Biomedical subjects

Chiara D'Ambrosio

Publications and source records attributed to Chiara D'Ambrosio.

At least 19 recordsLinked to original sources

PubMed2007

Oxidized transthyretin in amniotic fluid as an early marker of preeclampsia.

Preeclampsia is a pregnancy-specific hypertensive syndrome and a major cause of maternal and fetal morbidity and mortality. At the present time, no reliable screening tests to identify women at risk are available. We have compared the amniotic fluids (AF) proteomic maps of five preeclamptic patients with those of five controls. The analysis was carried out by two-dimensional electrophoresis followed by peptide mapping and tandem mass spectrometric analysis. Besides the implementation of the previously published AF proteomic maps, our results show that transthyretin (TTR), the protein responsible for transporting both the thyroid hormone tyroxine and the retinol binding protein, is present in the AF of both preeclamptic and control women as a mixture of dimeric and post-translationally modified monomeric forms. Although the nature of these forms is similar in both groups, the preeclamptic women showed a significant increase in the amount of monomeric proteins with respect to the control group. Since the TTR monomeric forms are the results of different oxidizing reactions, we hypothesize that the higher oxidative stress in preeclampsia is the major destabilizing factor of the TTR functional dimeric form in the preeclamptic women.

Amniocentesis↗

PubMed2006

Identification of the ligands of protein interaction domains through a functional approach.

The identification of protein-protein interaction networks has often given important information about the functions of specific proteins and on the cross-talk among metabolic and regulatory pathways. The availability of entire genome sequences has rendered feasible the systematic screening of collections of proteins, often of unknown function, aimed to find the cognate ligands. Once identified by genetic and/or biochemical approaches, the interaction between two proteins should be validated in the physiologic environment. Herein we describe an experimental strategy to screen collections of protein-protein interaction domains to find and validate candidate interactors. The approach is based on the assumption that the overexpression in cultured cells of protein-protein interaction domains, isolated from the context of the whole protein, could titrate the endogenous ligand and, in turn, exert a dominant negative effect. The identification of the ligand could provide us with a tool to check the relevance of the interaction because the contemporary overexpression of the isolated domain and of its ligand could rescue the dominant negative phenotype. We explored this approach by analyzing the possible dominant negative effects on the cell cycle progression of a collection of phosphotyrosine binding (PTB) domains of human proteins. Of 47 PTB domains, we found that the overexpression of 10 of them significantly interfered with the cell cycle progression of NIH3T3 cells. Four of them were used as baits to identify the cognate interactors. Among these proteins, CARM1, interacting with the PTB domain of RabGAP1, and EF1alpha, interacting with RGS12, were able to rescue the block of the cell cycle induced by the isolated PTB domain of the partner protein, thus confirming in vivo the relevance of the interaction. These results suggest that the described approach can be used for the systematic screening of the ligands of various protein-protein interaction domains also by using different biological assays.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Secretory proteins as potential semiochemical carriers in the horse.

Two soluble proteins were isolated as major secretory products of horse sweat and of the parotid gland and characterized for structural and functional properties. The first protein, lipocalin allergen EquC1, was characterized for its glycosylation sites and bound glycosidic moieties. Only one (Asn53) of the two putative glycosylation sites within the sequence was post-translationally modified; a different glycosylation pattern was determined with respect to data previously reported. When purified from horse sweat, this protein contained oleamide and other organic molecules as natural ligands. Ligand binding experiments indicated good protein selectivity toward volatile compounds having a straight chain structure of 9-11 carbon atoms, suggesting a role of this lipocalin in chemical communication. The second protein, here reported for the first time in the horse, belongs to the group of parotid secretory proteins, part of a large superfamily of binding proteins whose function in most cases is still unclear. This protein was sequenced and characterized for its post-translational modifications. Of the three cysteine residues present, two were involved in a disulfide bridge (Cys155-Cys198). A model, built up on the basis of similar proteins, indicated a general fold characterized by the presence of a long hydrophobic barrel. Binding experiments performed with a number of different organic compounds failed to identify ligands for this protein with a well-defined physiological role.

Amino Acid Sequence↗

PubMed2006

Analytical methodologies for the detection and structural characterization of phosphorylated proteins.

