Lilia Calabrese

Neurodegeneration in amyotrophic lateral sclerosis: the role of oxidative stress and altered homeostasis of metals

Amyotrophic lateral sclerosis is one of the most common neurodegenerative disorders, with an incidence of about 1/100,000. One of the typical features of this progressive, lethal disease, occurring both sporadically and as a familial disorder, is degeneration of cortical and spinal motor neurones. Present evidence indicates that loss of neurones in patients results from a complex interplay among oxidative injury, excitotoxic stimulation, dysfunction of critical proteins and genetic factors. This review focuses on existing evidence that oxidative stress is a major culprit in the pathogenesis of amyotrophic lateral sclerosis. An increase in reactive oxygen species and in products of oxidation has been observed both in post-mortem samples and in experimental models for ALS. This increase may be consequent to altered metabolism of copper and iron ions, that share the property to undergo redox cycling and generate reactive oxygen species. Metal-mediated oxidative stress would lead to several intracellular alterations and contribute to the induction of cell death pathways.

CCCTC-binding factor activates PARP-1 affecting DNA methylation machinery

Our previous data have shown that in L929 mouse fibroblasts the control of methylation pattern depends in part on poly(ADP-ribosyl)ation and that ADP-ribose polymers (PARs), both present on poly(ADP-ribosyl)ated PARP-1 and/or protein-free, have an inhibitory effect on Dnmt1 activity. Here we show that transient ectopic overexpression of CCCTC-binding factor (CTCF) induces PAR accumulation, PARP-1, and CTCF poly(ADP-ribosyl)ation in the same mouse fibroblasts. The persistence in time of a high PAR level affects the DNA methylation machinery; the DNA methyltransferase activity is inhibited with consequences for the methylation state of genome, which becomes diffusely hypomethylated affecting centromeric minor satellite and B1 DNA repeats. In vitro data show that CTCF is able to activate PARP-1 automodification even in the absence of nicked DNA. Our new finding that CTCF is able per se to activate PARP-1 automodification in vitro is of great interest as so far a burst of poly(ADP-ribosyl)ated PARP-1 has generally been found following introduction of DNA strand breaks. CTCF is unable to inhibit DNMT1 activity, whereas poly(ADP-ribosyl)ated PARP-1 plays this inhibitory role. These data suggest that CTCF is involved in the cross-talk between poly(ADP-ribosyl)ation and DNA methylation and underscore the importance of a rapid reversal of PARP activity, as DNA methylation pattern is responsible for an important epigenetic code.

Parp1 localizes within the Dnmt1 promoter and protects its unmethylated state by its enzymatic activity

Background Aberrant hypermethylation of CpG islands in housekeeping gene promoters and widespread genome hypomethylation are typical events occurring in cancer cells. The molecular mechanisms behind these cancer-related changes in DNA methylation patterns are not well understood. Two questions are particularly important: (i) how are CpG islands protected from methylation in normal cells, and how is this protection compromised in cancer cells, and (ii) how does the genome-wide demethylation in cancer cells occur. The latter question is especially intriguing since so far no DNA demethylase enzyme has been found. Methodology/Principal Findings Our data show that the absence of ADP-ribose polymers (PARs), caused by ectopic over-expression of poly(ADP-ribose) glycohydrolase (PARG) in L929 mouse fibroblast cells leads to aberrant methylation of the CpG island in the promoter of the Dnmt1 gene, which in turn shuts down its transcription. The transcriptional silencing of Dnmt1 may be responsible for the widespread passive hypomethylation of genomic DNA which we detect on the example of pericentromeric repeat sequences. Chromatin immunoprecipitation results show that in normal cells the Dnmt1 promoter is occupied by poly(ADP-ribosyl)ated Parp1, suggesting that PARylated Parp1 plays a role in protecting the promoter from methylation. Conclusions/Significance In conclusion, the genome methylation pattern following PARG over-expression mirrors the pattern characteristic of cancer cells, supporting our idea that the right balance between Parp/Parg activities maintains the DNA methylation patterns in normal cells. The finding that in normal cells Parp1 and ADP-ribose polymers localize on the Dnmt1 promoter raises the possibility that PARylated Parp1 marks those sequences in the genome that must remain unmethylated and protects them from methylation, thus playing a role in the epigenetic regulation of gene expression.

Erythrocuprein and singlet oxygen

McCord and Fridovich [ 1,2] have shown that erythrocuprein, the copper and zinc containing protein extracted from red blood cells, can enzymatically disproportionate superoxide radicals (Oh), an intermediate of the reduction of oxygen, the generation of which in some Ha 0s -producing biological oxidations has been demonstrated [3]. Khan has recently pointed out [4] that dismutation of Og gives rise to the production of the highly reactive singlet state of oxygen, which can be revealed by its own luminescence; indeed Arneson [5] observed that oxidation of xan’thine by xanthine oxidase produced luminescence, which was abolished by the presence of erythrocuprein. On the basis of these data, the main purpose of the following paper is to show that erythrocuprein has a quenching effect on luminescence phenomena which are reasonably ascribed to singlet oxygen produced even in the absence of superoxide intermediates. Thus the physiological role of erythrocuprein could be to catalyze the singlet-triplet conversion of the oxygen states: in other words to afford an enzyme-regulated dismutation liberating in solution as a product the inert ground state triplet oxygen, rather than simply accelerating the dismutation reaction.

