Specific Acceptor Proteins Research Articles

Poly(ADP-ribose) polymerase-1 hyperactivation and impairment of mitochondrial respiratory chain complex I function in reperfused mouse hearts

Poly(ADP-ribose) polymerase-1 (PARP-1), the most abundant member of the PARP family, is a nuclear enzyme that catalyzes ADP-ribose transfer from NAD+ to specific acceptor proteins in response to DNA damage. Excessive PARP-1 activation is an important cause of infarction and contractile dysfunction in heart tissue during interruptions of blood flow. The mechanisms by which PARP-1 inhibition and disruption dramatically improve metabolic recovery and reduce oxidative stress during cardiac reperfusion have not been fully explored. We developed a mouse heart experimental protocol to test the hypothesis that mitochondrial respiratory complex I is a downstream mediator of beneficial effects of PARP-1 inhibition or disruption. Pharmacological inhibition of PARP-1 activity produced no deterioration of hemodynamic function in C57BL/6 mouse hearts. Hearts from PARP-1 knockout mice also exhibited normal baseline contractility. Prolonged ischemia-reperfusion produced a selective defect in complex I function distal to the NADH dehydrogenase component. PARP-1 inhibition and PARP-1 gene disruption conferred equivalent protection against mitochondrial complex I injury and were strongly associated with improvement in myocardial energetics, contractility, and tissue viability. Interestingly, ischemic preconditioning abolished cardioprotection stimulated by PARP-1 gene disruption. Treatment with the antioxidant N-(2-mercaptopropionyl)-glycine or xanthine oxidase inhibitor allopurinol restored the function of preconditioned PARP-1 knockout hearts. This investigation establishes a strong association between PARP-1 hyperactivity and mitochondrial complex I dysfunction in cardiac myocytes. Our findings advance understanding of metabolic regulation in myocardium and identify potential therapeutic targets for prevention and treatment of ischemic heart disease.

Poly(ADP-ribose) glycohydrolase mediates oxidative and excitotoxic neuronal death.

Excessive activation of poly(ADP-ribose) polymerase 1 (PARP1) leads to NAD(+) depletion and cell death during ischemia and other conditions that generate extensive DNA damage. When activated by DNA strand breaks, PARP1 uses NAD(+) as substrate to form ADP-ribose polymers on specific acceptor proteins. These polymers are in turn rapidly degraded by poly(ADP-ribose) glycohydrolase (PARG), a ubiquitously expressed exo- and endoglycohydrolase. In this study, we examined the role of PARG in the PARP1-mediated cell death pathway. Mouse neuron and astrocyte cultures were exposed to hydrogen peroxide, N-methyl-d-aspartate (NMDA), or the DNA alkylating agent, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Cell death in each condition was markedly reduced by the PARP1 inhibitor benzamide and equally reduced by the PARG inhibitors gallotannin and nobotanin B. The PARP1 inhibitor benzamide and the PARG inhibitor gallotannin both prevented the NAD(+) depletion that otherwise results from PARP1 activation by MNNG or H(2)O(2). However, these agents had opposite effects on protein poly(ADP-ribosyl)ation. Immunostaining for poly(ADP-ribose) on Western blots and neuron cultures showed benzamide to decrease and gallotannin to increase poly(ADP-ribose) accumulation during MNNG exposure. These results suggest that PARG inhibitors do not inhibit PARP1 directly, but instead prevent PARP1-mediated cell death by slowing the turnover of poly(ADP-ribose) and thus slowing NAD(+) consumption. PARG appears to be a necessary component of the PARP-mediated cell death pathway, and PARG inhibitors may have promise as neuroprotective agents.

