Daniel S. Bejan

Ubiquitin (Ub) cooperates with other post-translational modifications to provide a tiered opportunity for protein regulation. Deltex E3 ligases were previously implicated in ubiquitylation of ADP-ribose (ADPr)-containing macromolecules in vitro, generating a noncanonical mono-ADPr-Ub ester (MARUbe). We previously identified mono-ADPr ubiquitylation (MARUbylation) on PARP7 in cells, which was extended with K11-linked polyUb, suggesting an intricately regulated, multilayered post-translational modification. Here, we show that the Deltex DTX2 ubiquitylates ADPr modifications on PARP7 in cells, which depends on PARP7 catalytic activity. We further identify RNF114 as the E3 ligase responsible for K11-linked polyUb extension on sites of PARP7 MARUbylation. Using a chemoenzymatic approach, we developed a fluorescent Ub-ADPr probe and find that RNF114 explicitly recognizes MARUbylated species. We used AlphaFold3 to examine the mechanisms of Ub-ADPr recognition and K11-linked polyUb extension by RNF114. We identify a tandem Di19-UIM module in RNF114 as a MARUbe-binding domain (M-UBD), thus providing a reader function that interfaces with K11-specific writer activity. Finally, we describe a small family of M-UBD-containing E3 ligases that demonstrate preference for Ub-ADPr, which we call MARUbe-Targeted Ligases (M-UTLs).

Ubiquitin (Ub) cooperation with other post-translational modifications provides a tiered opportunity for protein regulation. Small modifications to Ub such as phosphorylation, acetylation, or ADP-ribosylation have varying impacts on signaling. The Deltex family of E3 ligases was previously implicated in the ubiquitylation of ADP-ribose (ADPr) and ADPr-containing macromolecules. Our previous work found ester-linked mono-ADPr ubiquitylation (MARUbylation) on PARP7 and PARP10 in cells and that this mark is extended with K11 polyUb. We previously screened for E3 ligases that interact with PARP7 through three different approaches and identified six candidates, including the Deltex family member DTX2. One of these hits, RNF114, interacts with various other PARPs, leading us to hypothesize that RNF114 binds to sites of MARUbylation and extends K11 polyUb. Here, we show that DTX2 generates the initial MARUbe on PARP7 in cells, which depends on PARP7 catalytic activity. The MARUbe on PARP7 is extended with K11 polyUb by RNF114. To investigate the mechanism of RNF114 reader/writer function, we developed a click chemistry-inspired chemoenzymatic approach to create a novel fluorescent Ub-ADPr probe for studying its interaction with RNF114. Strikingly, we found that RNF114 has a weak affinity for ADPr and Ub separately but explicitly recognizes the linkage between Ub and ADPr present in MARUbylated species. We used AlphaFold3 modeling to examine the mechanisms of Ub-ADPr recognition and K11-linked polyUb extension by RNF114. We identified a tandem Di19-UIM module in RNF114 as a M AR U be- b inding d omain (MUBD), thus providing a reader function that interfaces with K11-specific writer activity. Finally, we described a small family of MUBD-containing E3 ligases that demonstrate preference for Ub-ADPr, which we call M ARUbe- T argeted L igases (MUTLs).

The prevailing view on post-translational modifications (PTMs) is that a single amino acid is modified with a single PTM at any given time. However, recent work has demonstrated crosstalk between different PTMs, some occurring on the same residue. Such interplay is seen with ADP-ribosylation and ubiquitylation. For example, DELTEX E3 ligases were reported to ubiquitylate a hydroxyl group on free NAD ⁺ and ADP-ribose in vitro, generating a noncanonical ubiquitin ester-linked species. In this report, we show, for the first time, that this dual PTM occurs in cells on mono-ADP-ribosylated (MARylated) PARP10 on Glu/Asp sites to form a MAR ubiquitin ester. We call this process mono-ADP-ribosyl ubiquitylation or MARUbylation. Using chemical and enzymatic treatments, including a newly characterized bacterial deubiquitinase with esterase-specific activity, we discovered that multiple PARPs are MARUbylated and extended with K11-linked polyubiquitin chains when exogenously expressed. Finally, we show that in response to type I interferon stimulation, MARUbylation can occur endogenously on PARP targets. Thus, MARUbylation represents a new dual PTM that broadens our understanding of the function of PARP-mediated ADP-ribosylation in cells.

The prevailing view on post-translational modifications (PTMs) is that amino acid side chains in proteins are modified with a single PTM at any given time. However, a growing body of work has demonstrated crosstalk between different PTMs, some occurring on the same residue. Such interplay is seen with ADP-ribosylation and ubiquitylation, where specialized E3 ligases ubiquitylate targets for proteasomal degradation in an ADP-ribosylation-dependent manner. More recently, the DELTEX family of E3 ligases was reported to catalyze ubiquitylation of the 3’- hydroxy group of the adenine-proximal ribose of free NAD ⁺ and ADP-ribose in vitro , generating a non-canonical ubiquitin ester-linked species. In this report, we show, for the first time, that this dual PTM occurs in cells on mono-ADP-ribosylated (MARylated) PARP10 on Glu/Asp sites to form a MAR ubiquitin ester (MARUbe). We term this process m ono- A DP-ribosyl ub iquit ylation or MARUbylation. Using chemical and enzymatic treatments, including a newly characterized bacterial deubiquitinase with esterase-specific activity, we discovered that PARP10 MARUbylation is extended with K11-linked polyubiquitin chains. Finally, mechanistic studies using proteasomal and ubiquitin-activating enzyme inhibitors demonstrated that PARP10 MARUbylation leads to its proteasomal degradation, providing a functional role for this new PTM in regulating protein turnover.

PARP16—the sole ER-resident PARP family member—is gaining attention as a potential therapeutic target for cancer treatment. Nevertheless, the precise function of the catalytic activity of PARP16 is poorly understood. This is primarily due to the lack of inhibitors that are selective for PARP16 over other PARP family members. Herein, we describe a structure-guided strategy for generating a selective PARP16 inhibitor by incorporating two selectivity determinants into a phthalazinone pan-PARP inhibitor scaffold: (i) an acrylamide-based inhibitor (DB008) designed to covalently react with a non-conserved cysteine (Cys169, human numbering) in the NAD⁺ binding pocket of PARP16 and (ii) a dual-purpose ethynyl group designed to bind in a unique hydrophobic cavity adjacent to the NAD⁺ binding pocket as well as serve as a click handle. DB008 exhibits good selectivity for PARP16 versus other PARP family members. Copper-catalyzed azide–alkyne cycloaddition (CuAAC) confirmed that covalent labeling of PARP16 by DB008 in cells is dependent on Cys169. DB008 exhibits excellent proteome-wide selectivity at concentrations required to achieve saturable labeling of endogenous PARP16. In-cell competition labeling experiments using DB008 provided a facile strategy for evaluating putative PARP16 inhibitors. Lastly, we found that PARP16 is sequestered into a detergent-insoluble fraction under prolonged amino acid starvation, and surprisingly, treatment with PARP16 inhibitors prevented this effect. These results suggest that the catalytic activity of PARP16 regulates its solubility in response to nutrient stress.