Caterina Grillo

DNA Damage and Repair: From Molecular Mechanisms to Health Implications

DNA is subjected to several modifications, resulting from endogenous and exogenous sources. The cell has developed a network of complementary DNA-repair mechanisms, and in the human genome, >130 genes have been found to be involved. Knowledge about the basic mechanisms for DNA repair has revealed an unexpected complexity, with overlapping specificity within the same pathway, as well as extensive functional interactions between proteins involved in repair pathways. Unrepaired or improperly repaired DNA lesions have serious potential consequences for the cell, leading to genomic instability and deregulation of cellular functions. A number of disorders or syndromes, including several cancer predispositions and accelerated aging, are linked to an inherited defect in one of the DNA-repair pathways. Genomic instability, a characteristic of most human malignancies, can also arise from acquired defects in DNA repair, and the specific pathway affected is predictive of types of mutations, tumor drug sensitivity, and treatment outcome. Although DNA repair has received little attention as a determinant of drug sensitivity, emerging knowledge of mutations and polymorphisms in key human DNA-repair genes may provide a rational basis for improved strategies for therapeutic interventions on a number of tumors and degenerative disorders.

ERp57/GRP58: A protein with multiple functions

The protein ERp57/GRP58 is a stress-responsive protein and a component of the protein disulfide isomerase family. Its functions in the endoplasmic reticulum are well known, concerning mainly the proper folding and quality control of glycoproteins, and participation in the assembly of the major histocompatibility complex class 1. However, ERp57 is present in many other subcellular locations, where it is involved in a variety of functions, primarily suggested by its participation in complexes with other proteins and even with DNA. While in some instances these roles need to be confirmed by further studies, a great number of observations support the participation of ERp57 in signal transduction from the cell surface, in regulatory processes taking place in the nucleus, and in multimeric protein complexes involved in DNA repair.

Endogenous reverse transcriptase as a mediator of ursolic acid’s anti-proliferative and differentiating effects in human cancer cell lines

Ursolic acid (UA) is a pentacyclic triterpenoid compound that is widely distributed in the plant kingdom and has a broad range of biological effects. Here, we examined the effects of UA on the proliferation and differentiation of human tumor cell lines from melanoma (A375), glioblastoma (U87) and thyroid anaplastic carcinoma (ARO), and on the proliferation of a non-transformed human fibroblast cell line (WI-38). The results show that UA inhibits tumor cell proliferation in a dose- and time-dependent manner. Consistent with this finding, UA treatment promotes differentiation of all of the analyzed tumor cell lines. Interestingly, we found that UA inhibits the endogenous reverse transcriptase (RT) activity in tumor cells, which has recently been shown to be involved in the control of proliferation and differentiation of neoplastic cells. Considering these findings, we suggest that the observed anti-proliferative and differentiating effects of UA may be related to this target.

The stress protein ERp57/GRP58 binds specific DNA sequences in HeLa cells

The protein ERp57/GRP58 is a member of the protein disulfide isomerase family and is also a glucose‐regulated protein, which, together with the other GRPs, is induced by a variety of cellular stress conditions. ERp57/GRP58 is mainly located in the endoplasmic reticulum (ER), but has also been found in the cytoplasm and in the nucleus, where it can bind DNA. In order to identify a possible correlation between the stress‐response and the nuclear location of ERp57/GRP58, its binding sites on DNA in HeLa cells have been searched by chromatin immunoprecipitation and cloning of the immunoprecipitated DNA fragments. Following sequencing of the cloned fragments, 10 DNA sequences have been securely identified as in vivo targets of ERp57/GRP58. Nine of them are present in the non‐coding regions of identified genes, and seven of these in introns. The features of some of these DNA sequences, that is, DNase hypersensitivity, proximity of MAR regions, and homology to the non‐coding regions of orthologue genes of mouse or rat, are compatible with a gene expression regulatory function. Considering the nature of the genes concerned, two of which code for DNA repair proteins, we would suggest that at least part of the mechanism of action of ERp57/GRP58 takes place through the regulation of these, and possibly other still unidentified, stress‐response genes. J. Cell. Physiol. 210: 343–351, 2007.