Phosphorylation of proteins is a frequent post-translational modification affecting a great number of fundamental cellular functions in living organisms. Because of its key role in many biological processes, much effort has been spent over the time on the development of analytical methodologies for characterizing phosphoproteins. In the past decade, mass spectrometry-based techniques have emerged as a viable alternative to more traditional methods of phosphorylation analysis, providing accurate information for a purified protein on the number of the occurring phosphate groups and their exact localization on the polypeptide sequence. This review summarizes the analytical methodologies currently available for the analysis of protein phosphorylation, emphasizing novel mass spectrometry (MS) technologies and dedicated biochemical procedures that have been recently introduced in this field. A formidable armamentarium is now available for selective enrichment, exaustive structural characterization and quantitative determination of the modification degree for phosphopeptides/phosphoproteins. These methodologies are now successfully applied to the global analysis of cellular proteome repertoire according a holistic approach, allowing the quantitative study of phosphoproteomes on a dynamic time-course basis. The enormous complexity of the protein phosphorylation pattern inside the cell and its dynamic modification will grant important challenges to future scientists, contributing significantly to deeper insights into cellular processes and cell regulation.

Electrophoresis, Gel, Two-Dimensional↗

PubMed2006

Cooperative activity of Ref-1/APE and ERp57 in reductive activation of transcription factors.

ERp57, a protein disulfide isomerase localized mainly in the endoplasmic reticulum, has also been found in lesser amounts in the cytosol and nucleus, where its function is still not characterized. We report here that ERp57 displays affinity for Ref-1, a protein involved in DNA repair as well as in the reduction and activation of transcription factors. Immunoprecipitation experiments showed that Ref-1 and ERp57 also interact in vivo in at least three types of cultured human cells, namely HepG2, M14, and Raji. Oxidative stress increased the amount of nuclear Ref-1 associated with ERp57. Moreover, ERp57 reduced by the thioredoxin-reductase/thioredoxin system stimulated the binding of AP-1 to its consensus sequence on DNA, and HeLa cells stably transfected and overexpressing ERp57 were protected against hydrogen peroxide-induced cell killing. Accordingly, ERp57 appears to cooperate with Ref-1 in the regulation of gene expression mediated by redox-sensitive transcription factors and in the adaptive response of the cell to oxidative insult.

Blotting, Western↗

PubMed2006

Nucleotide receptors stimulation by extracellular ATP controls Hsp90 expression through APE1/Ref-1 in thyroid cancer cells: a novel tumorigenic pathway.

Nucleotide receptors signaling affects cell proliferation, with possible implications on tumorigenic processes. However, molecular targets and action mechanisms of the extracellular nucleotides are still poorly elucidated. We have previously shown in ARO cells that APE1/Ref-1, a transcriptional coactivator responsible for the maintenance of the cellular proliferative rate, is functionally controlled by P2-mediated signaling. Here, we demonstrate that extracellular ATP has a mitogenic effect on ARO cells, increasing ERK phosphorylation, AP1 activation, and cyclin D1 expression. Using the ATP/ADPase apyrase and the P2 receptor antagonist suramin, we show that the extracellular ATP, physiologically released by ARO cells, exerts mitogenic effects. A differential proteomic approach was used to identify molecular events associated with the ATP-induced cell proliferation. Among other proteins, Hsp90 was found upregulated upon ATP stimulation. Pretreatment with suramin completely blocked the ATP-induced Hsp90 activation, confirming the involvement of cell-surface P2 nucleotide receptors in the ATP-mediated activation of ARO cells. Treatment of proliferating ARO cells with suramin and apyrase significantly reduced the intracellular levels of Hsp90, suggesting an autocrine/paracrine mechanism of control on Hsp90 expression by extracellular ATP. The influence of Hsp90 on ATP-induced cell proliferation was also demonstrated by its specific inhibition with 17-AAG. The molecular pathway by which ATP stimulates cell proliferation was further investigated by siRNA strategies showing that Hsp90 is a target of APE1/Ref-1 functional activation. Stimulation of ARO cells with specific nucleotide receptors agonists evidenced a major involvement of P2Y1 and P2Y2 receptors in controlling the Hsp90 activation. Accordingly, these two receptors resulted significantly upregulated in sample biopsies from different thyroid tumors.

Adenosine Triphosphate↗

PubMed2006

A study of Streptococcus thermophilus proteome by integrated analytical procedures and differential expression investigations.

