Spindle Checkpoint Silencing by TRIP13

The spindle checkpoint is important for maintaining genomic stability and preventing aneuploidy, a hallmark of cancer. The checkpoint ensures that chromosome segregation does not occur until all sister chromatids are correctly attached to the mitotic spindle during metaphase. When this requirement is met, the checkpoint must be silenced for the cell to proceed to anaphase. Thyroid hormone receptor interacting protein 13 (TRIP13) is a hexameric AAA+ ATPase involved in spindle checkpoint silencing. TRIP13 functions by initiating a conformational change in mitotic arrest deficient 2 (Mad2), a key component of the mitotic checkpoint complex (MCC). This TRIP13-mediated conformational change of Mad2 causes MCC disassembly and relieves inhibition of the anaphase promoting complex/cyclosome (APC/C). The interaction between TRIP13 and Mad2 is dependent on the p31comet adaptor protein. In my first project, I show that TRIP13-p31comet disrupts the MCC by local unfolding of Mad2. I identify a binding surface on human TRIP13 for p31comet-Mad2 and key TRIP13 residues involved in its conformational dynamics. I propose that the flexibility of the hinge region of TRIP13 is important for coupling its ATPase activity to substrate unfolding. The hinge region is conserved in other eukaryotic AAA+ ATPases, and may also be important for energetic coupling in those systems. I have also reconstituted the process of spindle checkpoint silencing in vitro. Importantly, I show that TRIP13 can disrupt the free MCC complex, but not MCC bound to APC/C, providing an explanation for the coordination of the multiple mechanisms that work together to achieve spindle checkpoint silencing. In my second project, to provide a tool for future mechanistic studies and to examine the oncogenic activity of TRIP13, I attempted to identify chemical inhibitors for TRIP13 through high-throughput screening. I identified a series of lead compounds that indirectly inhibited TRIP13 as pan-assay interference compounds. These compounds are redox cyclers that generate hydrogen peroxide, which covalently modifies protein residues such as cysteines and tryptophans. No other potent lead compounds were discovered. This study revealed that TRIP13 may be a difficult protein to target, and that large compound libraries should be prescreened for redox cyclers before they are used in high-throughput inhibitor screening.