Marianna Caterino

Analysis of the Ribosomal Protein S19 Interactome

Ribosomal protein S19 (RPS19) is a 16-kDa protein found mainly as a component of the ribosomal 40 S subunit. Its mutations are responsible for Diamond Blackfan anemia, a congenital disease characterized by defective erythroid progenitor maturation. Dysregulation of RPS19 has therefore been implicated in this defective erythropoiesis, although the link between them is still unclear. Two not mutually exclusive hypotheses have been proposed: altered protein synthesis and loss of unknown functions not directly connected with the structural role of RPS19 in the ribosome. A role in rRNA processing has been surmised for the yeast ortholog, whereas the extracellular RPS19 dimer has a monocyte chemotactic activity. Three proteins are known to interact with RPS19: FGF2, complement component 5 receptor 1, and a nucleolar protein called RPS19-binding protein. We have used a yeast two-hybrid approach to identify a fourth protein: the serine-threonine kinase PIM1. The present study describes our use of proteomics strategies to look for proteins interacting with RPS19 to determine its functions. Proteins were isolated by affinity purification with a GST-RPS19 recombinant protein and identified using LCMS/MS analysis coupled to bioinformatics tools. We identified 159 proteins from the following Gene Ontology categories: NTPases (ATPases and GTPases; five proteins), hydrolases/helicases (19 proteins), isomerases (two proteins), kinases (three proteins), splicing factors (five proteins), structural constituents of ribosome (29 proteins), transcription factors (11 proteins), transferases (five proteins), transporters (nine proteins), DNA/RNA-binding protein species (53 proteins), other (one dehydrogenase protein, one ligase protein, one peptidase protein, one receptor protein, and one translation elongation factor), and 13 proteins of still unknown function. Proteomics results were validated by affinity purification and Western blotting. These interactions were further confirmed by co-immunoprecipitation using a monoclonal RPS19 antibody. Many interactors are nucleolar proteins and thus are expected to take part in the RPS19 interactome; however, some proteins suggest additional functional roles for RPS19.

Transcription factor TBX1 overexpression induces downregulation of proteins involved in retinoic acid metabolism: a comparative proteomic analysis.

TBX1 haploinsufficiency is considered a major contributor to the del22q11.2/DiGeorge syndrome (DGS) phenotype. We have used proteomic tools to look at all the major proteins involved in the TBX1-mediated pathways in an attempt to better understand the molecular interactions instrumental to its cellular functions. We found more than 90 proteins that could be targeted by TBX1 through different mechanisms. The most interesting observation is that overexpression of TBX1 results in down-regulation of two proteins involved in retinoic acid metabolism.

Maternal vitamin B12 deficiency detected in expanded newborn screening

OBJECTIVES Besides the inherited form, vitamin B(12) deficiency may be due to diet restrictions or abnormal absorption. The spread of newborn screening programs worldwide has pointed out that non-inherited conditions are mainly secondary to a maternal deficiency. The aim of our work was to study seven cases of acquired vitamin B12 deficiency detected during our newborn screening project. Moreover, we aimed to evaluate vitamin B(12) and related biochemical parameters status on delivering female to verify the consequences on newborns of eventually altered parameters. DESIGN AND METHODS 35,000 newborns were screened; those showing altered propionyl carnitine (C3) underwent second-tier test for methylmalonic acid (MMA) on dried blood spot (DBS). Subsequently, newborns positive to the presence of MMA on DBS and their respective mothers underwent further tests: serum vitamin B(12), holo-transcobalamin (Holo-TC), folate and homocysteine; newborns were also tested for urinary MMA content. A control study was conducted on 203 females that were tested for the same parameters when admitted to hospital for delivery. RESULTS Approximately 10% of the examined newborns showed altered C3. Among these, seven cases of acquired vitamin B(12) deficiency were identified (70% of the MMA-positive cases). Moreover, our data show a high frequency of vitamin B(12) deficiency in delivering female (approximately 48% of examined pregnants). CONCLUSIONS We suggest to monitor vitamin B(12) and Holo-TC until delivery and to reconsider the reference interval of vitamin B(12) for a better identification of cases at risk. Finally, newborns from mothers with low or borderline levels of vitamin B(12) should undergo second-tier test for MMA; in the presence of MMA they should be supplemented with vitamin B(12) to prevent adverse effects related to vitamin B(12) deficiency.

