Design of linked-domain protein inhibitors of UBE2D as tools to study cellular ubiquitination
Bukhari, Z.; Gu, L.; Nederstigt, A. E.; Cope, L. J.; Bolhuis, D. L.; Harvey, K.; Allen, T.; Hill, S.; Yang, Y.; Lawson, G.; Lu, C.; Tran, T.; Leah, P.; Low, L.; Chiang, A.; Song, J.; Fong, M. V.; Rangel, V. M.; Chan, W. K.; Kleiger, G.; Goldfarb, D.; Vierra, C. A.; Brown, N. G.; Harrison, J. S.
Show abstract
Ubiquitin (Ub) is a post-translational modification that largely controls proteostasis through mechanisms spanning transcription, translation, and notably, protein degradation. Ub conjugation occurs through a hierarchical cascade of three enzyme classes (E1, E2, and E3s) involving >1000 proteins that regulate the ubiquitination of proteins. The E2 Ub-conjugating enzymes are the midpoint, yet their cellular roles remain under-characterized, partly due to a lack of inhibitors. For example, the cellular roles of the promiscuous E2 UBE2D/UBCH5 are not well described. Here, we develop a highly selective, multivalent, engineered protein inhibitor for the UBE2D family that simultaneously targets the RING- and backside-binding sites. In HeLa cells, these inhibitors phenocopy knockdown of UBE2D by reducing the IC50 to cisplatin and whole-cell proteomics reveal an increased abundance of [~]20% of the identified proteins, consistent with reduced Ub degradation and proteotoxic stress. These precision tools will enable new studies probing UBE2Ds central role in proteome management.
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