Mechanistic insights into the three steps of poly(ADP-ribosylation) reversal.

Poly(ADP-ribosyl)ation (PAR) is a versatile and complex posttranslational modification composed of repeating units of ADP-ribose arranged into linear or branched polymers. This scaffold is linked to the regulation of many of cellular processes including the DNA damage response, alteration of chromatin structure and Wnt signalling. Despite decades of research, the principles and mechanisms underlying all steps of PAR removal remain actively studied. In this work, we synthesise well-defined PAR branch point molecules and demonstrate that PARG, but not ARH3, can resolve this distinct PAR architecture. Structural analysis of ARH3 in complex with dimeric ADP-ribose as well as an ADP-ribosylated peptide reveal the molecular basis for the hydrolysis of linear and terminal ADP-ribose linkages. We find that ARH3-dependent hydrolysis requires both rearrangement of a catalytic glutamate and induction of an unusual, square-pyramidal magnesium coordination geometry.

Unrestrained poly-ADP-ribosylation provides insights into chromatin regulation and human disease.

ARH3/ADPRHL2 and PARG are the primary enzymes reversing ADP-ribosylation in vertebrates, yet their functions in vivo remain unclear. ARH3 is the only hydrolase able to remove serine-linked mono(ADP-ribose) (MAR) but is much less efficient than PARG against poly(ADP-ribose) (PAR) chains in vitro. Here, by using ARH3-deficient cells, we demonstrate that endogenous MARylation persists on chromatin throughout the cell cycle, including mitosis, and is surprisingly well tolerated. Conversely, persistent PARylation is highly toxic and has distinct physiological effects, in particular on active transcription histone marks such as H3K9ac and H3K27ac. Furthermore, we reveal a synthetic lethal interaction between ARH3 and PARG and identify loss of ARH3 as a mechanism of PARP inhibitor resistance, both of which can be exploited in cancer therapy. Finally, we extend our findings to neurodegeneration, suggesting that patients with inherited ARH3 deficiency suffer from stress-induced pathogenic increase in PARylation that can be mitigated by PARP inhibition.

Real-time monitoring of PARP1-dependent PARylation by ATR-FTIR spectroscopy.

Poly-ADP-ribosylation (PARylation) is a fully reversible post-translational modification with key roles in cellular physiology. Due to the multi-domain structure of poly(ADP-ribose) polymerase-1 (PARP1) and the highly dynamic nature of the PARylation reaction, studies on the biochemical mechanism and structural dynamics remain challenging. Here, we report label-free, time-resolved monitoring of PARP1-dependent PARylation using ATR-FTIR spectroscopy. This includes PARP1 activation by binding to DNA strand break models, NAD+ substrate binding, PAR formation, and dissociation of automodified PARP1 from DNA. Analyses of PARP1 activation at different DNA models demonstrate a strong positive correlation of PARylation and PARP1 dissociation, with the strongest effects observed for DNA nicks and 3' phosphorylated ends. Moreover, by examining dynamic structural changes of PARP1, we reveal changes in the secondary structure of PARP1 induced by NAD+ and PARP inhibitor binding. In summary, this approach enables holistic and dynamic insights into PARP1-dependent PARylation with molecular and temporal resolution.

Tankyrase inhibition preserves osteoarthritic cartilage by coordinating cartilage matrix anabolism via effects on SOX9 PARylation.

Osteoarthritis (OA) is a prevalent degenerative disease, which involves progressive and irreversible destruction of cartilage matrix. Despite efforts to reconstruct cartilage matrix in osteoarthritic joints, it has been a difficult task as adult cartilage exhibits marginal repair capacity. Here we report the identification of tankyrase as a regulator of the cartilage anabolism axis based on systems-level factor analysis of mouse reference populations. Tankyrase inhibition drives the expression of a cartilage-signature matrisome and elicits a transcriptomic pattern that is inversely correlated with OA progression. Furthermore, tankyrase inhibitors ameliorate surgically induced OA in mice, and stem cell transplantation coupled with tankyrase knockdown results in superior regeneration of cartilage lesions. Mechanistically, the pro-regenerative features of tankyrase inhibition are mainly triggered by uncoupling SOX9 from a poly(ADP-ribosyl)ation (PARylation)-dependent protein degradation pathway. Our findings provide insights into the development of future OA therapies aimed at reconstruction of articular cartilage.