Features of ceruloplasmin in the cerebrospinal fluid of Alzheimer’s disease patients

The level of the apo-form of the copper enzyme ceruloplasmin (CP) is an established peripheral marker in diseases associated with copper imbalance. In view of the proposal that disturbances of copper homeostasis may contribute to neurodegeneration associated with Alzheimer’s disease (AD), the present work investigates, by Western blot and non-reducing SDS-PAGE followed by activity staining, the features of CP protein, and the copper/CP relationship in cerebrospinal fluid (CSF) and serum of AD patients. Results show that only a fraction of total copper is associated with CP in the CSF, at variance with serum, both in affected and in healthy individuals. Furthermore, a conspicuous amount of apo-ceruloplasmin and a decrease of CP oxidase activity characterize the CSF of the affected individuals, and confirm that an impairment of copper metabolism occurs in their central nervous system. In the CSF of AD patients the decrease of active CP, associated with the increase in the pool of copper not sequestered by this protein, may play a role in the neurodegenerative process.

Cobalt erythrocuprein: Preparation and properties

Abstract Co(II) has been substituted for zinc in erythrocuprein. Cobalt erythrocuprein is fully active as a superoxide dismutase. The optical spectrum of the cobalt derivative shows three peaks at 532, 558 and 592 nm. The extinction coefficient at 592 nm is approximately 600 M −1 ·cm −1 . The intensity of the EPR signal of the copper is remarkably lower in the cobalt erythrocuprein than that expected on the basis of the chemically determined copper, indicating magnetic interaction between the two paramagnetic ions. This result suggests that the zinc and copper sites are very close to each other.

Ceruloplasmin impairs endothelium-dependent relaxation of rabbit aorta

This study evaluated the effects of ceruloplasmin, the copper-containing blue oxidase of vertebrate plasma, on the relaxation of rabbit aortic rings after endothelial release of nitric oxide (NO). Ceruloplasmin at physiological, i.e., micromolar, concentrations inhibited relaxation of rabbit aorta induced by endothelium-dependent agonists like acetylcholine or ADP, whereas it was ineffective toward vasodilation due to direct stimulation of smooth muscle cells by nitroglycerin. The effect was reversible and specific for native, fully metalated ceruloplasmin, since relaxation was not impaired by the heat-treated or metal-depleted derivatives. A trapping mechanism, involving a direct interaction of NO or other NO-containing species (like nitrosothiols and iron-dinitrosyls) with the copper sites and/or with the free thiol of ceruloplasmin, could be safely excluded on the basis of spectroscopic and chemical analyses of the protein exposed to authentic NO, nitrosothiols, or iron-dinitrosyls. The data presented in this paper constitute the first evidence of impairment of the endothelium-dependent vasodilatation by a plasma protein and may shed some light on the still uncertain physiological role of ceruloplasmin.

pH dependence of the nuclear magnetic relaxation rate of solvent water protons in solutions of bovine superoxide dismutase

Abstract The nuclear magnetic relaxation rate of solvent water protons has been studied in solutions of the copper enzyme bovine superoxide dismutase at pH values between pH 6 and 13. An increase of relaxivity is observed between pH 10 and 12.5, followed by a steep decrease above pH 12.5. The first effect is reversible, while the second one is not. Comparison with theory and supplementary measurements suggests that the relaxivity increase is due to a decrease of the copper-proton internuclear distance ν, which occurs at the active site of the enzyme in the corresponding pH region. The results are discussed in view of the parallel changes of the EPR spectrum and of the enzyme activity. A deprotonation of the copper-bound H2O to OH− appears to be a likely explanation of the observed facts.

On the lability and functional significance of the type 1 copper pool in ceruloplasmin.

Abstract The possibility that ceruloplasmin (CP) functions as a copper transferase has fueled a continuing interest in studies of the copper release process. The principal goal of the current investigation has been to identify the most labile copper centers in sheep protein. In fact, subjecting the enzyme to a slow flux of cyanide at pH 5.2 under nitrogen in the presence of ascorbate and a phenanthroline ligand produces partially demetalated forms of the protein. By standard chromatographic techniques it is possible to isolate protein with a Cu/CP ratio of ∼4 or ∼5 as opposed to the native protein which has Cu/CP=5.8. In contrast to other blue oxidases, analysis suggests that CP preferentially loses its type 1 coppers under these conditions. Thus, the spectroscopic signals from the type 1 centers exhibit a loss of intensity while the EPR signal of the type 2 copper becomes stronger. Furthermore, the Cu/CP≈4 and Cu/CP≈5 components retain about 50% of the activity of the native protein, consistent with an intact type 2/type 3 cluster. All three type 1 copper sites appear to suffer copper loss. Reconstitution with a copper(I) reagent restores the spectroscopic properties of the native protein and 90% of the original activity. The results suggest a possible functional significance for the presence of three type 1 coppers in CP. By employing a pool of redox-active but relatively labile type 1 copper centers, the enzyme can serve as a copper donor, if necessary, without completely sacrificing its oxidase activity.

Substitution of arginine for lysine 134 alters electrostatic parameters of the active site in shark Cu,Zn superoxide dismutase.

The complete amino acid sequence was determined for the Cu,Zn superoxide dismutase from the shark Prionace glauca. The active site region shows the substitution of an Arg for Lys at position 134, which is important for electrostatic facilitation of the diffusion of O− 2 to the catalytically active copper. This change may be related to observed alterations of electrostatic parameters of the enzyme (pK of the pH dependence of the enzyme activity, rate of inactivation by H2O2), although it preserves a high efficiency of dismutation at neutral pH.