A Colorimetric Substrate for Poly(ADP‐Ribose) Polymerase‐1, VPARP, and Tankyrase‐1
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Color me yellow: Poly(ADP-ribose) polymerases (PARPs) play a major role in cellular survival and maintenance of energy stores after genotoxic insult. The colorimetric PARP substrate ADP-ribose-pNP can be used to monitor PARP activity. By monitoring the production of p-nitrophenolate, the kinetic parameters of PARP-1, tankyrase, and PARP-4 could be evaluated. ADP=adenosine diphosphate, pNP=p-nitrophenoxy.Keywords:
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Abstract We have characterized the biochemical association of two DNA damage‐dependent enzymes, poly(ADP‐ribose) polymerase‐1 (PARP‐1) [EC 2.4.2.30] and DNA polymerase β (pol β ) [2.7.7.7]. We reproducibly observed that pol β is an efficient covalent target for ADP‐ribose polymers under standard conditions of enzymatically catalyzed ADP‐ribosylation of β NAD + as a substrate. The efficiency of poly(ADP‐ribosyl)ation increased as a function of the pol β and β NAD + concentrations. To further characterize the molecular interactions between these two unique polymerases, we also subjected human recombinant PARP‐1 to peptide‐specific enzymatic degradation with either caspase‐3 or caspase‐7 in vitro. This proteolytic treatment, commonly referred to as ‘a hallmark of apoptosis’, generated the two physiologically relevant peptide fragments of PARP‐1, e.g. , a 24‐kDa amino‐terminus and an 89‐kDa carboxy‐terminal domain. Interestingly, co‐incubation of the two peptide fragments of PARP‐1 with full‐length pol β resulted in their domain‐specific molecular association as determined by co‐immunoprecipitation and reciprocal immunoblotting. Therefore, our data strongly suggest that, once PARP‐1 is proteolyzed by either caspase‐3 or caspase‐7 during cell death, the specific association of its apoptotic fragments with DNA repair enzymes, such as pol β , may serve a regulatory molecular role in the execution phase of apoptosis.
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Poly(ADP-ribose) polymerase (PARP) polyADP-ribosylates proteins involved in various physiological processes. Accumulated evidence suggests not only protein-conjugated poly(ADP-ribose) but also protein-free poly(ADP-ribose) function in various physiological processes. There are increasing occasions that require protein-free poly(ADP-ribose) to study the function and dynamics of poly(ADP-ribose) in cells. However, the availability of poly(ADP-ribose) is still limited because a chemical synthesis method has not been established. Here, we describe an improved method for the preparation of protein-free poly(ADP-ribose), synthesized enzymatically by using a recombinant PARP-1 expression system and purified with an anion-exchange column chromatography. This method will be useful for biochemical and biological investigation of poly(ADP-ribose) functions and dynamics.
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Declines in cellular nicotinamide adenine dinucleotide (NAD) contribute to metabolic dysfunction, increase susceptibility to disease, and occur as a result of pathogenic infection. The enzymatic cleavage of NAD + transfers ADP-ribose (ADPr) to substrate proteins generating mono-ADP-ribose (MAR), poly-ADP-ribose (PAR) or O-acetyl-ADP-ribose (OAADPr). These important post-translational modifications have roles in both immune response activation and the advancement of infection. In particular, emergent data show viral infection stimulates activation of poly (ADP-ribose) polymerase (PARP) mediated NAD + depletion and stimulates hydrolysis of existing ADP-ribosylation modifications. These studies are important for us to better understand the value of NAD + maintenance upon the biology of infection. This review focuses specifically upon the NAD + utilising enzymes, discusses existing knowledge surrounding their roles in infection, their NAD + depletion capability and their influence within pathogenic infection.
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Poly(ADP-ribose) polymerase-1 consumes NAD+ to catalyze poly(ADP-ribosyl)ation of target proteins, which modulates various biological functions. However, excessive poly(ADP-ribose) polymerase-1 activation mediates oxidative cell death. Our recent studies have indicated that NAD+ can enter into astrocytes to prevent poly(ADP-ribose) polymerase-1 cytotoxicity. In this study, we show that NADH can also enter into astrocytes, which can significantly decrease poly(ADP-ribose) polymerase-1-induced astrocyte death even when applied 3–4 h after poly(ADP-ribose) polymerase-1 activation. The protective effects can be produced by 10 μM NADH, which is significantly lower than that required for NAD+ to be protective. These results provide novel information suggesting that NADH can be used for decreasing poly(ADP-ribose) polymerase-1 toxicity, and extracellular NADH can enter into astrocytes to influence cellular functions.
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Poly( ADP-ribose) polymerase 1 (PARP-1) plays an essential role in DNA repair and maintenance of homeostasis and genome stability. Increased PARP-1 activity has been reported in various forms of cancer. Thus the absence of PARP-1 or the use of its inhibitors depresses DNA repair function, sensitizes cancer cells to DNA damage and enhances the therapeutic effect of radio-or chemotherapy. PARP-1 is potentially a therapeutic target of malignant tumors.
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Poly(ADP-ribose) polymerases; DNA repair enzymes; Neoplasms; Poly (ADP-ribose) polymerases inhibitors
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