OGG1 Inhibition Reduces Acinar Cell Injury in a Mouse Model of Acute Pancreatitis

Acute pancreatitis (AP) is a potentially life-threatening gastrointestinal disease with a complex pathology including oxidative stress. Oxidative stress triggers oxidative DNA lesions such as formation of 7,8-dihydro-8-oxo-2′-oxoguanine (8-oxoG) and also causes DNA strand breaks. DNA breaks can activate the nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP1) which contributes to AP pathology. 8-oxoG is recognized by 8-oxoG glycosylase 1 (OGG1) resulting in the removal of 8-oxoG from DNA as an initial step of base excision repair. Since OGG1 also possesses a DNA nicking activity, OGG1 activation may also trigger PARP1 activation. In the present study we investigated the role played by OGG1 in AP. We found that the OGG1 inhibitor compound TH5487 reduced edema formation, inflammatory cell migration and necrosis in a cerulein-induced AP model in mice. Moreover, TH5487 caused 8-oxoG accumulation and reduced tissue poly(ADP-ribose) levels. Consistent with the indirect PARP inhibitory effect, TH5487 shifted necrotic cell death (LDH release and Sytox green uptake) towards apoptosis (caspase activity) in isolated pancreatic acinar cells. In the in vivo AP model, TH5487 treatment suppressed the expression of various cytokine and chemokine mRNAs such as those of TNF, IL-1β, IL1ra, IL6, IL16, IL23, CSF, CCL2, CCL4, CCL12, IL10 and TREM as measured with a cytokine array and verified by RT-qPCR. As a potential mechanism underlying the transcriptional inhibitory effect of the OGG1 inhibitor we showed that while 8-oxoG accumulation in the DNA facilitates NF-κB binding to its consensus sequence, when OGG1 is inhibited, target site occupancy of NF-κB is impaired. In summary, OGG1 inhibition provides protection from tissue injury in AP and these effects are likely due to interference with the PARP1 and NF-κB activation pathways

The PARP Enzyme Family and the Hallmarks of Cancer Part 1. Cell Intrinsic Hallmarks

The 17-member poly (ADP-ribose) polymerase enzyme family, also known as the ADP-ribosyl transferase diphtheria toxin-like (ARTD) enzyme family, contains DNA damage-responsive and nonresponsive members. Only PARP1, 2, 5a, and 5b are capable of modifying their targets with poly ADP-ribose (PAR) polymers; the other PARP family members function as mono-ADP-ribosyl transferases. In the last decade, PARP1 has taken center stage in oncology treatments. New PARP inhibitors (PARPi) have been introduced for the targeted treatment of breast cancer 1 or 2 (BRCA1/2)-deficient ovarian and breast cancers, and this novel therapy represents the prototype of the synthetic lethality paradigm. Much less attention has been paid to other PARPs and their potential roles in cancer biology. In this review, we summarize the roles played by all PARP enzyme family members in six intrinsic hallmarks of cancer: uncontrolled proliferation, evasion of growth suppressors, cell death resistance, genome instability, reprogrammed energy metabolism, and escape from replicative senescence. In a companion paper, we will discuss the roles of PARP enzymes in cancer hallmarks related to cancer-host interactions, including angiogenesis, invasion and metastasis, evasion of the anticancer immune response, and tumor-promoting inflammation. While PARP1 is clearly involved in all ten cancer hallmarks, an increasing body of evidence supports the role of other PARPs in modifying these cancer hallmarks (e.g., PARP5a and 5b in replicative immortality and PARP2 in cancer metabolism). We also highlight controversies, open questions, and discuss prospects of recent developments related to the wide range of roles played by PARPs in cancer biology. Some of the summarized findings may explain resistance to PARPi therapy or highlight novel biological roles of PARPs that can be therapeutically exploited in novel anticancer treatment paradigms

OGG1 Inhibition Reduces Acinar Cell Injury in a Mouse Model of Acute Pancreatitis

