Oxidation-induced structural changes of ceruloplasmin foster NGR motif deamidation that promotes integrin binding and signaling.

Asparagine deamidation occurs spontaneously in proteins during aging; deamidation of Asn-Gly-Arg (NGR) sites can lead to the formation of isoAsp-Gly-Arg (isoDGR), a motif that can recognize the RGD-binding site of integrins. Ceruloplasmin (Cp), a ferroxidase present in the cerebrospinal fluid (CSF), contains two NGR sites in its sequence: one exposed on the protein surface ((568)NGR) and the other buried in the tertiary structure ((962)NGR). Considering that Cp can undergo oxidative modifications in the CSF of neurodegenerative diseases, we investigated the effect of oxidation on the deamidation of both NGR motifs and, consequently, on the acquisition of integrin binding properties. We observed that the exposed (568)NGR site can deamidate under conditions mimicking accelerated Asn aging. In contrast, the hidden (962)NGR site can deamidate exclusively when aging occurs under oxidative conditions, suggesting that oxidation-induced structural changes foster deamidation at this site. NGR deamidation in Cp was associated with gain of integrin-binding function, intracellular signaling, and cell pro-adhesive activity. Finally, Cp aging in the CSF from Alzheimer disease patients, but not in control CSF, causes Cp deamidation with gain of integrin-binding function, suggesting that this transition might also occur in pathological conditions. In conclusion, both Cp NGR sites can deamidate during aging under oxidative conditions, likely as a consequence of oxidative-induced structural changes, thereby promoting a gain of function in integrin binding, signaling, and cell adhesion.

Ceruloplasmin potentiates nitric oxide synthase activity and cytokine secretion in activated microglia.

BACKGROUND: Ceruloplasmin is a ferroxidase expressed in the central nervous system both as soluble form in the cerebrospinal fluid (CSF) and as membrane-bound GPI-anchored isoform on astrocytes, where it plays a role in iron homeostasis and antioxidant defense. It has been proposed that ceruloplasmin is also able to activate microglial cells with ensuing nitric oxide (NO) production, thereby contributing to neuroinflammatory conditions. In light of the possible role of ceruloplasmin in neurodegenerative diseases, we were prompted to investigate how this protein could contribute to microglial activation in either its native form, as well as in its oxidized form, recently found generated in the CSF of patients with Parkinson's and Alzheimer's diseases. METHODS: Primary rat microglial-enriched cultures were treated with either ceruloplasmin or oxidized-ceruloplasmin, alone or in combination with lipopolysaccharide (LPS). Production of NO and expression of inducible nitric oxide synthase (iNOS) were evaluated by Griess assay and Western blot analysis, respectively. The productions of the pro-inflammatory cytokine IL-6 and the chemokine MIP-1α were assessed by quantitative RT-PCR and ELISA. RESULTS: Regardless of its oxidative status, ceruloplasmin by itself was not able to activate primary rat microglia. However, ceruloplasmin reinforced the LPS-induced microglial activation, promoting an increase of NO production, as well as the induction of IL-6 and MIP-1α. Interestingly, the ceruloplasmin-mediated effects were observed in the absence of an additional induction of iNOS expression. The evaluation of iNOS activity in primary glial cultures and in vitro suggested that the increased NO production induced by the combined LPS and ceruloplasmin treatment is mediated by a potentiation of the enzymatic activity. CONCLUSIONS: Ceruloplasmin potentiates iNOS activity in microglial cells activated by a pro-inflammatory stimulus, without affecting iNOS expression levels. This action might be mediated by the activation of a yet unknown Cp receptor that triggers intracellular signaling that cross-talks with the response elicited by LPS or other pro-inflammatory stimuli. Therefore, ceruloplasmin might contribute to pathological conditions in the central nervous system by exacerbating neuroinflammation.

Amino acid deprivation triggers a novel GCN2-independent response leading to the transcriptional reactivation of non-native DNA sequences.

In a variety of species, reduced food intake, and in particular protein or amino acid (AA) restriction, extends lifespan and healthspan. However, the underlying epigenetic and/or transcriptional mechanisms are largely unknown, and dissection of specific pathways in cultured cells may contribute to filling this gap. We have previously shown that, in mammalian cells, deprivation of essential AAs (methionine/cysteine or tyrosine) leads to the transcriptional reactivation of integrated silenced transgenes, including plasmid and retroviral vectors and latent HIV-1 provirus, by a process involving epigenetic chromatic remodeling and histone acetylation. Here we show that the deprivation of methionine/cysteine also leads to the transcriptional upregulation of endogenous retroviruses, suggesting that essential AA starvation affects the expression not only of exogenous non-native DNA sequences, but also of endogenous anciently-integrated and silenced parasitic elements of the genome. Moreover, we show that the transgene reactivation response is highly conserved in different mammalian cell types, and it is reproducible with deprivation of most essential AAs. The General Control Non-derepressible 2 (GCN2) kinase and the downstream integrated stress response represent the best candidates mediating this process; however, by pharmacological approaches, RNA interference and genomic editing, we demonstrate that they are not implicated. Instead, the response requires MEK/ERK and/or JNK activity and is reproduced by ribosomal inhibitors, suggesting that it is triggered by a novel nutrient-sensing and signaling pathway, initiated by translational block at the ribosome, and independent of mTOR and GCN2. Overall, these findings point to a general transcriptional response to essential AA deprivation, which affects the expression of non-native genomic sequences, with relevant implications for the epigenetic/transcriptional effects of AA restriction in health and disease.

Ceruloplasmin functional changes in Parkinson's disease-cerebrospinal fluid.

BACKGROUND: Ceruloplasmin, a ferroxidase present in cerebrospinal fluid (CSF), plays a role in iron homeostasis protecting tissues from oxidative damage. Its reduced enzymatic activity was reported in Parkinson's disease (PD) contributing to the pathological iron accumulation. We previously showed that ceruloplasmin is modified by oxidation in vivo, and, in addition, in vitro by deamidation of specific NGR-motifs that foster the gain of integrin-binding function. Here we investigated whether the loss of ceruloplasmin ferroxidase activity in the CSF of PD patients was accompanied by NGR-motifs deamidation and gain of function. RESULTS: We have found that endogenous ceruloplasmin in the CSF of PD patients showed structural changes, deamidation of the (962)NGR-motif which is usually hidden within the ceruloplasmin structure, and the gain of integrin-binding function. These effects occur owing to the presence of abnormal levels of hydrogen peroxide we detected in the CSF of PD patients. Interestingly, the pathological CSF's environment of PD patients promoted the same modifications in the exogenously added ceruloplasmin, which in turn resulted in loss of ferroxidase-activity and acquisition of integrin-binding properties. CONCLUSIONS: We show that in pathological oxidative environment of PD-CSF the endogenous ceruloplasmin, in addition to loss-of-ferroxidase function, is modified as to gain integrin-binding function. These findings, beside the known role of ceruloplasmin in iron homeostasis, might have important pathogenic implications due to the potential triggering of signals mediated by the unusual integrin binding in cells of central nervous system. Furthermore, there are pharmacological implications because, based on data obtained in murine models, the administration of ceruloplasmin has been proposed as potential therapeutic treatment of PD, however, the observed CSF's pro-oxidant properties raise the possibility that in human the ceruloplasmin-based therapeutic approach might not be efficacious.