Abstract
PolyADP-ribosylation is a post-translational modification that plays key roles in cellular physiological functions and DNA damage responses. PolyADP-ribosylation is finely and dynamically regulated by various enzymes and factors involved in the synthesis and degradation of poly(ADP-ribose) (PAR). To better understand the function of polyADP-ribosylation, it is necessary to quantify and monitor the change of the in vivo level of PAR, the product of polyADP-ribosylation, which is rapidly turning over and kept in quite low level in cells or in organs. Recent developments of potent inhibitors of polyADP-ribosylation is expected to kill BRCA1/2-mutated breast cancer cells and ovarian cancer cells (synthetic lethality). To know the efficacy of these inhibitors in vivo, it is necessary to develop highly sensitive and reproducible methods to know PAR levels within cells or organs. However there have been several difficulties in measuring the physiologically low level of PAR without artefacts. Our experiments recently clarified that the method of sample preparation is very important in addition to the sensitivity and specificity. From reviewing the literature, including ours, we would like to emphasize the importance of the procedures of sample preparation for the assay, in addition to the sensitivity by comparing the reported PAR levels in vivo.
Highlights
PAR contained much higher molecular weight PAR with branches). Using this polyclonal antibody they showed that there was an immunoreactive principle in the extract of calf thymus nuclei and its reactivity was lost by prior incubation with PAR glycohydrolase (PARG) or snake venom phosphodiesterase, but not with DNase I, pancreatic RNase, nuclease P1, pronase E or 0.5 N NaOH treatment for 18 h at 37 ◦ C
Since various biological functions have been proposed as described in the first part of this review, if we know more about quantitative view such as that on PAR level, we will be much closer to inventing effective methods to control or prevent diseases
Had performed the cited experiments, which motivated to write this review; M.M., K.K., M.Ts. and M.Ta. collected the references and analyzed the data of sample preparation of the cited references on the in vivo level of PAR;
Summary
PolyADP-ribosylation is a post-translational modification that adds a long polymer of poly(ADP-ribose) (PAR) chain to the acceptor proteins [1,2]. 2 (ART2), are responsible for synthesis of PAR attached to acceptor proteins using βNAD+ as the substrate [4,5,6,7]. PAR glycohydrolase (PARG) and ADP-ribosyl hydrolase 3 (ARH3). Can degrade the PAR polymer from acceptor proteins [8,9,10]. ADP-ribosyl protein lyase, macrodomain-containing proteins, terminal ADP-ribose protein glycohydrolase 1 (TARG 1) and ARH3 can remove the proximal ADP-ribose residue from the acceptor protein [11,12,13,14] (Figure 1). This article is intended to emphasize the importance of measuring the actual amount of PAR found in vivo and to discuss the methods and the resulting PAR levels in vivo
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