CD38 - ADP-ribosyl cyclase/cyclic ADP-ribose …

ADP-ribosylation is the addition of one or more ADP-ribose moieties to a protein

CD38 - ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase …

Cyclic ADP-ribose (cADPR) is a Ca2+-mobilizing cyclic nucleotide derived from NAD+. Accumulating evidence indicates that it is an endogenous modulator of the Ca2+-induced Ca2+ release mechanism in cells. In this study, we show that ADP-ribosyl cyclase catalyzes the cyclization of not only NAD+ but also several of its analogs with various purine bases (guanine, hypoxanthine, or xanthine) substituting for adenine. Unlike cADPR, the resulting cyclic products are fluorescent. Comparisons with various model compounds indicate that only 7-methyl substituted purine nucleosides and nucleotides are fluorescent, and the pH-dependence of their UV spectra is most similar to that of the fluorescent cADPR analogs, indicating that the site of cyclization of these analogs is at the NT-position of the purine ring. This finding is novel since the site of cyclization is at the N1-position for cADPR as determined by X-ray crystallography. That a single enzyme can cyclize a variety of substrates at two different sites has important implications mechanistically, and a model is proposed to account for these novel catalytic properties. Among the analogs synthesized, cyclic GDP-ribose is highly resistant to hydrolysis, while cyclic IDP-ribose can be readily hydrolyzed by CD38, a bifunctional enzyme involved in the metabolism of cADPR. These unique properties of the analogs can be used to develop fluorimetric assays for monitoring separately the cyclization and hydrolytic reactions catalyzed by the metabolic enzymes of cADPR. The convenience of the method in measuring kinetic parameters, pH-dependence, and modulator activity of the metabolic enzymes of cADPR is illustrated.

The Zn3 Domain of Human Poly(ADP-ribose) …

Poly(ADP-ribose) synthesis and degradation in …

We imagined that our ADPR-peptide conjugates could be useful in the identification of novel ADPR-binding proteins from crude cellular lysates. The affinity of free ADPR for mH2A1.1 is modest, in the μM range. Thus, we were concerned that affinity-capture methods might not be fruitful. Indeed, peptide 2a was unable to pull-down low concentrations of mH2A1.1 doped into HeLa S3 nuclear lysates (, lane 6). One approach to overcome this problem would be to incorporate a photocrosslinker into the conjugate, thereby covalently linking the probe to the target. With this in mind, we synthesized ADPR-peptide conjugate 8a featuring benzophenone crosslinker 12 appended to the N-terminus of the H2B sequence (). We also synthesized peptide 7, which contains the crosslinker but lacks the ADPR moiety. Incubation of peptide 8a, but not 7, with HeLa S3 nuclear lysates doped with mH2A1.1 followed by UV irradiation, resulted in the generation of a robust crosslink to the macro domain (). This effect was observed at two macro domain concentrations, differing by 5-fold. At the higher concentration, peptide 8a was able to pull down both crosslinked mH2A1.1 and non-crosslinked mH2A1.1 (upper and lower bands, respectively, in the α-His blot of the precipitated material, ). Since the mH2A1.1 macro domain can form dimers, this data suggests that the crosslinked species may co-precipitate interacting proteins in their native state. In addition, PARP9, a known ADP-ribose binding protein, could be enriched from Farage nuclear lysates after crosslinking with peptide 8a, but not with 7 (). Based on these observations, incorporation of photocrosslinkers and ADPR into peptides could be useful for enriching, or probing for ADPR binding proteins.

