Papers by Michela Di Michele
Journal of Proteomics, May 1, 2011
Molecular & Cellular Proteomics, 2012
Journal of Thrombosis and Haemostasis, Jul 1, 2011
Human Molecular Genetics, Oct 15, 2020
Current Medicinal Chemistry, Oct 1, 2012
Expert Review of Proteomics, Aug 1, 2012
Journal of Proteomics, Jun 1, 2013
Journal of Thrombosis and Haemostasis, Jul 16, 2011
Journal of Thrombosis and Haemostasis, Jul 1, 2010
Platelets, Jul 7, 2011
Smoking accelerates atherosclerosis and is a well-known risk factor for acute cardiovascular comp... more Smoking accelerates atherosclerosis and is a well-known risk factor for acute cardiovascular complications; however, the mechanisms of these effects have not been completely clarified. Recently developed proteomic approaches may offer new clues when combined with well-established functional tests. Platelet proteome of healthy smokers and non-smokers was resolved by two-dimensional difference gel electrophoresis, compared by Decyder software and identified by mass spectrometry analysis (nano-LC-MS/MS). In smokers, three proteins (Factor XIII-A subunit, platelet glycoprotein IIb and beta-actin) were significantly up-regulated, whereas WDR1 protein and chaperonine HSP60 were down-regulated. Furthermore, the highest scored network derived by Ingenuity Pathway Analysis using the modulated proteins as input showed the involvement of several proteins to be related to inflammation and apoptosis. Platelet function tests and the levels of markers of platelet and leukocyte activation were not different in smokers vs. non-smoker subjects. The platelet proteomic approach confirms that cigarette smoking triggers several inflammatory reactions and may help clarify some of the molecular mechanisms of smoke effect on cellular systems relevant for vascular integrity and human health.
Class III β-tubulin (TUBB3) has been discovered as a marker of drug resistance in human cancer. T... more Class III β-tubulin (TUBB3) has been discovered as a marker of drug resistance in human cancer. To get insights into the mechanisms by which this protein is involved in drug resistance, we analyzed TUBB3 in a panel of drug-sensitive and drug-resistant cell lines. We identified two main different isoforms of TUBB3 having a specific electrophoretic profile. We showed that the apparently higher molecular weight isoform is glycosylated and phosphorylated and it is localized in the cytoskeleton. The apparently lower molecular weight isoform is instead found exclusively in mitochondria. We observed that levels of phosphorylation and glycosylation of TUBB3 are associated with the resistant phenotype and compartmentalization into cytoskeleton. By two-dimensional nonreduced/reduced SDS-PAGE analysis, we also found that TUBB3 protein in vivo forms protein complexes through intermolecular disulfide bridges. Through TUBB3 immunoprecipitation, we isolated protein species able to interact with TU...