Nuclear acceptor sites for steroid hormone receptors: Comparisons of steroids and antisteroids

The respective chromatin binding sites (acceptor sites) for both the avian oviduct progesterone receptor (PR) and the rabbit uterine estrogen receptor (ER) reported by two separate laboratories are compared. Support for a saturable, high affinity binding to the chromatin acceptor sites by both receptors is described. Nonradiolabelled PR or ER compete with their homologous radiolabelled receptors for binding. However, there is no competition between the heterologous ER and PR for the nuclear binding sites. In both receptor systems, evidence for a receptor dependent, receptor specific binding, which mimics the binding measured in vivo, is reported. With both the PR and ER, evidence for extensive masking in chromatin of many of the acceptor sites is described. The PR and ER acceptor sites appear to be composed of specific acceptor proteins bound to DNA. The dissociation of these proteins from DNA causes a loss of specific binding. Reannealing of these same chromatin protein fractions, but not other protein fractions, back to the DNA reinstates the specific PR binding. Antibodies against the PR acceptors proteins are described. These antibodies block PR but not ER binding to the chromatin acceptor sites, supporting the steroid receptor specific acceptor sites. In the rabbit uterine system, the ER acceptor proteins dissociate from chromatin in three distinct fractions according to their affinity for the DNA. Each of these fractions contains acceptor activity in that each can be reannealed to the DNA to reconstitute specific ER binding sites, i.e. those which are saturable, high affinity, and receptor dependent. Interestingly, the antiestrogen receptor complexes do not bind to one fraction of acceptor proteins which does bind the native estrogen receptor complex. This ER acceptor protein fraction contains two regions of acceptor activity in the molecular mass range of 50,000 and 12,000 daltons. The latter species is in the size range of the PR acceptor proteins. This difference in binding to chromatin acceptor sites by the estrogen receptor, complexed with an estrogen or antiestrogen, may explain the differential regulation of gene expression and the differential biological response to antiestrogens compared to the native estrogens.

The interaction between the presynaptic phospholipase neurotoxins beta-bungarotoxin and crotoxin and mixed detergent-phosphatidylcholine micelles. A comparison with non-neurotoxic snake venom phospholipases A2.

Certain phospholipase A2 enzymes (E.C.3.1.1.4) selectively inhibit neurotransmitter release from cholinergic nerve terminals. Both specific acceptor proteins and the physical state of nerve terminal phospholipids have been implicated in studies of the mechanism of phospholipase neurotoxin action. Here we have examined the effects of charge on a micellar phospholipid substrate by comparing the enzyme activity and binding of two neurotoxic phospholipases (beta-bungarotoxin and crotoxin) with other non-neurotoxic phospholipases. This has been achieved by altering either the phospholipid or the ionic charge of the detergent in the mixed phospholipid micelle. The neurotoxic phospholipases were only active on negatively charged micelles, whereas the non-neurotoxic enzymes were equally active in hydrolyzing neutral micelles. This distinction was also reflected in binding studies; the non-neurotoxic phospholipases bound to both types of substrate, whereas beta-bungarotoxin and crotoxin selectively bound to negatively charged micellar structures. These experiments suggest that, in addition to the existence of any specific acceptor proteins, neurotoxin binding is also governed by the charge on the lipid phase of the nerve terminal membrane.

Effects of hydrocortisone and aspirin on protein synthesis and post-translational protein modification in cultured cells

Arginyl-tRNA transferase is suggested to function as a post-translational modifier of proteins through the addition of arginine to the NH 2-terminus of specific acceptor proteins. Both hydrocortisone and aspirin produced an age-dependent stimulation of protein synthesis in normal human fibroblasts (IMR90), while producing an inhibition of protein synthesis in SV40 virus-transformed IMR90 cells. The effect of aspirin was confined primarily to the cytoplasmic compartment, whereas hydrocortisone produced its effect at both cytoplasmic and nuclear levels. Neither hydrocortisone nor aspirin had a direct effect on arginyl-tRNA transferase activity in vitro; however, hydrocortisone resulted in a marked increase in the availability of chromosomal proteins subject to modification by arginyl-tRNA transferase. This stimulatory effect was attenuated by increasing culture age. The modified chromosomal proteins were found to be dissociated from native chromatin, suggesting that arginylation either triggered their release or prevented reassociation with chromatin thereafter. Hydrocortisone produced a moderate decrease in the availability of chromosomal proteins for arginylation in SV40 virus-transformed cells, and this effect was not modulated by aging phenomena.