Insulin‐like growth factor 1 receptor signaling induced by supraphysiological doses of IGF‐1 in human peripheral blood lymphocytes

Insulin‐like growth factor‐1 (IGF‐1) mediates some of growth hormone anabolic functions through its receptor, IGF‐1R. Following ligand binding, intracellular signaling pathways are activated favouring proliferation, cell survival, tissue growth, development, and differentiation. IGF‐1 is included in the World Anti‐Doping Agency Prohibited List. While the evidence for IGF‐1 as performance‐enhancing substrate in healthy humans is still weak, clinical studies demonstrated that the endogenous growth hormone/IGF‐1 excess is associated with cardiovascular implications. Previously, we demonstrated that human peripheral blood lymphocytes represent a suitable system to identify a gene signature, related to dihydrotestosterone or IGF‐1 abuse, independent from the type of sport. In addition, in a proteomic study, we demonstrated that dihydrotestosterone hyperdosage affects cell motility and apoptosis. Here, we investigate the doping action of IGF‐1 by means of a differential proteomic approach and specific protein arrays, revealing an active cytoskeletal reorganization mediated by Stat‐1; moreover, IGF‐1 stimulation produces a sustained activation of different signaling pathways as well as an overproduction of cytokines positively related to immune response and inflammation. In conclusion, these data indicate that, following IGF‐1 hyperdosage, circulating peripheral blood lymphocytes could be more prone to transendothelial migration.

The secretome signature of colon cancer cell lines

The definition of the secretome signature of a cancer cell line can be considered a potential tool to investigate tumor aggressiveness and a preclinical exploratory study required to optimize the search of cancer biomarkers. Dealing with a cell‐specific secretome limits the contamination by the major components of the human serum and reduces the range of dynamic concentrations among the secreted proteins, thus favouring under‐represented tissue‐specific species. The aim of the present study is to characterize the secretome of two human colon carcinoma cell lines, CaCo‐2 and HCT‐GEO, in order to evaluate differences and similarities of two colorectal cancer model systems. In this study, we identified more than 170 protein species, 64 more expressed in the secretome of CaCo‐2 cells and 54 more expressed in the secretome of HCT‐GEO cells; 58 proteins were shared by the two systems. Among them, more than 50% were deemed to be secretory according to their Gene Ontology annotation and/or to their SignalP or SecretomeP scores. Such a characterization allowed corroborating the potential of a cell culture‐based model in order to describe the cell‐specific invasive properties and to provide a list of putative cancer biomarkers. J. Cell. Biochem. 114: 2577–2587, 2013.

The proteome of cblC defect: in vivo elucidation of altered cellular pathways in humans

Methylmalonic acidemia with homocystinuria, cobalamin deficiency type C (cblC) (MMACHC) is the most common inborn error of cobalamin metabolism. Despite a multidrug treatment, the long-term follow-up of early-onset patients is often unsatisfactory, with progression of neurological and ocular impairment. Here, the in-vivo proteome of control and MMACHC lymphocytes (obtained from patients under standard treatment with OHCbl, betaine, folate and L-carnitine) was quantitatively examined by two dimensional differential in-gel electrophoresis (2D-DIGE) and mass spectrometry. Twenty three proteins were found up-regulated and 38 proteins were down-regulated. Consistent with in vivo studies showing disturbance of glutathione metabolism, a deregulation in proteins involved in cellular detoxification, especially in glutathione metabolism was found. In addition, relevant changes were observed in the expression levels of proteins involved in intracellular trafficking and protein folding, energy metabolism, cytoskeleton organization and assembly. This study demonstrates relevant changes in the proteome profile of circulating lymphocytes isolated from treated cblC patients. Some results confirm previous observations in vivo on fibroblast, thus concluding that some dysregulation is ubiquitous. On the other hand, new findings could be tissue-specific. These observations expand our current understanding of the cblC disease and may ignite new research and therapeutic strategies to treat this disorder.