Thrombosis Research, 2014
International Journal of Oncology, Jun 23, 2010
Molecular Autism, Nov 4, 2013
Background: Neurobeachin (NBEA) has been identified as a candidate gene for autism spectrum disor... more Background: Neurobeachin (NBEA) has been identified as a candidate gene for autism spectrum disorders (ASD) in several unrelated patients with alterations in the NBEA gene. The exact function of NBEA, a multidomain scaffolding protein, is currently unknown. It contains an A-kinase anchoring protein (AKAP) domain which binds the regulatory subunit of protein kinase A (PKA) thereby confining its activity to specific subcellular regions. NBEA has been implicated in post-Golgi membrane trafficking and in regulated secretion. The mechanism of regulated secretion is largely conserved between neurons and platelets, and the morphology of platelet dense granules was found to be abnormal in several ASD patients, including one with NBEA haploinsufficiency. Platelet dense granules are secreted upon vascular injury when platelets are exposed to for instance collagen. Dense granules contain serotonin, ATP and ADP, which are necessary for platelet plug formation and vascular contraction. Methods: To further investigate possible roles for NBEA in secretion or dense granule morphology, platelets from Nbea +/− mice were analyzed morphometrically, functionally and biochemically. A differential proteomics and peptidomics screen was performed between Nbea +/− and Nbea +/+ mice, in which altered Talin-1 cleavage was further investigated and validated in brain samples. Finally, the phosphorylation pattern of PKA substrates was analyzed. Results: Platelet dense granules of Nbea +/− mice had a reduced surface area and abnormal dense-core halo, but normal serotonin-content. Nbea haploinsufficiency did not affect platelet aggregation and ATP secretion after collagen stimulation, although the platelet shape change was more pronounced. Furthermore, peptidomics revealed that Nbea +/− platelets contain significantly reduced levels of several actin-interacting peptides. Decreased levels were detected of the actin-binding head and rod domain of Talin-1, which are cleavage products of Calpain-2. This is most likely due to increased PKA-mediated phosphorylation of Calpain-2, which renders the enzyme less active. Analysis of other PKA substrates revealed both increased and reduced phosphorylation. Conclusion: Our results show the pleiotropic effects of alterations in PKA activity due to Nbea haploinsufficiency, highlighting the important function of the AKAP domain in Nbea in regulating and confining PKA activity. Furthermore, these results suggest a role for Nbea in remodeling the actin cytoskeleton of platelets.
Heredity, May 14, 2008
Plant biodiversity must be safeguarded because it constitutes a resource of genes that may be use... more Plant biodiversity must be safeguarded because it constitutes a resource of genes that may be used, for instance, in breeding programs. Lentil (Lens culinaris Medik.) is one of the most ancient crops of the Mediterranean region. Extensive differentiation of L. culinaris over millennia has resulted in a myriad of different landraces. However, in more recent times many landraces have disappeared consequent to environmental and socioeconomic changes. To promote the survival of endangered lentil landraces, we have investigated the genetic relationship between two ancient landrace cultivated in Capracotta and Conca Casale (Molise, south-central Italy) and widely spread commercial varieties using an integrated approach consisting of studies at morphological, DNA and protein level. Seeds of these two landraces were collected from local farmers and conserved in the Molise germoplasm bank. The two local landraces were well differentiated from each other, and the Conca Casale landrace was separated from the commercial varieties at morphological, protein and DNA level. The Capracotta landrace, was well separated from the commercial varieties, except Castelluccio di Norcia, at DNA level showing a more complex and heterogeneous segregation at morphological and biochemical level. The correlation between morphological, DNA and protein data, illustrates that proteomics is a powerful tool with which to complement the analysis of biodiversity in ecotypes of a single plant species and to identify physiological and/or environmental markers.
Environmental and Experimental Botany, Sep 1, 2011
Medicago marina (L.) is a Mediterranean species whose seeds show strong dormancy that prevents ge... more Medicago marina (L.) is a Mediterranean species whose seeds show strong dormancy that prevents germination. We used an integrated approach of physiological analyses and proteomics to investigate the mechanisms that control M. marina dormancy/germination and that underlie stress tolerance. First, we evaluated the effects on dormancy breaking of the following treatments: mechanical scarification, freezing at −20 • C, storage for 4 months and heating at 100 • C for 1 h. Mechanical scarification and freezing were the most effective treatments in overcoming dormancy. The role of abscisic acid (ABA) in M. marina dormancy was studied by ELISA immuno-enzymatic assay. The ABA content of germinated and nongerminated mature (control) and treated seeds was determined. The level of ABA was higher in treated seeds than in control seeds; the most significant increase occurred in the heated seeds. A comparison of the ABA level in the germinated, control and treated seeds suggests that different mechanisms modulate ABA content in response to different stresses, and that a specific ABA-signalling pathway regulates germination. Proteomic analysis revealed 46 proteins differentially expressed between treated and untreated seeds; 14 of these proteins were subsequently identified by mass spectrometry. Several of the proteins identified are important factors in the stress response, and are involved in such diverse functions as lipid metabolism, protein folding and chromatin protection. Lastly, an analysis of the phosphoproteome maps showed that the function of many proteins in seeds subjected to temperature treatment is modulated through post-translational modifications.