Effects of UVB-induced oxidative stress on protein expression and specific protein oxidation in normal human epithelial keratinocytes: A proteomic approach

BackgroundThe UVB component of solar ultraviolet irradiation is one of the major risk factors for the development of skin cancer in humans. UVB exposure elicits an increased generation of reactive oxygen species (ROS), which are responsible for oxidative damage to proteins, DNA, RNA and lipids. In order to examine the biological impact of UVB irradiation on skin cells, we used a parallel proteomics approach to analyze the protein expression profile and to identify oxidatively modified proteins in normal human epithelial keratinocytes.ResultsThe expression levels of fifteen proteins - involved in maintaining the cytoskeleton integrity, removal of damaged proteins and heat shock response - were differentially regulated in UVB-exposed cells, indicating that an appropriate response is developed in order to counteract/neutralize the toxic effects of UVB-raised ROS. On the other side, the redox proteomics approach revealed that seven proteins - involved in cellular adhesion, cell-cell interaction and protein folding - were selectively oxidized.ConclusionsDespite a wide and well orchestrated cellular response, a relevant oxidation of specific proteins concomitantly occurs in UVB-irradiated human epithelial Keratinocytes. These modified (i.e. likely dysfunctional) proteins might result in cell homeostasis impairment and therefore eventually promote cellular degeneration, senescence or carcinogenesis.

Role of ERp57 in the signaling and transcriptional activity of STAT3 in a melanoma cell line

Chromatin immunoprecipitation in M14 melanoma cells showed that the protein ERp57 (endoplasmic reticulum protein 57) binds to DNA in the proximity of STAT3 in a subset of STAT3-regulated genes. In the same cells, IL-6 induced a significant increase of the expression of one of these genes, i.e. CRP. Upon depletion of ERp57 by RNA interference, the phosphorylation of STAT3 on tyrosine 705 was decreased, and the IL-6-induced activation of CRP expression was completely suppressed. In vitro experiments showed that ERp57 is also required for the binding of STAT3 to its consensus sequence on DNA. Thus ERp57, previously shown to associate with STAT3 in the cytosol and in the nuclear STAT3-containing enhanceosome, is a necessary cofactor for the regulation of at least a subset of STAT3-dependent genes, probably intervening both at the site of STAT3 phosphorylation and at the nuclear level.

Altered Expression of the CB1 Cannabinoid Receptor in the Triple Transgenic Mouse Model of Alzheimer's Disease

The endocannabinoid system has gained much attention as a new potential pharmacotherapeutic target in various neurodegenerative diseases, including Alzheimers disease (AD). However, the association between CB1 alterations and the development of AD neuropathology is unclear and often contradictory. In this study, brain CB1 mRNA and CB1 protein levels were analyzed in 3 × Tg-AD mice and compared to wild-type littermates at 2, 6 and 12 months of age, using in-situ hybridization and immunohistochemistry, respectively. Semiquantitative analysis of CB1 expression focused on the prefrontal cortex (PFC), prelimbic cortex, dorsal hippocampus (DH), basolateral amygdala complex (BLA), and ventral hippocampus (VH), all areas with high CB1 densities that are strongly affected by neuropathology in 3 × Tg-AD mice. At 2 months of age, there was no change in CB1 mRNA and protein levels in 3 × Tg-AD mice compared to Non-Tg mice in all brain areas analyzed. However, at 6 and 12 months of age, CB1 mRNA levels were significantly higher in PFC, DH, and BLA, and lower in VH in 3 × Tg-AD mice compared to wild-type littermates. CB1 immunohistochemistry revealed that CB1 protein expression was unchanged in 3 × Tg-AD at 2 and 6 months of age, while a significant decrease in CB1 receptor immunoreactivity was detected in the BLA and DH of 12-month-old 3 × Tg-AD mice, with no sign of alteration in other brain areas. The altered CB1 levels appear, rather, to be age-and/or pathology-dependent, indicating an involvement of the endocannabinoid system in AD pathology and supporting the ECS as a potential novel therapeutic target for treatment of AD.

