Oligomerization mediated by the D2 domain of DTX3L is critical for DTX3L-PARP9 reading function of mono-ADP-ribosylated androgen receptor.

Deltex proteins are a family of E3 ubiquitin ligases that encode C-terminal RING and DTC domains that mediate interactions with E2 ubiquitin-conjugating enzymes and recognize ubiquitination substrates. DTX3L is unique among the Deltex proteins based on its N-terminal domain architecture. The N-terminal D1 and D2 domains of DTX3L mediate homo-oligomerization, and the D3 domain interacts with PARP9, a protein that contains tandem macrodomains with ADP-ribose reader function. While DTX3L and PARP9 are known to heterodimerize, and assemble into a high molecular weight oligomeric complex, the nature of the oligomeric structure, including whether this contributes to the ADP-ribose reader function is unknown. Here, we report a crystal structure of the DTX3L N-terminal D2 domain and show that it forms a tetramer with, conveniently, D2 symmetry. We identified two interfaces in the structure: a major, conserved interface with a surface of 973 Å2 and a smaller one of 415 Å2. Using native mass spectrometry, we observed molecular species that correspond to monomers, dimers and tetramers of the D2 domain. Reconstitution of DTX3L knockout cells with a D1-D2 deletion mutant showed the domain is dispensable for DTX3L-PARP9 heterodimer formation, but necessary to assemble an oligomeric complex with efficient reader function for ADP-ribosylated androgen receptor. Our results suggest that homo-oligomerization of DTX3L is important for the DTX3L-PARP9 complex to read mono-ADP-ribosylation on a ligand-regulated transcription factor.

Oligomerisation mediated by the D2 domain of DTX3L is critical for DTX3L-PARP9 reading function of mono-ADP-ribosylated androgen receptor.

Deltex proteins are a family of E3 ubiquitin ligases that encode C-terminal RING and DTC domains that mediate interactions with E2 ubiquitin-conjugating enzymes and recognise ubiquitination substrates. DTX3L is unique among the Deltex proteins based on its N-terminal domain architecture. The N-terminal D1 and D2 domains of DTX3L mediate homo-oligomerisation, and the D3 domain interacts with PARP9, a protein that contains tandem macrodomains with ADP-ribose reader function. While DTX3L and PARP9 are known to heterodimerize, they assemble into a high molecular weight oligomeric complex, but the nature of the oligomeric structure, including whether this contributes to the ADP-ribose reader function is unknown. Here, we report a crystal structure of the DTX3L N-terminal D2 domain and show that it forms a tetramer with, conveniently, D2 symmetry. We identified two interfaces in the structure: a major, conserved interface with a surface of 973 Å2 and a smaller one of 415 Å2. Using native mass spectrometry, we observed molecular species that correspond to monomers, dimers and tetramers of the D2 domain. Reconstitution of DTX3L knockout cells with a D1-D2 deletion mutant showed the domain is dispensable for DTX3L-PARP9 heterodimer formation, but necessary to assemble an oligomeric complex with efficient reader function for ADP-ribosylated androgen receptor. Our results suggest that homo-oligomerisation of DTX3L is important for mono-ADP-ribosylation reading by the DTX3L-PARP9 complex and to a ligand-regulated transcription factor.

Synthetic Dual Cysteine-ADP Ribosylated Peptides from the Androgen Receptor are Recognized by the DTX3L/PARP9 Complex.

Androgen signaling in prostate cancer cells involves multisite cysteine ADP-ribosylation of the androgen receptor (AR) by PARP7. The AR modification is read by ADP-ribosyl binding macrodomains in PARP9, but the reason that multiple cysteines are modified is unknown. Here, we use synthetic peptides to show that dual ADP-ribosylation of closely spaced cysteines mediates recognition by the DTX3L/PARP9 complex. Mono and dual ADP-ribosylated cysteine peptides were prepared using a novel solid-phase synthetic strategy utilizing a key, Boc-protected, ribofuranosylcysteine building block. This synthetic strategy allowed us to synthesize fluorescently labeled peptides containing a dual ADP-ribosylation motif. It was found that the DTX3L/PARP9 complex recognizes the dual ADP-ribosylated AR peptide (Kd = 80.5 nM) with significantly higher affinity than peptides with a single ADP-ribose. Moreover, oligomerization of the DTX3L/PARP9 complex proved crucial for ADP-ribosyl-peptide interaction since a deletion mutant of the complex that prevents its oligomer formation dramatically reduced peptide binding. Our data show that features of the substrate modification and the reader contribute to the efficiency of the interaction and imply that multivalent interactions are important for AR-DTX3L/PARP9 assembly.

