We propose a model in which the natural product parthenolide covalently binds Cys54 of BUGZ, inhibiting its ability to facilitate proper kinetochore-microtubule attachment required for accurate chromosome segregation in mitosis. 10/11

Finally, we analyzed PTL-bound BUGZ using mass spectrometry and identified Cys54 of BUGZ as the binding site of PTL. 9/11

...and alkyne parthenolide was strongly reduced at the kinetochores of BUGZ-depleted cells, indicating that BUGZ is the main target of parthenolide at the kinetochores! 8/11

Indeed, BUGZ depletion showed a similar mitotic phenotype as observed in parthenolide-treated cells... 7/11

Next, the pull-downs from alkyne parthenolide-treated cells were clicked to the azide agarose beads and analyzed by quantitative mass spectrometry. Using this approach, we identified ZNF207/BUGZ as the most promising candidate involved in the regulation of kinetochore-microtubule attachments. 6/11

First, we generated a clickable derivative of PTL with an alkyne group, clicked it to an azide-fluorophore and monitored intracellular localization of PTL by microscopy. In line with its mitotic effects, we found that PTL localizes at kinetochores in prometaphase cells! 5/11

To identify mitotic targets of PTL, we combined click chemistry with quantitative mass spectrometry and microscopy. 4/11

We found out that PTL does not alter microtubule dynamics directly but rather obstructs the formation of kinetochore-microtubule attachment in early mitosis. 3/11

When added to cells prior to mitosis, the natural product parthenolide (PTL) interferes with chromosome congression to the metaphase plate, causing a strong mitotic arrest. 2/11

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