PKD-dependent PARP12-catalyzed mono-ADP-ribosylation of Golgin-97 is required for E-cadherin transport from Golgi to plasma membrane

Adenosine diphosphate (ADP)-ribosylation is a posttranslational modification involved in key regulatory events catalyzed by ADP-ribosyltransferases (ARTs). Substrate identification and localization of the mono-ADP-ribosyltransferase PARP12 at the trans-Golgi network (TGN) hinted at the involvement of ARTs in intracellular traffic. We find that Golgin-97, a TGN protein required for the formation and transport of a specific class of basolateral cargoes (e.g., E-cadherin and vesicular stomatitis virus G protein [VSVG]), is a PARP12 substrate. PARP12 targets an acidic cluster in the Golgin-97 coiled-coil domain essential for function. Its mutation or PARP12 depletion, delays E-cadherin and VSVG export and leads to a defect in carrier fission, hence in transport, with consequent accumulation of cargoes in a trans-Golgi/Rab11–positive intermediate compartment. In contrast, PARP12 does not control the Golgin-245–dependent traffic of cargoes such as tumor necrosis factor alpha (TNFα). Thus, the transport of different basolateral proteins to the plasma membrane is differentially regulated by Golgin-97 mono-ADP-ribosylation by PARP12. This identifies a selective regulatory mechanism acting on the transport of Golgin-97– vs. Golgin-245–dependent cargoes. Of note, PARP12 enzymatic activity, and consequently Golgin-97 mono-ADP-ribosylation, depends on the activation of protein kinase D (PKD) at the TGN during traffic. PARP12 is directly phosphorylated by PKD, and this is essential to stimulate PARP12 catalytic activity. PARP12 is therefore a component of the PKD-driven regulatory cascade that selectively controls a major branch of the basolateral transport pathway. We propose that through this mechanism, PARP12 contributes to the maintenance of E-cadherin–mediated cell polarity and cell–cell junctions.

Mono-ADP-ribosylation sites of human CD73 inhibit its adenosine-generating enzymatic activity

CD73-derived adenosine plays a major role in damage-induced tissue responses by inhibiting inflammation. Damage-associated stimuli, such as hypoxia and mechanical stress, induce the cellular release of ATP and NAD+ and upregulate the expression of the nucleotide-degrading purinergic ectoenzyme cascade, including adenosine-generating CD73. Extracellular NAD+ also serves as substrate for mono-ADP-ribosylation of cell surface proteins, which in human cells is mediated by ecto-ADP-ribosyltransferase 1 (ARTC1). Here we explored, whether human CD73 enzymatic activity is regulated by mono-ADP-ribosylation, using recombinant human CD73 in the presence of ARTC1 with etheno-labelled NAD+ as substrate. Multi-colour immunoblotting with an anti-etheno-adenosine antibody showed ARTC1-mediated transfer of ADP-ribose together with the etheno label to CD73. HPLC analysis of the enzymatic activity of in vitro-ribosylated CD73 revealed strong inhibition of adenosine generation in comparison to non-ribosylated CD73. Mass spectrometry of in vitro-ribosylated CD73 identified six ribosylation sites. 3D model analysis indicated that three of them (R328, R354, R545) can interfere with CD73 enzymatic activity. Our study identifies human CD73 as target for ARTC1-mediated mono-ADP-ribosylation, which can profoundly modulate its adenosine-generating activity. Thus, in settings with enhanced release of NAD+ as substrate for ARTC1, assessment of CD73 protein expression in human tissues may not be predictive of adenosine formation resulting in anti-inflammatory activity.

Serine ADP-ribosylation marks nucleosomes for ALC1-dependent chromatin remodeling

Serine ADP-ribosylation (ADPr) is a DNA damage-induced post-translational modification catalyzed by the PARP1/2:HPF1 complex. As the list of PARP1/2:HPF1 substrates continues to expand, there is a need for technologies to prepare mono- and poly-ADP-ribosylated proteins for biochemical interrogation. Here we investigate the unique peptide ADPr activities catalyzed by PARP1 in the absence and presence of HPF1. We then exploit these activities to develop a method that facilitates installation of ADP-ribose polymers onto peptides with precise control over chain length and modification site. Importantly, the enzymatically mono- and poly-ADP-ribosylated peptides are fully compatible with protein ligation technologies. This chemoenzymatic protein synthesis strategy was employed to assemble a series of full-length, ADP-ribosylated histones and show that ADPr at H2BS6 or H3S10 converts nucleosomes into robust substrates for the chromatin remodeler ALC1. We found ALC1 preferentially remodels 'activated' substrates within heterogeneous mononucleosome populations and asymmetrically ADP-ribosylated dinucleosome substrates, and that nucleosome serine ADPr is sufficient to stimulate ALC1 activity in nuclear extracts. Our study identifies a biochemical function for nucleosome serine ADPr and describes a new, highly modular approach to explore the impact that site-specific serine mono- and poly-ADPr have on protein function.