The Role of PARPs in Inflammation-and Metabolic-Related Diseases: Molecular Mechanisms and Beyond.

Poly(ADP-ribosyl)ation (PARylation) is an essential post-translational modification catalyzed by poly(ADP-ribose) polymerase (PARP) enzymes. Poly(ADP-ribose) polymerase 1 (PARP1) is a well-characterized member of the PARP family. PARP1 plays a crucial role in multiple biological processes and PARP1 activation contributes to the development of various inflammatory and malignant disorders, including lung inflammatory disorders, cardiovascular disease, ovarian cancer, breast cancer, and diabetes. In this review, we will focus on the role and molecular mechanisms of PARPs enzymes in inflammation- and metabolic-related diseases. Specifically, we discuss the molecular mechanisms and signaling pathways that PARP1 is associated with in the regulation of pathogenesis. Recently, increasing evidence suggests that PARP inhibition is a promising strategy for intervention of some diseases. Thus, our in-depth understanding of the mechanism of how PARPs are activated and how their signaling downstream effecters can provide more potential therapeutic targets for the treatment of the related diseases in the future is crucial.

Poly(ADP-ribosyl)ation and DNA repair synthesis in the extracts of naked mole rat, mouse, and human cells.

DNA repair capacity in cells of naked mole rat (Hgl), a species known for its longevity and resistance to cancer, is still poorly characterized. Here, using the whole-cell extracts (WCEs) of Hgl, mouse and human cells, we studied the interrelation between DNA synthesis on the substrates of base excision repair and the activity of poly(ADP-ribose) polymerases (PARPs) responsible for the transfer of the ADP-ribose moieties onto different targets. The level of PAR synthesis was more than ten-fold higher in human WCE as compared to rodent WCEs, while the efficiency of DNA synthesis was comparable. Under conditions of PAR synthesis, the efficiency of DNA synthesis was only slightly enhanced in all extracts and in mouse WCEs unusual products of the primer elongation were detected. The results obtained with WCEs, recombinant proteins and recently found ability of PARPs to attach the ADP-ribose moieties to DNA allowed us to attribute these products to primer mono(ADP-ribosyl)ation (MARylation) at the 5'-terminal phosphate by PARP3 during the DNA synthesis. PARP1/PARP2 can then transfer the ADP-ribose moieties onto initial ADP-ribose. Our results suggest that MARylation/PARylation of DNA in the extracts depends on the ratios between PARPs and can be controlled by DNA-binding proteins.

Poly(ADP-Ribose)-Dependent Chromatin Remodeling in DNA Repair.

The tightly packed and dynamic structure of chromatin can undergo major reorganization in response to endogenous or exogenous stimuli, such as the regulation of transcription or the cell cycle, or following DNA damage. A fast and local chromatin decondensation is observed upon DNA damage induced by laser micro-irradiation. This decondensation is under the control of poly(ADP-ribosyl)ation (PARylation) by PARP1, one of the first proteins recruited at the DNA damage sites. This chapter provides a step-by-step guide to perform and analyze chromatin decondensation upon DNA damage induction. The protocol is based on fluorescence microscopy of live cells expressing a core histone tagged with a photoactivatable fluorophore. Laser micro-irradiation is used to simultaneously induce DNA damage and activate the fluorescence signal within the irradiated area. This photo-perturbation experiment can be easily implemented on any confocal laser-scanning microscope equipped with a photoperturbation module. The experimental framework can also be used to follow chromatin relaxation in parallel with the recruitment kinetics of a protein of interest at DNA lesions in cells co-expressing the tagged histones and a second protein of interest fused to a different fluorescent tag.

Inputs and outputs of poly(ADP-ribosyl)ation: Relevance to oxidative stress.

Oxidative stress can cause DNA breaks which induce activation of the DNA nick sensor enzyme poly(ADP-ribose) polymerase-1 (PARP-1), part of the 17 member PARP enzyme family. PARP-1 modifies target proteins by attaching to them several NAD-derived ADP-ribose units forming poly(ADP-ribose) (PAR) polymers. PARylation controls many cellular processes while intense PARylation may also lead to cell death by various mechanisms. Here we summarize the modes of activation, inhibitors and modulators of PARP-1 and review the cellular functions regulated by the enzyme.