Dissecting Mechanisms of Ubiquitination and Deubiquitination in Cell Cycle Contro

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DESCRIPTION (provided by applicant): Posttranslational modification with ubiquitin chains is essential for cell division in all eukaryotes. Ubiquitin chains are synthesized by a cascade of E1 ubiquitin-activating, E2 ubiquitin-conjugating, and E3 ubiquitin ligase enzymes, with the latter defining the substrate specificity of this modification. Among ~600 human E3s, the anaphase-promoting complex (APC/C) is essential for mitosis, and the aberrant expression of APC/C- regulators or substrates has been tightly linked to tumorigenesis. Moreover, the indirect inhibition of the APC/C by paclitaxel, a small molecule that activates the APC/C-inhibitory spindle checkpoint, has become a mainstay of chemotherapy. We have recently discovered that the APC/C assembles chains of a novel topology, linked through Lys11 of ubiquitin. The APC/C achieves K11-linkage formation by employing an initiating E2, Ube2C, and an elongating E2, Ube2S. Similar to the APC/C, K11-linked chains and the E2-module composed of Ube2C and Ube2S are required for cell division. Therefore, dissecting the APC/C-dependent assembly and function of K11-linked chains is critical to our understanding of cell division. Here, we propose t dissect the mechanism of K11-linked ubiquitin chain formation by using a set of unique biochemical and structural assays combined with ubiquitin- and E2-mutants derived from extensive biochemical screens. We will directly test for functions of K11-linked ubiquitin chains by employing the novel approach of E3-reprogramming that allows us to switch the topology of ubiquitin chains that are assembled by the APC/C in cells. Finally, we will investigate the regulation of K11-linked chain formation, using a group of cancer-related APC/C-substrates, the spindle assembly factors HURP, NuSAP, and Tpx2, as our model system. Together, we expect that our studies will reveal core principles of ubiquitylation and cell cycle regulation. In this manner, our work will likely provide guidance for the development of novel classes of chemotherapeutics that target essential E2 enzymes.