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

Ubiquitination is a reversible protein post-translational modification in which consequent enzymatic activity results in the covalent linking of ubiquitin to a target protein. Once ubiquitinated, a protein can undergo multiple rounds of ubiquitination on multiple sites or form poly-ubiquitin chains. Ubiquitination regulates various cellular processes, and dysregulation of ubiquitination has been associated with more than one type of cancer. Therefore, efforts have been carried out to identify modulators of the ubiquitination cascade. Herein, we present the development of a FRET-based assay that allows us to monitor ubiquitination activity of DTX3L, a RING-type E3 ubiquitin ligase. Our method shows a good signal window with a robust average Z' factor of 0.76 on 384-well microplates, indicating a good assay for screening inhibitors in a high-throughput setting. From a validatory screening experiment, we have identified the first molecules that inhibit DTX3L with potencies in the low micromolar range. We also demonstrate that the method can be expanded to study deubiquitinases, such as USP28, that reduce FRET due to hydrolysis of fluorescent poly-ubiquitin chains.

Activities and binding partners of E3 ubiquitin ligase DTX3L and its roles in cancer.

Ubiquitination is a protein post-translational modification that affects protein localisation, stability and interactions. E3 ubiquitin ligases regulate the final step of the ubiquitination reaction by recognising target proteins and mediating the ubiquitin transfer from an E2 enzyme. DTX3L is a multi-domain E3 ubiquitin ligase in which the N-terminus mediates protein oligomerisation, a middle D3 domain mediates the interaction with PARP9, a RING domain responsible for recognising E2 ∼ Ub and a DTC domain has the dual activity of ADP-ribosylating ubiquitin and mediating ubiquitination. The activity of DTX3L is known to be modulated by at least two different factors: the concentration of NAD+, which dictates if the enzyme acts as a ligase or as an ADP-ribosyltransferase, and its binding partners, which affect DTX3L activity through yet unknown mechanisms. In light of recent findings it is possible that DTX3L could ubiquitinate ADP-ribose attached to proteins. Different DTX3L-protein complexes have been found to be part of multiple signalling pathways through which they promote the adhesion, proliferation, migration and chemoresistance of e.g. lymphoma, glioma, melanoma, and prostate cancer. In this review, we have covered the literature available for the molecular functions of DTX3L especially in the context of cancer biology, different pathways it regulates and how these relate to its function as an oncoprotein.

Reconstitution of the DTX3L-PARP9 complex reveals determinants for high-affinity heterodimerization and multimeric assembly.

Ubiquitination and ADP-ribosylation are post-translational modifications that play major roles in pathways including the DNA damage response and viral infection. The enzymes responsible for these modifications are therefore potential targets for therapeutic intervention. DTX3L is an E3 Ubiquitin ligase that forms a heterodimer with PARP9. In addition to its ubiquitin ligase activity, DTX3L-PARP9 also acts as an ADP-ribosyl transferase for Gly76 on the C-terminus of ubiquitin. NAD+-dependent ADP-ribosylation of ubiquitin by DTX3L-PARP9 prevents ubiquitin from conjugating to protein substrates. To gain insight into how DTX3L-PARP9 generates these post-translational modifications, we produced recombinant forms of DTX3L and PARP9 and studied their physical interactions. We show the DTX3L D3 domain (230-510) mediates the interaction with PARP9 with nanomolar affinity and an apparent 1 : 1 stoichiometry. We also show that DTX3L and PARP9 assemble into a higher molecular weight oligomer, and that this is mediated by the DTX3L N-terminal region (1-200). Lastly, we show that ADP-ribosylation of ubiquitin at Gly76 is reversible in vitro by several Macrodomain-type hydrolases. Our study provides a framework to understand how DTX3L-PARP9 mediates ADP-ribosylation and ubiquitination through both intra- and inter-subunit interactions.

A FRET-based high-throughput screening platform for the discovery of chemical probes targeting the scaffolding functions of human tankyrases.

Tankyrases catalyse poly-ADP-ribosylation of their binding partners and the modification serves as a signal for the subsequent proteasomal degradation of these proteins. Tankyrases thereby regulate the turnover of many proteins involved in multiple and diverse cellular processes, such as mitotic spindle formation, telomere homeostasis and Wnt/ß-catenin signalling. In recent years, tankyrases have become attractive targets for the development of inhibitors as potential therapeutics against cancer and fibrosis. Further, it has become clear that tankyrases are not only enzymes, but also act as scaffolding proteins forming large cellular signalling complexes. While many potent and selective tankyrase inhibitors of the poly-ADP-ribosylation function exist, the inhibition of tankyrase scaffolding functions remains scarcely explored. In this work we present a robust, simple and cost-effective high-throughput screening platform based on FRET for the discovery of small molecule probes targeting the protein-protein interactions of tankyrases. Validatory screening with the platform led to the identification of two compounds with modest binding affinity to the tankyrase 2 ARC4 domain, demonstrating the applicability of this approach. The platform will facilitate identification of small molecules binding to tankyrase ARC or SAM domains and help to advance a structure-guided development of improved chemical probes targeting tankyrase oligomerization and substrate protein interactions.