Acute pancreatitis (AP) is a potentially life-threatening gastrointestinal disease with a complex pathology including oxidative stress. Oxidative stress triggers oxidative DNA lesions such as formation of 7,8-dihydro-8-oxo-2′-oxoguanine (8-oxoG) and also causes DNA strand breaks. DNA breaks can activate the nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP1) which contributes to AP pathology. 8-oxoG is recognized by 8-oxoG glycosylase 1 (OGG1) resulting in the removal of 8-oxoG from DNA as an initial step of base excision repair. Since OGG1 also possesses a DNA nicking activity, OGG1 activation may also trigger PARP1 activation. In the present study we investigated the role played by OGG1 in AP. We found that the OGG1 inhibitor compound TH5487 reduced edema formation, inflammatory cell migration and necrosis in a cerulein-induced AP model in mice. Moreover, TH5487 caused 8-oxoG accumulation and reduced tissue poly(ADP-ribose) levels. Consistent with the indirect PARP inhibitory effect, TH5487 shifted necrotic cell death (LDH release and Sytox green uptake) towards apoptosis (caspase activity) in isolated pancreatic acinar cells. In the in vivo AP model, TH5487 treatment suppressed the expression of various cytokine and chemokine mRNAs such as those of TNF, IL-1β, IL1ra, IL6, IL16, IL23, CSF, CCL2, CCL4, CCL12, IL10 and TREM as measured with a cytokine array and verified by RT-qPCR. As a potential mechanism underlying the transcriptional inhibitory effect of the OGG1 inhibitor we showed that while 8-oxoG accumulation in the DNA facilitates NF-κB binding to its consensus sequence, when OGG1 is inhibited, target site occupancy of NF-κB is impaired. In summary, OGG1 inhibition provides protection from tissue injury in AP and these effects are likely due to interference with the PARP1 and NF-κB activation pathways

Genomic variants reveal differential evolutionary constraints on human transglutaminases and point towards unrecognized significance of transglutaminase 2.

Transglutaminases (TGMs) catalyze Ca2+-dependent transamidation of proteins with specified roles in blood clotting (F13a) and in cornification (TGM1, TGM3). The ubiquitous TGM2 has well described enzymatic and non-enzymatic functions but in-spite of numerous studies its physiological function in humans has not been defined. We compared data on non-synonymous single nucleotide variations (nsSNVs) and loss-of-function variants on TGM1-7 and F13a from the Exome aggregation consortium dataset, and used computational and biochemical analysis to reveal the roles of damaging nsSNVs of TGM2. TGM2 and F13a display rarer damaging nsSNV sites than other TGMs and sequence of TGM2, F13a and TGM1 are evolutionary constrained. TGM2 nsSNVs are predicted to destabilize protein structure, influence Ca2+ and GTP regulation, and non-enzymatic interactions, but none coincide with conserved functional sites. We have experimentally characterized six TGM2 allelic variants detected so far in homozygous form, out of which only one, p.Arg222Gln, has decreased activities. Published exome sequencing data from various populations have not uncovered individuals with homozygous loss-of-function variants for TGM2, TGM3 and TGM7. Thus it can be concluded that human transglutaminases differ in harboring damaging variants and TGM2 is under purifying selection suggesting that it may have so far not revealed physiological functions

Summary of population frequencies of nsSNVs alleles of transglutaminases in the ExAC database covering 60,706 individuals.

<p>Summary of population frequencies of nsSNVs alleles of transglutaminases in the ExAC database covering 60,706 individuals.</p

Damaging nsSNVs located in ID regions embedding SLiMs in TGM2.

<p>Damaging nsSNVs located in ID regions embedding SLiMs in TGM2.</p

Analyses of non-synonymous variants in the transglutaminase family.