The Making and Breaking of Serine-ADP-Ribosylation in the DNA Damage Response

ADP-ribosylation is a widespread posttranslational modification that is of particular therapeutic relevance due to its involvement in DNA repair. In response to DNA damage, PARP1 and 2 are the main enzymes that catalyze ADP-ribosylation at damage sites. Recently, serine was identified as the primary amino acid acceptor of the ADP-ribosyl moiety following DNA damage and appears to act as seed for chain elongation in this context. Serine-ADP-ribosylation strictly depends on HPF1, an auxiliary factor of PARP1/2, which facilitates this modification by completing the PARP1/2 active site. The signal is terminated by initial poly(ADP-ribose) chain degradation, primarily carried out by PARG, while another enzyme, (ADP-ribosyl)hydrolase 3 (ARH3), specifically cleaves the terminal seryl-ADP-ribosyl bond, thus completing the chain degradation initiated by PARG. This review summarizes recent findings in the field of serine-ADP-ribosylation, its mechanisms, possible functions and potential for therapeutic targeting through HPF1 and ARH3 inhibition.

Poly ADP-ribosylation of SET8 leads to aberrant H4K20me1 domains in mammalian cells

In mammalian cells, SET8 mediated Histone H4 Lys 20 monomethylation (H4K20me1) has been implicated in regulating mitotic condensation, DNA replication, DNA damage response, and gene expression. Here we show SET8, the only known enzyme for H4K20me1 is post-translationally poly ADP-ribosylated by PARP1 on lysine residues. PARP1 interacts with SET8 in a cell cycle-dependent manner. Poly ADP-ribosylation on SET8 renders it catalytically compromised and it undergoes degradation via ubiquitylation pathway. Knockdown of PARP1 shifted the relative dynamic equilibrium of H4K20me2 to H4k20me3 in cells. Overexpression or knockdown of PARP1 led to aberrant H4K20me1 domains genome-wide, impacting Wnt signaling pathways genes and transcription factor binding site enrichment. Therefore, SET8 mediated chromatin remodeling and gene expression in mammalian cells are influenced by poly ADP-ribosylation by PARP1.

Sirtuin inhibition is synthetic lethal with BRCA1 or BRCA2 deficiency

PARP enzymes utilise NAD+ as a co-substrate for their enzymatic activity. Inhibition of PARP1 is synthetic lethal with defects in either BRCA1 or BRCA2. In order to assess whether other genes implicated in NAD+ metabolism were synthetic lethal with BRCA1 or BRCA2 gene defects, we carried out a genetic screen, which identified a synthetic lethality between BRCA1 and genetic inhibition of either of two sirtuin (SIRT) enzymes, SIRT1 or SIRT6. This synthetic lethal interaction was replicated using small-molecule SIRT inhibitors and was associated with replication stress and increased cellular PARylation, in contrast to the decreased PARylation associated with BRCA-gene/PARP inhibitor synthetic lethality. SIRT/BRCA1 synthetic lethality was reversed by genetic ablation of either PARP1 or the histone PARylation factor-coding gene HPF1, implicating PARP1/HPF1-mediated serine ADP-ribosylation as part of the mechanistic basis of this synthetic lethal effect. These observations suggest that PARP1/HPF1-mediated serine ADP-ribosylation, when driven by SIRT inhibition, can inadvertently inhibit the growth of BRCA-gene mutant cells.

Interplay between ADP-ribosyltransferases and essential cell signaling pathways controls cellular responses

Signaling cascades provide integrative and interactive frameworks that allow the cell to respond to signals from its environment and/or from within the cell itself. The dynamic regulation of mammalian cell signaling pathways is often modulated by cascades of protein post-translational modifications (PTMs). ADP-ribosylation is a PTM that is catalyzed by ADP-ribosyltransferases and manifests as mono- (MARylation) or poly- (PARylation) ADP-ribosylation depending on the addition of one or multiple ADP-ribose units to protein substrates. ADP-ribosylation has recently emerged as an important cell regulator that impacts a plethora of cellular processes, including many intracellular signaling events. Here, we provide an overview of the interplay between the intracellular diphtheria toxin-like ADP-ribosyltransferase (ARTD) family members and five selected signaling pathways (including NF-κB, JAK/STAT, Wnt-β-catenin, MAPK, PI3K/AKT), which are frequently described to control or to be controlled by ADP-ribosyltransferases and how these interactions impact the cellular responses.