Streptococcus thermophilus is a Gram-positive bacterium belonging to the group of lactic acid bacteria, among which several genera play an essential role in manufacture of food products. Recently, a genomic consortium sequenced and annotated its entire genome, which has been demonstrated to contain 1900 coding sequences. In this study, we have revealed the expression products of almost 200 different genes using a proteomic strategy combining 2-DE plus MALDI-TOF PMF and differential 1-DE plus muLC-ESI-IT-MS/MS. Thus, a number of cellular pathways related to important physiological processes were described at the proteomic level. Almost 50 genes were related to multiple electrophoretic species, whose heterogeneity was mainly due to variability in pI values. A 2-DE reference map obtained for lactose-grown cells was compared with those obtained after heat, cold, acid, oxidative and starvation stresses. Protein up/down-regulation measurements demonstrated that adaptation to different environmental challenges may involve the contribution of unique as well as combined physiological mechanisms. Common regulatory sites in the promoter region of genes whose expression was induced after stress were identified. These results provide a better comprehension of biochemical processes related to stress resistance in S. thermophilus, allowing defining the molecular bases of adaptative responses or markers for the identification of strains with potential industrial applications.

Bacterial Proteins↗

PubMed2006

Proteomic analysis of liver tissues subjected to early ischemia/reperfusion injury during human orthotopic liver transplantation.

Knowledge of early molecular events occurring upon ischemia/reperfusion (I/R) during liver transplantation (LT) is of great importance to improve the therapeutic intervention of surgical treatment. However, nowadays, few data are available on early protein targets of I/R injury. To identify these proteins, we used a differential proteomics approach in the characterization human liver biopsies during I/R upon LT. Analyses were performed on nine donor livers during LT. By using 2-DE and MALDI-TOF MS, we identified 36 proteins which resulted significantly altered upon I/R injury. The majority of these proteins are functionally involved in lipid and energy metabolism, in different metabolic pathways, in redox signalling and in oxidative-stress response. Our data represent the first global approach in the study of I/R injury in liver.

Adult↗

PubMed2006

A proteomic study on human osteoblastic cells proliferation and differentiation.

Changes in expression profiles for 17 proteins were ascertained in human mature osteoblasts compared to pre-osteoblasts (differentiation markers). A differential approach was used to highlight proteomic changes between human osteosarcoma cells and mature osteoblasts, showing a relative over-expression of 8 proteins (proliferation and tumor indicators), as well as under-expression of proteins also found down-regulated in pre-osteoblasts (specific markers of osteoblast differentiation). Our findings confirmed the differences between cell lines and primary human cell cultures and suggested caution on the use of osteosarcoma to study anti-osteoporotic drugs in humans.

Adult↗

PubMed2006

Proteomic analysis of tomato fruits from two ecotypes during ripening.

Ripening of climacteric fruits is a complex physiological phenomenon, which makes berries attractive/palatable and also determines their nutritional characteristics. We report here a comparative proteomic investigation on tomato fruits from a regional and commercial elite ecotype during maturation. Several hundreds of protein components were resolved on 2-DE gels (pH range 4-7) stained with colloidal Coomassie; almost 57% presented overlapping gel coordinates between the two compared varieties. Specific proteins were recognized in each ecotype as differentially expressed during ripening. Constant and variable components were excised, in-gel digested and analysed by MALDI-TOF-MS and microLC-ESI-IT-MS/MS approaches. Peptide MS and MS/MS data were searched against publicly available protein and EST databases, and 83 protein spots were unambiguously identified by MS. Gel matching procedures allowed further identification of proteins between ecotypes. In general, common variably expressed proteins in both ecotypes during maturation were associated to important physiological processes such as redox status control, defence, stress, carbon metabolism, energy production and cellular signalling. Protein components differentially expressed in each variety were also identified. The role of some of the identified proteins in the berries is discussed in relation to tomato fruit ripening. Accordingly, this investigation provides the first characterization of the tomato fruit proteome and description of its variation during maturation.

Gene Expression Profiling↗

PubMed2006

Selective ion tracing and MSn analysis of peptide digests from FSBA-treated kinases for the analysis of protein ATP-binding sites.

Kinases play a key role in many cellular processes by catalyzing the transfer of phosphoryl groups from ATP to a broad number of substrates, including amino acids on target proteins. The reagent 5'-fluorosulfonylbenzoyl-5'-adenosine (FSBA) has been widely used to identify ATP-binding sites in kinases since it reacts with nucleophilic amino acids occurring within these motifs, determining a mass increase of 433 Da. In this study, we present a versatile MS approach that has been developed to recognize labeled peptides generated after enzymatic digestion of FSBA-treated kinases. Using selective ion tracing and MS(2)/MS(3) experiments, we were able to easily identify peptides occurring at protein ATP-binding sites, also affording a complete characterization of the modified amino acids. This methodology may be used in the development of future parent ion scanning-based applications directed to large scale analysis of kinases within complex protein mixtures.

