Mukhopadhyay, U. | Search bioRxiv

bioRxiv2023

Structural Insights into The Role of MAGEA4 in RAD18 Regulation: Implications for Ubiquitin Ligase-Binding across the MAGE Protein Family

Melanoma associated antigen 4 (MAGEA4) is a cancer-testis antigen (CTA) that is primarily expressed in the testes of healthy adults but is aberrantly overexpressed and also a poor prognostic marker in several human cancers. In its cancer-specific role, MAGEA4 interacts with RAD18 and activates trans-lesion DNA synthesis (TLS), potentially favouring tumour evolution. However, the precise mode of interaction between RAD18 and MAGEA4 and its implications on the ubiquitination activity of RAD18 are unknown. Here, we employed NMR and AlphaFold2 (AF) to reveal that the ubiquitin-conjugating enzyme RAD6-binding domain (R6BD) of RAD18 interacts with a groove in the C-terminal winged-helix subdomain (WH) of MAGEA4. Using cross-linking mass spectrometry (XL-MS), we found that MAGEA4 displaces RAD6 from the R6BD of RAD18 and inhibits degradative autoubiquitination of RAD18, which could be countered by a competing short peptide of the RAD18 R6B region. AF and XL-MS also revealed an evolutionary invariant intramolecular interaction within RAD18 between the catalytic RING and the DNA-binding SAP domains that is essential for the ubiquitination of PCNA. Using interaction proteomics, we revealed that another Type-I MAGE, MAGE-C2, interacts with the RING ubiquitin ligase TRIM28 in a similar fashion as the MAGEA4/RAD18 complex. We propose that the peptide-binding groove identified in the C-terminal WH of MAGEA4 exists in other type-I MAGE proteins and serves as a ligase-binding cleft. Our data reveal crucial insights into RAD18-mediated ubiquitination of PCNA and its regulation by MAGE-A4.

biochemistry↗

bioRxiv2023

A ubiquitin-specific, proximity-based labeling approach for the identification of ubiquitin ligase substrates

Ubiquitination of proteins is central to protein homeostasis and other cellular processes including DNA repair, vesicular transport, cell-division etc. The process of ubiquitination is conserved from yeast to humans and is carried out by the sequential action of three enzymes: E1, E2 and E3. There are an estimated >600 E3 ligases in humans that execute ubiquitination of specific target proteins in a spatio-temporal manner to elicit desired signaling effects. Here, we developed a ubiquitin-specific proximity-based labeling method to selectively biotinylate substrates of a given ubiquitin ligase. Our method exploits the proximity and the relative orientation of the E3-ligase catalytic domain with respect to ubiquitin observed in the enzymatic intermediate-state structures of E3-E2[~]Ub. By fusing the biotin ligase BirA and an Avi-tag variant to the candidate E3 ligase and ubiquitin, respectively, we were able to specifically enrich bona fide substrates and potential new substrates of a ligase using a one-step streptavidin pulldown under denaturing conditions. As proof-of-principle, we applied our method, which we named Ub-POD, to the RING E3 ligase RAD18. RAD18 ubiquitinates DNA-sliding clamp PCNA upon UV-induced DNA damage. We identified PCNA and several other critical players in the DNA damage repair pathway in a single RAD18 Ub-POD experiment. We went on to validate DNA replicase POLE as a possible new substrate of RAD18. Through RAD18 Ub-POD, we were also able to pin down the cellular localization of RAD18-mediated ubiquitination to the damaged DNA nuclear puncta using streptavidin immunofluorescence. Furthermore, we applied Ub-POD to TRAF6, another RING ubiquitin ligase involved in NF-{kappa}B signaling and successfully identified known and potentially new TRAF6 substrates. Finally, we adapted our method to the U-box-type E3 ubiquitin ligase CHIP to demonstrate that we can identify substrates of two major classes of mammalian ubiquitin ligases. We anticipate that our method and principle could be widely adapted to all classes of ubiquitin ligases to identify substrates and localize the cellular site(s) of ubiquitination.

biochemistry↗