Next we sought to demonstrate that the peptide ADPR conjugates could interact with a known ADPR binding protein. Several so-called macro domains have been shown to bind ADPR, and it is postulated that ADP-ribosylated proteins may also interact with these domains., Indeed, a recent report suggests that the macro domain containing histone, mH2A1.1, can bind poly-ADP-ribosylated PARP1. Since histones H2B and mH2A1.1 exist in the same nuclear environment (i.e. within nucleosome core particles of chromatin), we asked if mH2A1.1 could bind the synthetic ADPR conjugated peptides using a peptide pull-down assay. A splice-variant, mH2A1.2, which does not bind ADPR, was used as a negative control in this experiment. Peptides 1, 2a and 9a were assessed for their ability to interact with mH2A1.1 and mH2A1.2 (). ADPR-peptide conjugates 2a and 9a were able to pull-down mH2A1.1, but not mH2A1.2. Control peptide 1 did not interact with mH2A1.1, suggesting that the ADPR moiety is important for the interaction. Consistent with this, peptide 2a did not pull-down with a point-mutant of mH2A1.1 (G224E) () which abolishes ADPR binding. Interestingly, peptide 2a pulls down more mH2A1.1 than peptide 9a, suggesting that the H2B peptide sequence in 2a also contributes to the interaction. Additional studies will be needed to confirm this. The key result here is that the ADPR-peptide conjugates afforded by our strategy are able to interact with the macro domain. To our knowledge, this is the first demonstration that an ADPR binding domain can engage the modification when it is covalently linked to a peptide carrier.


Poly(ADP-Ribose) polymerase 1 (PARP-1) ..

ADP-ribosylation involves the transfer of ADP-ribose (ADPR) from β-NAD+ onto protein substrates. A family of enzymes termed poly(ADP-ribose) polymerases (PARPs), which generate protein-linked ADPR monomers (mono-ADP-ribosylation) or polymers (poly-ADP-ribosylation), catalyze this reaction. ADP-ribosylation occurs on several residues including glutamic acid where the attachment is through an ester linkage. The functional roles of ADP-ribosylation, in particular mono-ADP-ribosylation, are poorly understood. The study of ADP-ribosylation has proven difficult for many reasons. For example, the ester-linked ADPR generated by PARPs is unstable at basic pH (t1/2 ), and endogenous enzymes rapidly cleave poly-ADP-ribose polymers (e.g. poly(ADP-ribose) glycohydrolase; t1/2 0.6-6 min). There is also a lack of commercial antibodies specific for mono-ADP-ribose, mono-ADP-ribose protein-linkages, or poly-ADP-ribose branching. The ability to site-specifically attach ADPR to peptides/proteins would provide a useful tool for studying the biochemical effects of this modification. Such a technique, using a protected mono-ADP-ribosylated asparagine building block for peptide synthesis has recently been described. In this report, we demonstrate the use of aminooxy-functionalized amino acids to enable site-specific attachment of ADPR onto peptides. Using this technology, we show that an ADP-ribosylated version of the histone H2B tail can interact with the ADPR binding protein, macroH2A1.1 (mH2A1.1), and demonstrate that incorporation of photocrosslinkers into these peptides improves their ability to detect ADPR binding proteins.

Glutaminase and poly(ADP-ribose) polymerase …

ADP-ribosylation is an important post-translational modification involved in processes including cellular replication, DNA repair, and cell death. Despite these roles, the functions of ADP-ribosylation, in particular mono-ADP-ribosylation, remain poorly understood. The development of a technique to generate large amounts of site-specific, ADP-ribosylated peptides would provide a useful tool for deconvoluting the biochemical roles of ADP-ribosylation. Here we demonstrate that synthetic histone H2B tail peptides, incorporating aminooxy or N-methyl aminooxy functionalized amino acids, can be site-specifically conjugated to ADP-ribose. These peptides are recognized as substrates by the ADP-ribosylation biochemical machinery (PARP1), can interact with the ADP-ribose binding proteins macroH2A1.1 and PARP9, and demonstrate superior enzymatic and chemical stability when compared to ester-linked ADP-ribose. In addition, the incorporation of benzophenone photocrosslinkers into these peptides is demonstrated to provide a means to probe for, and enrich ADP-ribose binding proteins.