Adenosine Triphosphate↗

PubMed2005

Hyperphosphorylation of JNK-interacting protein 1, a protein associated with Alzheimer disease.

The c-Jun N-terminal kinase (JNK) group of mitogen-activated protein (MAP) kinases are activated by pleiotropic signals including environmental stresses, growth factors, and hormones. JNK-interacting protein 1 (JIP1) is a scaffold protein that assembles and facilitates the activation of the mixed lineage kinase-dependent JNK module and also establishes an interaction with beta-amyloid precursor protein that has been partially characterized. Here we show that, similarly to other proteins involved in various neurological diseases, JIP1 becomes hyperphosphorylated following activation of stress-activated and MAP kinases. By immobilized metal affinity chromatography and a combined microcapillary LC/MALDI-TOF/ESI-ion trap mass spectrometry approach, we identified 35 sites of mitotic phosphorylation within JIP1, among which eight were present within (Ser/Thr)-Pro sequence. This motif is modified by various kinases in aggregates of the microtubule-associated protein tau, which generates typical intraneuronal lesions occurring in Alzheimer disease. Most of the post-translational modifications found were located within the JNK, MAP kinase kinase, and RAC-alpha Ser/Thr protein kinase binding regions; no modifications occurred in protein Src homology 3 and phosphotyrosine interaction domains, which are essential for binding to kinesin, beta-amyloid precursor protein, and MAP kinase kinase kinase. Protein phosphorylation is known to affect stability and protein-protein interactions. Thus, the findings that JIP1 is extensively phosphorylated after activation of stress-activated and MAP kinases indicate that these signaling pathways might modulate JIP1 signaling by regulating its stability and association with some, but not all, interacting proteins.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Fibromodulin gene transcription is induced by ultraviolet irradiation, and its regulation is impaired in senescent human fibroblasts.

Cells undergoing replicative senescence display an altered pattern of gene expression. Senescent fibroblasts show significant changes in the expression of mRNAs encoding extracellular matrix-remodeling proteins; among these mRNAs, the mRNA encoding fibromodulin is highly decreased in these cells. To understand the molecular basis of this phenomenon, we explored the regulatory mechanisms of the human fibromodulin gene. We found that fibromodulin gene promoter contains a cis-element, crucial for its basal expression, that forms a DNA-protein complex when exposed to nuclear extracts from exponentially growing human fibroblasts and not to extracts from cells undergoing senescence by repeated in vitro passages or by mild oxidative stress. The purification of this complex showed that it contains the damage-specific DNA-binding protein DDB-1. The latter is known to be induced by UV irradiation; therefore we checked whether fibromodulin gene promoter is regulated upon the exposure of the cells to UV rays. The results showed that, in exponentially growing fibroblasts, the promoter efficiency is increased by UV irradiation and the DDB-1-containing complex is robustly enriched in cells exposed to UV light. Accordingly, in these experimental conditions the endogenous fibromodulin mRNA accumulates to very high levels. On the contrary, senescent cells did not show any activation of the fibromodulin gene promoter, any induction of the DDB-1-containing complex, or any accumulation of fibromodulin mRNA. These phenomena are accompanied in senescent cells by a decrease of the UV-damaged DNA binding activity.

Cell Line↗

PubMed2005

Comparative proteomic analysis of mammalian animal tissues and body fluids: bovine proteome database.

Characterizing the complete proteome of multicellular organisms is a challenging task using the currently available technologies. With the increasing degree of genetic complexity, animals acquire a broader repertoire of options to meet environmental challenges. Mammalian cells from different tissues/body fluids express different thousands of proteins with a predicted dynamic range of up to five to six orders of magnitude, thus necessitating the whole arsenal of dedicated analytical strategies for a detailed proteome characterization. Nevertheless, 2D-E analysis of whole cellular lysates still remains the most used initial approach for the proteomic description of specialized cells. It enables to obtain an overview of the main soluble protein components of a specific tissue/body fluid, allowing comparison between different cellular types and molecular description of organ specialization. Massive proteomic investigations have been reported mainly in the case of human, mouse and rat, allowing comparative analysis. For this reason, a research project focused on the 2D-E characterization of tissues and biological fluids from other domestic mammals has been undertaken in our laboratory. A number of high-resolution reference electrophoretic maps have been established for liver, kidney, muscle, plasma and red blood cells samples from Holstein Friesian bovine female individuals. Among the 1863 distinct protein features detected, 534 species were identified and associated to 209 different genes by a combination of MALDI-TOF mass fingerprint, capillary LC-ESI-IT-MS-MS and image gel matching procedures. Identified polypeptide species and differences in expression profiles between various tissues/fluids clearly reflected organ biochemical specialization. This experimental output allowed establishing a 2D-E bovine database accessible at the URL address for image comparison.

