Biology subjects

Bejan, D. S.

Publications and source records attributed to Bejan, D. S..

3 recordsLinked to original sources

bioRxiv2025

A family of E3 ligases extend K11 polyubiquitin on sites of MARUbylation

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 MARUbe-binding domain (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 MARUbe-Targeted Ligases (MUTLs).

biochemistry↗

bioRxiv2024

Discovery of ester-linked ubiquitylation of PARP10 mono-ADP-ribosylation in cells: a dual post-translational modification on Glu/Asp side chains

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 mono-ADP-ribosyl ubiquitylation 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.

cell biology↗

bioRxiv2024

PARP14 is pro- and anti-viral host factor that promotes IFN production and affects the replication of multiple viruses

PARP14 is a 203 kDa multi-domain protein that is primarily known as an ADP-ribosyltransferase, and is involved in a variety of cellular functions including DNA damage, microglial activation, inflammation, and cancer progression. In addition, PARP14 is upregulated by interferon (IFN), indicating a role in the antiviral response. Furthermore, PARP14 has evolved under positive selection, again indicating that it is involved in host-pathogen conflict. We found that PARP14 is required for increased IFN-I production in response to coronavirus infection lacking ADP-ribosylhydrolase (ARH) activity and poly(I:C), however, whether it has direct antiviral function remains unclear. Here we demonstrate that the catalytic activity of PARP14 enhances IFN-{beta} and IFN-{lambda} responses and restricts ARH-deficient murine hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication. To determine if PARP14s antiviral functions extended beyond CoVs, we tested the ability of herpes simplex virus 1 (HSV-1), a DNA virus, vesicular stomatitis virus (VSV), a negative-sense RNA virus, and lymphocytic choriomeningitis virus (LCMV), an ambisense RNA virus, to infect A549 PARP14 knockout (KO) cells. While LCMV infection was unaffected, HSV-1 replication was increased in PARP14 KO cells and VSV replication was decreased. These results indicate that PARP14 restricts HSV-1 replication but enhances the replication of VSV. A PARP14 active site inhibitor had no impact on HSV-1 or VSV replication, indicating that its effect on these viruses was independent of its catalytic activity. These data demonstrate that PARP14 promotes IFN production and has both proviral and antiviral functions targeting multiple viruses. IMPORTANCEThe antiviral response is largely regulated by post-translation modifications (PTM), including ADP-ribosylation. PARP14 is an ADP-ribosyltransferase that is upregulated by interferon and is under positive selection, indicating that it is involved in host-pathogen conflict. However, no anti-viral function has been described for PARP14. Here, we found that PARP14 represses both coronavirus and HSV-1 replication, demonstrating that PARP14 has antiviral functions. Surprisingly, we also found that PARP14 also has pro-viral functions, as it was critical for the efficient replication of VSV. These data indicate that PARP14 has both proviral and antiviral functions. Defining the mechanisms used by PARP14 to both repress and promote virus replication will provide new insights into how PARPs regulate virus infection.

microbiology↗