Nanomolar Melatonin Enhances nNOS Expression and Controls HaCaT-cells Bioenergetics

A novel role of melatonin was unveiled, using immortalized human keratinocyte cells (HaCaT) as a model system. Within a time window compatible with its circadian rhythm, melatonin at nanomolar concentration raised both the expression level of the neuronal nitric oxide synthase mRNA and the nitric oxide oxidation products, nitrite and nitrate. On the same time scale, a depression of the mitochondrial membrane potential was detected together with a decrease of the oxidative phosphorylation efficiency, compensated by glycolysis as testified by an increased production of lactate. The melatonin concentration, ∼ nmolar, inducing the bioenergetic effects and their time dependence, both suggest that the observed nitric oxide‐induced mitochondrial changes might play a role in the metabolic pathways characterizing the circadian melatonin chemistry.

Green tea catechins can bind and modify ERp57/PDIA3 activity

BACKGROUND Green tea is a rich source of polyphenols, mainly catechins (flavanols), which significantly contribute to the beneficial health effects of green tea in the prevention and treatment of various diseases. In this study the effects of four green tea catechins on protein ERp57, also known as protein disulfide isomerase isoform A3 (PDIA3), have been investigated in an in vitro model. METHODS The interaction of catechins with ERp57 was explored by fluorescence quenching and surface plasmon resonance techniques and their effect on ERp57 activities was investigated. RESULTS A higher affinity was observed for galloylated cathechins, which bind close to the thioredoxin-like redox-sensitive active sites of the protein, with a preference for the oxidized form. The effects of these catechins on ERp57 properties were also investigated and a moderate inhibition of the reductase activity of ERp57 was observed as well as a strong inhibition of ERp57 DNA binding activity. CONCLUSIONS Considering the high affinity of galloylated catechins for ERp57 and their capability to inhibit ERp57 binding to other macromolecular ligands, some effects of catechins interaction with this protein on eukaryotic cells may be expected. GENERAL SIGNIFICANCE This study provides information to better understand the molecular mechanisms underlying the biological activities of catechins and to design new polyphenol-based ERp57-specific inhibitors.

DNA-binding Activity of the ERp57 C-terminal Domain Is Related to a Redox-dependent Conformational Change

ERp57, a member of the protein-disulfide isomerase family, although mainly localized in the endoplasmic reticulum is here shown to have a nuclear distribution. We previously showed the DNA-binding properties of ERp57, its association with the internal nuclear matrix, and identified the C-terminal region, containing the a′ domain, as being directly involved in the DNA-binding activity. In this work, we demonstrate that its DNA-binding properties are strongly dependent on the redox state of the a′ domain active site. Site-directed mutagenesis experiments on the first cysteine residue of the -CGHC-thioredoxin-like active site lead to a mutant domain (C406S) lacking DNA-binding activity. Biochemical studies on the recombinant domain revealed a conformational change associated with the redox-dependent formation of a homodimer, having two disulfide bridges between the cysteine residues of two a′ domain active sites. The formation of intermolecular disulfide bridges rather than intramolecular oxidation of active site cysteines is important to generate species with DNA-binding properties. Thus, in the absence of any dedicated motif within the protein sequence, this structural rearrangement might be responsible for the DNA-binding properties of the C-terminal domain. Moreover, NADH-dependent thioredoxin reductase is active on intermolecular disulfides of the a′ domain, allowing the control of dimeric protein content as well as its DNA-binding activity. A similar behavior was also observed for whole ERp57.