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AI26 inhibits the ADP-ribosylhydrolase ARH3 and suppresses DNA damage repair.

ABSTRACT: The ADP-ribosylhydrolase ARH3 plays a key role in DNA damage repair, digesting poly(ADP-ribose) and removing ADP-ribose from serine residues of the substrates. Specific inhibitors that selectively target ARH3 would be a useful tool to examine DNA damage repair, as well as a possible strategy for tumor suppression. However, efforts to date have not identified any suitable compounds. Here, we used in silico and biochemistry screening to search for ARH3 inhibitors. We discovered a small molecule compound named ARH3 inhibitor 26 (AI26) as, to our knowledge, the first ARH3 inhibitor. AI26 binds to the catalytic pocket of ARH3 and inhibits the enzymatic activity of ARH3 with an estimated IC₅₀ of ∼2.41 μm in vitro Moreover, hydrolysis of DNA damage-induced ADP-ribosylation was clearly inhibited when cells were pretreated with AI26, leading to defects in DNA damage repair. In addition, tumor cells with DNA damage repair defects were hypersensitive to AI26 treatment, as well as combinations of AI26 and other DNA-damaging agents such as camptothecin and doxorubicin. Collectively, these results reveal not only a chemical probe to study ARH3-mediated DNA damage repair but also a chemotherapeutic strategy for tumor suppression.

SUBMITTER: Liu X

PROVIDER: S-EPMC7535916 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Proteomic analyses identify ARH3 as a serine mono-ADP-ribosylhydrolase.

Project description:ADP-ribosylation is a posttranslational modification that exists in monomeric and polymeric forms. Whereas the writers (e.g. ARTD1/PARP1) and erasers (e.g. PARG, ARH3) of poly-ADP-ribosylation (PARylation) are relatively well described, the enzymes involved in mono-ADP-ribosylation (MARylation) have been less well investigated. While erasers for the MARylation of glutamate/aspartate and arginine have been identified, the respective enzymes with specificity for serine were missing. Here we report that, in vitro, ARH3 specifically binds and demodifies proteins and peptides that are MARylated. Molecular modeling and site-directed mutagenesis of ARH3 revealed that numerous residues are critical for both the mono- and the poly-ADP-ribosylhydrolase activity of ARH3. Notably, a mass spectrometric approach showed that ARH3-deficient mouse embryonic fibroblasts are characterized by a specific increase in serine-ADP-ribosylation in vivo under untreated conditions as well as following hydrogen peroxide stress. Together, our results establish ARH3 as a serine mono-ADP-ribosylhydrolase and as an important regulator of the basal and stress-induced ADP-ribosylome.

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The structure of human ADP-ribosylhydrolase 3 (ARH3) provides insights into the reversibility of protein ADP-ribosylation.

Project description:Posttranslational modifications are used by cells from all kingdoms of life to control enzymatic activity and to regulate protein function. For many cellular processes, including DNA repair, spindle function, and apoptosis, reversible mono- and polyADP-ribosylation constitutes a very important regulatory mechanism. Moreover, many pathogenic bacteria secrete toxins which ADP-ribosylate human proteins, causing diseases such as whooping cough, cholera, and diphtheria. Whereas the 3D structures of numerous ADP-ribosylating toxins and related mammalian enzymes have been elucidated, virtually nothing is known about the structure of protein de-ADP-ribosylating enzymes. Here, we report the 3Dstructure of human ADP-ribosylhydrolase 3 (hARH3). The molecular architecture of hARH3 constitutes the archetype of an all-alpha-helical protein fold and provides insights into the reversibility of protein ADP-ribosylation. Two magnesium ions flanked by highly conserved amino acids pinpoint the active-site crevice. Recombinant hARH3 binds free ADP-ribose with micromolar affinity and efficiently de-ADP-ribosylates poly- but not monoADP-ribosylated proteins. Docking experiments indicate a possible binding mode for ADP-ribose polymers and suggest a reaction mechanism. Our results underscore the importance of endogenous ADP-ribosylation cycles and provide a basis for structure-based design of ADP-ribosylhydrolase inhibitors.

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ADP-ribosylhydrolase 3 (ARH3), not poly(ADP-ribose) glycohydrolase (PARG) isoforms, is responsible for degradation of mitochondrial matrix-associated poly(ADP-ribose).

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Pathogenic ARH3 mutations result in ADP-ribose chromatin scars during DNA strand break repair.

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Proteomic analyses identify ARH3 as a serine mono-ADP-ribosylhydrolase

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2017-12-14 | PXD008083 | Pride

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