<p><b>(A) Amino acid residues polymorphic to non-synonymous variants.</b> The percentage of polymorphic residues was obtained as a ratio of the total number of amino acid residues polymorphic to nsSNVs and the sequence length. The percentage of polymorphic residues is shown. <b>(B</b>) <b>Proportion of damaging and benign non-synonymous variants by the PolyPhen score.</b> Ratio of damaging nsSNVs: F13a (42.8%), TGM1 (60.8%), TGM2 (43.7%), TGM3 (52.2%), TGM4 (51%), TGM5 (55.3%), TGM6 (52.5%), and TGM7 (49.5%). <b>(C) Location of damaging nsSNVs in amino acid sequence of TGM2 by PolyPhen/SIFT scores.</b> Lane 1: sequence of human TGM2, Lane 2: damaging nsSNVs in human TGM2. Functional regions of human TGM2: Intrinsically disordered regions (dark red) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172189#pone.0172189.ref004" target="_blank">4</a>], amino acid clusters in light blue [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172189#pone.0172189.ref030" target="_blank">30</a>], fibronectin binding sites (FN) (green) K30, R116, and H134 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172189#pone.0172189.ref031" target="_blank">31</a>], GDP binding residues (orange) S171, K173, R476, R478, V479, R580, Y583 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172189#pone.0172189.ref032" target="_blank">32</a>], catalytic residues (pink); non-canonical Ca²⁺-binding sites: S4: 149–159, S1: 228–236, S3A: 305–311, S3B: 326–333, S2A: 395–401, S5: 432–440, and S2B: 445–455 (underlined and bolded) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172189#pone.0172189.ref033" target="_blank">33</a>]. Domains of human TGM2 are presented vertically: β sandwich (1–139), catalytic core (147–460), β-barrel 1 (472–583), and β-barrel 2 (584–687).</p

The 12 human TGM2 homozygotes related to 6 nsSNVs.

<p>The 12 human TGM2 homozygotes related to 6 nsSNVs.</p

Allele frequencies of three most frequent non-synonymous variants in transglutaminase family genes.

<p>Values were calculated based on the ExAC database. For each family, nsSNVs with top three allele frequency (%) values are shown. <b>F13a</b>: p.Pro565Leu (21%), p.Glu652Gln (20.8%), p.Val35Leu (20.6%); <b>TGM1</b>: p.Val518Met (1.04%), p.Glu520Gly (0.56%), p.Ser42Tyr (0.40%); <b>TGM2</b>: see in text; <b>TGM3</b>: p.Gly654Arg (28.7%), p.Thr13Lys (19%), p.Ser249Asn (11.2%); <b>TGM4</b>: p.Glu313Lys (49%), p.Val409Ile (44.7%), p.Arg372Cys (42.8%); <b>TGM5</b>: p.Ala352Gly (14.8%), p.Val504Met (1.4%), p.Gln521Arg (1.25%); <b>TGM6</b>: p.Met58Val (9.2%), p.Arg448Trp (2.80%), p.Ala141Glu (0.9%); <b>TGM7</b>: p.Pro564Leu (3.4%), p.Val103Leu (1.07%), p.Val515Leu (0.82%).</p

Structural interpretation of the effect of the p.Arg222Gln variation on enzyme activity.

<p>We analyzed the interactions of R222 in a homology model of TGM2 containing three bound calcium ions (pink spheres), which supposedly corresponds to the active form (cyan: N-terminal, grey: catalytic, green and red: first and second beta barrel domains respectively). The squared area in the left panel is magnified on the right. R222 is located in the middle of the solvent accessible surface of the α-helix leading up to calcium binding site S1 (226–233). R222 is at the core of an H-bond network (light blue dashed lines) that serves to bundle neighboring structural elements of TGM2 together. Upon binding of calcium to site 1, H-bonds between E232, N229 and backbone atoms of Y369, and H-bonds of R222 to S365, E366, G372, and D389 cooperatively tether the flexible loop, P359-G372. The changing conformation of this loop leads to reorganization of another non-covalent interaction network near calcium binding site 2, including a directly calcium binding residue, N306, for metal binding, and to honing of the charge relay duad, E305-E363 that has recently identified importance for catalysis [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172189#pone.0172189.ref026" target="_blank">26</a>]. The Q222 variant (wheat) fails to establish the critical H-bonds with S365, E366, and E389, thus the calcium binding of both sites are impaired and the charge relay system is also negatively affected. The same loop, most probably, also contributes residues to the amine substrate binding surface and controls access to the active site (C277/H335/D358), likely explaining that the Q222 enzyme has conserved transamidase activity for a small molecular amine, but compromised cross-linking activity towards a protein amine donor and lost isopeptidase activity for a protein-peptide conjugate.</p