The E3 ubiquitin ligase DTX3L and the deubiquitinase USP28 fine-tune DNA double strand repair through mutual regulation of their protein levels

Abstract

The DNA damage response involves a complex protein network with members mediating different post-translational modifications such as ubiquitination and deubiquitination. Thereby the E3 ubiquitin ligase DTX3L as well as the deubiquitinase USP28 are recruited especially to DNA double strand breaks (DSBs) suggesting mutual functional interactions. Here we present evidence for the existence of such crosstalk. Mechanistically we show that DTX3L interacts directly with USP28 and ubiquitinates it, which leads to its proteasomal degradation. Vice versa, USP28 can remove those polyubiquitin chains from itself as well as from autoubiquitinated DTX3L. Consequently, these mutual regulatory interactions between DTX3L and USP28 affected DSB repair activities. Analysis of distinct DSB repair pathways reveals synthetic dysfunction of canonical non-homologues end joining (NHEJ) and homologous recombination (HR), upon USP28 and DTX3L double knockdown, suggesting cooperation between these proteins. Conversely, error-prone microhomology-mediated end joining (MMEJ) requires USP28 to counterbalance the antagonistic DTX3L effect. Together, the present data indicate that DTX3L and USP28 are under mutual control to fine-tune the capacity and quality of the cellular responses to stresses such as DNA damage.

Discovery of DTX3L inhibitors through a homogeneous FRET-based assay that monitors formation and removal of poly-ubiquitin chains

“…As a final confirmation that the assay was able to detect inhibition of USP28, we measured the IC 50 value of a known dual inhibitor for USP28 and USP25 (AZ1) using the FRET-based assay ( Figure 4E ). We previously showed that in cells this compound is able to affect DTX3L in a similar fashion as knock down of USP28 (Ashok et al ., 2023) and the IC 50 value determined with our method (IC 50 = 8 μM) is close to the binding affinity (K d value of 3.7 μM) measured previously with microscale thermophoresis (MST) (Wrigley et al ., 2017). This indicates that our system can be adopted to identify and characterize inhibitors also for the DUB.…”

“…We have previously reported that there is a mutual regulation between USP28 and DTX3L in which the DUB hydrolyses poly-Ub chains generated by DTX3L. Ubiquitination of USP28 by DTX3L was also demonstrated with a catalytically inactive USP28 C171A mutant (Ashok et al, 2023). We therefore sought to test if addition of USP28 to pre-formed poly-Ub chains on DTX3L would lead to reduction in rFRET due to hydrolysis poly-Ub chains.…”

“…DUBs are classified in the groups of cysteine proteinases and metalloproteases with distinct reaction mechanisms (Snyder and Silva, 2021). We have previously identified that at least Ubiquitin Specific Protease 28 (USP28) can remove the Ub chains generated by DTX3L both from DTX3L itself and from USP28 in vitro as well as in cellular context thereby regulating the DNA repair pathways (Ashok et al ., 2023).…”