Animals↗

PubMed2005

Helicobacter pylori immunoproteomes in case reports of rosacea and chronic urticaria.

Rosacea and chronic urticaria are two common skin disorders existing in idiopathic forms. A role of Helicobacter pylori bacterium infection in the aetiopathogenesis of rosacea or chronic urticaria has been suggested although still controversial. The aim of the present study was to establish a relationship between H. pylori infection and rosacea chronic urticaria by means of an immunoproteomic investigation. We analyzed immunoglobulin A (IgA)-, IgG-, and IgE-mediated immune-responses against H. pylori antigens and we identified some bacterial immunoresponsive proteins. A general IgA- and IgE-mediated immune response against antioxidative bacterial proteins was observed. A correlation between the bacterial occurrence and skin diseases pathogenesis is discussed.

Adult↗

PubMed2005

Transcription regulation by the adaptor protein Fe65 and the nucleosome assembly factor SET.

Fe65 protein interacts with the cytosolic domain of the amyloid precursor APP. Its possible involvement in gene regulation is suggested by numerous observations, including those demonstrating that it activates transcription. Here, we show that the Fe65 transcription activation domain overlaps with the WW domain of Fe65 and binds to the nucleosome assembly factor SET. This protein is required for the Fe65-mediated transactivation of a reporter gene. Two-step chromatin immunoprecipitation experiments demonstrate that a complex including Fe65/AICD/Tip60 and SET is associated with the KAI1 gene promoter. Suppression of SET levels by RNA interference shows that this protein is required for full levels of basal transcription of the KAI1 gene. These results further support the function of Fe65 and APP in gene regulation and show a new role for the SET factor.

Amino Acid Sequence↗

PubMed2005

Overoxidation of peroxiredoxins as an immediate and sensitive marker of oxidative stress in HepG2 cells and its application to the redox effects induced by ischemia/reperfusion in human liver.

Oxidative stress is a major pathogenetic event occurring in several liver disorders and is a major cause of liver damage due to Ischemia/Reperfusion (I/R) during liver transplantation. While several markers of chronic oxidative stress are well known, early protein targets of oxidative injury are not well defined. In order to identify these proteins, we used a differential proteomics approach to HepG2 human liver cells treated for 10 min with 500 microM H(2)O(2). This dose was sufficient to induce a slight decrease of total GSH and total protein thiol content without affecting cell viability. By performing Differential Proteomic analysis, by means of two-dimensional gel electrophoresis and MALDI-TOF mass spectrometry, we identified four proteins which resulted sensitive to H(2)O(2) treatment. The main changes were due to post-translational modifications of native polypeptides. Three of these proteins belong to the Peroxiredoxin family of hydroperoxide scavengers, namely PrxI, PrxII and PrxVI, that showed changes in their pI as result of overoxidation. Mass mapping experiments demonstrated the specific modification of peroxiredoxins active site thiol into sulphinic and/or sulphonic acid, thus explaining the increase in negative charge measured for these proteins. The oxidation kinetic of all peroxiredoxins was extremely rapid and sensitive, occurring at H(2)O(2) doses unable to affect the common markers of cellular oxidative stress. Recovery experiments demonstrated a quite different behaviour between 1-Cys and 2-Cys containing Prxs as their retroreduction features is concerned, thus suggesting a functional difference between different class of Prxs. The in vivo relevance of our study is demonstrated by the finding that overoxidation of PrxI occurs during I/R upon liver transplantation and is dependent on the time of warm ischemia. Our present data could be of relevance in setting up more standardized procedures to preserve organs for transplantations.

Biomarkers↗

PubMed2005

Differential proteomic analysis in the study of prokaryotes stress resistance.

Proteomic technologies are powerful tools to study the physiological response of bacteria to various environmental stress conditions. Complex protein toolkits coordinated by regulatory networks have evolved to accommodate bacterial survival under several stressing conditions, such as varying temperature or pH, and changes in nutrient availability. This manuscript provides an overview of the major achievements obtained by 2D gel based and non-gel based proteomic approaches for the study of adaptive response in bacteria. As an example, a study carried out on Streptococcus thermophilus response to various stresses is here reported. In this investigation, we used combined proteomic strategies to define proteomic maps useful to highlight protein profile variations following environmental challenges.

Adaptation, Physiological↗