Differential proteomic analysis in human cells subjected to ribosomal stress.

The biochemical phenotype of cells affected by ribosomal stress has not yet been studied in detail. Here we report a comparative proteomic analysis of cell lines silenced for the RPS19 gene versus cell lines transfected with scramble shRNA cells performed using the DIGE technology integrated to bioinformatics tools. Importantly, to achieve the broadest possible understanding of the outcome, we carried out two independent DIGE experiments using two different pH ranges, thus, allowing the identification of 106 proteins. Our data revealed the deregulation of proteins involved in cytoskeleton reorganization, PTMs, and translation process. A subset (26.9%) of these proteins is translated from transcripts that include internal ribosome entry site motifs. This supports the hypothesis that during ribosomal stress translation of specific messenger RNAs is altered.

Characterization of red cell membrane proteins as a function of red cell density: Annexin VII in different forms of hereditary spherocytosis

Annexin VII was present in reticulocytes but was then lost as the cells matured. A different pattern was found in band 3‐deficient samples: annexin VII was in fact present in both mature and immature red cell membranes. Cytoskeletal anomalies may then influence the turn‐over of annexin VII during erythrocyte maturation.

Proteome analysis of human amniotic mesenchymal stem cells (hA-MSCs) reveals impaired antioxidant ability, cytoskeleton and metabolic functionality in maternal obesity

Maternal obesity increases the risk of obesity and/or obesity-related diseases in the offspring of animal models. The aim of this study was to identify metabolic dysfunctions that could represent an enhanced risk for human obesity or obesity-related diseases in newborn or in adult life, similar to what occurs in animal models. To this aim, we studied the proteome of 12 obese (Ob-) and 6 non-obese (Co-) human amniotic mesenchymal stem cells (hA-MSCs) obtained from women at delivery by cesarean section (pre-pregnancy body mass index [mean ± SD]: 42.7 ± 7.7 and 21.3 ± 3.3 kg/m2, respectively). The proteome, investigated by two-dimensional fluorescence difference gel electrophoresis/mass spectrometry, revealed 62 differently expressed proteins in Ob- vs Co-hA-MSCs (P < 0.05), nine of which were confirmed by western blotting. Bioinformatics analysis showed that these 62 proteins are involved in several statistically significant pathways (P < 0.05), including the stress response, cytoskeleton and metabolic pathways. Oxidative stress was shown to be an early triggering factor of tissue fat accumulation and obesity-related disorders in the offspring of obese animal models. Our finding of a reduced stress response in Ob-hA-MSCs suggests that a similar mechanism could occur also in humans. Long-term follow-up studies of newborns of obese mothers are required to verify this hypothesis.

Synergistic effect of DHT and IGF-1 hyperstimulation in human peripheral blood lymphocytes

The abuse of mixed or combined performance‐enhancing drugs is widespread among athletes and amateurs, adults and adolescents. Clinical studies demonstrated that misuse of these doping agents is associated with serious adverse effects to many organs in human. Previously, we demonstrated in human peripheral blood lymphocytes that high doses of anabolic androgenic steroids, such as dihydrotestosterone (DHT) and growth factors, such as insulin‐like growth factor‐1 (IGF‐1), have effects at gene and protein levels. Supraphysiological treatments of DHT and IGF‐1 affected the expression of genes involved in skeletal muscle disorders as well as in cell‐mediated immunological response. At protein level, DHT hyperdosage affects cell motility and apoptosis; IGF‐1 hyperstimulation triggers an active cytoskeletal reorganization and an overproduction of immune response‐ and inflammation‐related cytokines. In this study, we investigate the combined effects of DHT and IGF‐1 hyperdosage in peripheral blood lymphocytes using a differential proteomic approach. DHT and IGF‐1 combined treatment affects cell adhesion, migration, and survival through modulation of expression levels of cytokines and paxillin‐signaling‐related proteins, and activation of several pathways downstream focal adhesion kinase. Our results indicate a synergistic effect of DHT and IGF‐1 which has potential implications for health risk factors.