ADP-ribosylation is an important post-translational protein modification (PTM) that regulates diverse biological processes. ADP-ribosyltransferase diphtheria toxin-like 10 (ARTD10, also known as PARP10) mono-ADP-ribosylates acidic side chains and is one of eighteen ADP-ribosyltransferases that catalyze mono- or poly-ADP-ribosylation of target proteins. Currently, no enzyme is known that reverses ARTD10-catalyzed mono-ADP-ribosylation. Here we report that ARTD10-modified targets are substrates for the macrodomain proteins MacroD1, MacroD2 and C6orf130 from Homo sapiens as well as for the macrodomain protein Af1521 from archaebacteria. Structural modeling and mutagenesis of MacroD1 and MacroD2 revealed a common core structure with Asp102 and His106 of MacroD2 implicated in the hydrolytic reaction. Notably, MacroD2 reversed the ARTD10-catalyzed, mono-ADP-ribose-mediated inhibition of glycogen synthase kinase 3β (GSK3β) in vitro and in cells, thus underlining the physiological and regulatory importance of mono-ADP-ribosylhydrolase activity. Our results establish macrodomain-containing proteins as mono-ADP-ribosylhydrolases and define a class of enzymes that renders mono-ADP-ribosylation a reversible modification.

The DNA damage response (DDR) generates transient repair compartments to concentrate repair proteins and activate signaling factors. The physicochemical properties of these spatially confined compartments and their function remain poorly understood. Here, we establish, based on live cell microscopy and CRISPR/Cas9‐mediated endogenous protein tagging, that 53BP1‐marked repair compartments are dynamic, show droplet‐like behavior, and undergo frequent fusion and fission events. 53BP1 assembly, but not the upstream accumulation of γH2AX and MDC1, is highly sensitive to changes in osmotic pressure, temperature, salt concentration and to disruption of hydrophobic interactions. Phase separation of 53BP1 is substantiated by optoDroplet experiments, which further allowed dissection of the 53BP1 sequence elements that cooperate for light‐induced clustering. Moreover, we found the tumor suppressor protein p53 to be enriched within 53BP1 optoDroplets, and conditions that disrupt 53BP1 phase separation impair 53BP1‐dependent induction of p53 and diminish p53 target gene expression. We thus suggest that 53BP1 phase separation integrates localized DNA damage recognition and repair factor assembly with global p53‐dependent gene activation and cell fate decisions.

The mammalian poly(ADP-ribose) polymerase 1 (PARP-1)

Poly(ADP-ribose) polymerase-1 (PARP-1) and nuclear factor B (NF-B) have both been demonstrated to play a pathophysiological role in a number of inflammatory disorders. We recently presented evidence that PARP-1 can act as a promoter-specific coactivator of NF-B in vivo independent of its enzymatic activity. PARP-1 directly interacts with p300 and both subunits of NF-B (p65 and p50) and synergistically coactivates NF-B-dependent transcription. Here we show that PARP-1 is acetylated in vivo at specific lysine residues by p300/CREB-binding protein upon stimulation. Furthermore, acetylation of PARP-1 at these residues is required for the interaction of PARP-1 with p50 and synergistic coactivation of NF-B by p300 and the Mediator complex in response to inflammatory stimuli. PARP-1 physically interacts with the Mediator. Interestingly, PARP-1 interacts in vivo with histone deacetylases (HDACs) 1-3 but not with HDACs 4 -6 and might be deacetylated in vivo by HDACs 1-3. Thus, acetylation of PARP-1 by p300/CREB-binding protein plays an important regulatory role in NF-B-dependent gene activation by enhancing its functional interaction with p300 and the Mediator complex.Nuclear factor B (NF-B) is a widely expressed transcription factor of particular importance to the regulation of cells of the immune system (1). NF-B encompasses a family of inducible transcription factors including RelA/p65, RelB, c-Rel, p50, and p52 (1). These proteins share a conserved 300-amino acid region within their amino termini, designated Rel-homology domain (RHD). This domain is responsible for dimerization, nuclear translocation, DNA binding, and interaction with heterologous transcription factors (1). NF-B is composed of homo-or heterodimers with a range of DNA binding and activation potentials. The most abundant and best-studied form of NF-B in cells is a heterodimer consisting of the two subunits, p50 (NF-B1) and p65 (RelA). NF-B plays a key role in the regulation of many genes involved in mammalian immune and inflammatory responses, apoptosis, cell proliferation, and differentiation (1, 2). NF-B has additionally been associated with neurodegenerative processes and cancer (3, 4). In unstimulated cells, NF-B is sequestered in the cytoplasm as an inactive transcription factor complex by its physical association with one of several inhibitors of NF-B (IBs) 2 (5). Treatment of cells with extracellular stimuli including cytokines, bacterial lipopolysaccharides (LPS), phorbol esters, or potent oxidants leads to rapid phosphorylation of IB␣, which results in ubiquitination of IB␣ and subsequent degradation by the 26 S proteasome (4, 5). Dissociation of NF-B unmasks the nuclear localization sequences of p65 and p50 subunits, which leads to nuclear translocation and binding of NF-B to specific B consensus sequences in the chromatin and activation of specific subsets of genes (3).NF-B-dependent gene expression requires growing families of transcriptional coactivators (6, 7). The two key coactivators of NF-B, histone acetyltransferases p300 and its homo...

SUMMARY We previously demonstrated that the deletion of the poly(ADP-ribose)polymerase (Parp)-1 gene in mice enhances oxidative metabolism, thereby protecting against diet-induced obesity. However, the therapeutic use of PARP inhibitors to enhance mitochondrial function remains to be explored. Here, we show tight negative correlation between Parp-1 expression and energy expenditure in heterogeneous mouse populations, indicating that variations in PARP-1 activity have an impact on metabolic homeostasis. Notably, these genetic correlations can be translated into pharmacological applications. Long-term treatment with PARP inhibitors enhances fitness in mice by increasing the abundance of mitochondrial respiratory complexes and boosting mitochondrial respiratory capacity. Furthermore, PARP inhibitors reverse mitochondrial defects in primary myotubes of obese humans and attenuate genetic defects of mitochondrial metabolism in human fibroblasts and C. elegans. Overall, our work validates in worm, mouse and human models that PARP inhibition may be used to treat both genetic and acquired muscle dysfunction linked to defective mitochondrial function.