Plant Science, May 1, 2006
The stability of plants growing on a slope may be affected by factors that tend to uproot the pla... more The stability of plants growing on a slope may be affected by factors that tend to uproot the plant. To avoid uprooting, roots undergo complex, finely regulated changes. Spanish broom (Spartium junceum L.) is a widespread Mediterranean legume that frequently grows on slopes. Using twodimensional polyacrylamide gel electrophoresis (2D-PAGE) we compared the protein patterns of S. junceum roots grown in a greenhouse in either upright or tilted pots. We obtained about 1000 protein spots that were highly reproducible across an isoelectric focusing range 4-7. The protein profile of 141 spots differed significantly (p < 0.001) between the two conditions. Based on spot quantity and quality, and the expression rate in the two conditions, 34 protein spots (20 up-regulated and 14 down-regulated in the slope condition) were selected for tandem mass spectrometry analysis. The peptide sequences were obtained by querying the sequences of two databases: an EST database containing DNA sequences of approximately 550,000 ESTs of seven species of the Fabaceae family and the SwissProt database. The function of 18 proteins was assigned by homology; the remaining proteins did not show any alignment with the sequences of the two databases. Most of the proteins whose expression differed between the two conditions showed homology with proteins involved in cell structure organisation and in several stress responses of diverse plant species. The data from this investigation are available at
Journal of Experimental Botany, Nov 28, 2003
Linker histone protein variants are expressed in different tissues, at various developmental stag... more Linker histone protein variants are expressed in different tissues, at various developmental stages or induced by speci®c environmental conditions in many plant species. In most cases, the function of these proteins remains unknown. In the work presented here an antisense strategy has been used to study the function of the drought-induced linker histone, H1-S of tomato. Three independent H1-S antisense tomato mutants, selected for their inability to accumulate H1-S in response to water stress, were studied. These mutants have been characterized at the physiological and morphological levels. Histone H1-S antisense transgenic plants developed normally indicating that H1-S does not play an important role in the basal functions of tomato development. No differences were detected in chromatin organization, excluding a structural role for H1-S in chromatin organization. However, differences between the wildtype and antisense plants were observed in leaf anatomy and physiological activities. This analysis indicates that H1-S has more than one function, at different times, in controlling plant water status, highlighting the complexity of the water stress response.
Electrophoresis, 2010
Lentil (Lens culinaris Medik.) is one of the most ancient crops of the Mediterranean region used ... more Lentil (Lens culinaris Medik.) is one of the most ancient crops of the Mediterranean region used for human nutrition; an extensive differentiation of L. culinaris over millennia has resulted in a number of different landraces. As a consequence of environmental and socioeconomic issues, the disappearance of many of them occurred in more recent times. To investigate the potential of proteomics as a tool in phylogenetic studies, testing the possibility to identify specific markers of different plant landraces, 2-D gel electrophoretic maps of mature seeds were obtained from seven lentil populations belonging to a local ecotype (Capracotta) and five commercial varieties (Turca Rossa, Canadese, Castelluccio di Norcia, Rascino and Colfiorito). 2-DE analysis resolved hundreds of protein species in each lentil sample, among which only 122 were further identified by MALDI-TOF PMF and/or nanoLC-ESI-LIT-MS/MS, probably as a result of the poor information available on L. culinaris genome. A comparison of these maps revealed that 103 protein spots were differentially expressed within and between populations. The multivariate statistical analyses carried out on these variably expressed spots showed that 24 protein species were essential for population discrimination, thus determining their proposition as landrace markers. Besides providing the first reference map of mature lentil seeds, our data confirm previous studies based on morphological/ genetic observations and further support the valuable use of proteomic techniques as phylogenetic tool in plant studies.
Uploads
Papers by Michela Di Michele