characterized Lipin1 and Lyric as novel ?-TRCP substrates. This finding provides a novel link between ?-TRCP and tissue-specific metabolic phenotypes observed in ?-TRCP1-/- mice. Therefore, another major focus is to explore mechanistically how ?-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 ?-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 ?-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.

Public Health Relevance

This project mainly focuses on elucidating how upstream signaling pathways including OTUD3 modulate the stability and the cellular functions of ?-TRCP in part via deubiquitinating ?-TRCP, as well as characterizing novel physiological tissue context-dependent roles of ?-TRCP in lipid metabolism and tumorigenesis by promoting the ubiquitination and subsequent degradation of different subset of downstream substrates such as Lipin1 or Lyric in different tissues including hepatocytes and enterocytes, respectively. These proposed studies should significantly extend our current understanding of ?-TRCP biology by uncovering a novel tissue context-dependent role for ?-TRCP in lipid metabolic regulation, and further implicate that aberrant regulation of ?-TRCP signaling pathway may lead to human diseases including cancer and lipid homeostasis disorders.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Cancer Etiology Study Section (CE)
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Spalholz, Barbara A
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Beth Israel Deaconess Medical Center
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