[Show abstract][Hide abstract] ABSTRACT: Neurological disorders can be associated with protein glycosylation abnormalities. Rett syndrome is a devastating genetic brain disorder, mainly caused by de novo loss-of-function mutations in the methyl-CpG binding protein 2 (MECP2) gene. Although its pathogenesis appears to be closely associated with a redox imbalance, no information on glycosylation is available. Glycoprotein detection strategies (i.e., lectin-blotting) were applied to identify target glycosylation changes in the whole brain of Mecp2 mutant murine models of the disease. Remarkable glycosylation pattern changes for a peculiar 50kDa protein i.e., the N-linked brain nucleotide pyrophosphatase-5 were evidenced, with decreased N-glycosylation in the presymptomatic and symptomatic mutant mice. Glycosylation changes were rescued by selected brain Mecp2 reactivation. Our findings indicate that there is a causal link between the amount of Mecp2 and the N-glycosylation of NPP-5.
Neuroscience Research 10/2015; DOI:10.1016/j.neures.2015.10.002 · 1.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background: Meat derives from muscle, but they are extremely different. The slaughtered muscle undergoes a number of biological changes during the maturation period, which is pivotal for the transformations that permit to obtain the final marketable product. Scope and approach: In this review a general process driving muscle-to-meat conversion is described, despite all the factors that can affect and diversify every individual process. We focus our attention on the switch from the normal, aerobic metabolism to the post-slaughter, anaerobic one, underlining all the consequences in terms of muscle reactions driving and influencing the transformation. The massive production of ROS is the pivotal event of the muscle-to-meat conversion, and muscle cells are stimulated to react as to cope with the oxidative stress. Despite the mobilization of defensive machineries, it soon becomes overwhelming and unsustainable: muscle cells are forced to die. Key findings and conclusions: ROS can induce both autophagy and apoptosis. Their role in muscle conversion is not completely clear, despite their differences have large influence on meat maturation and final product. A deeper understanding is pivotal on this argument as to better manage meat production.
[Show abstract][Hide abstract] ABSTRACT: Short p63 isoform, ΔNp63, is crucial for epidermis formation, and it plays a pivotal role in controlling the turnover of basal keratinocytes by regulating the expression of a subset of genes involved in cell cycle and cell adhesion programs. The glycolytic enzyme hexokinase 2 (HK2) represents the first step of glucose utilization in cells. The family of HKs has four isoforms that differ mainly in their tissue and subcellular distribution. The preferential mitochondrial localization of HK2 at voltage-dependent anion channels provides access to ATP generated by oxidative phosphorylation and generates an ADP/ATP recycling mechanism to maintain high respiration rates and low electron leak. Here, we report that ΔNp63 depletion in human keratinocytes impairs mitochondrial basal respiration and increases mitochondrial membrane polarization and intracellular reactive oxygen species. We show ΔNp63-dependent regulation of HK2 expression, and we use ChIP, validated by p63-Chip sequencing genomewide profiling analysis, and luciferase assays to demonstrate the presence of one p63-specific responsive element within the 15th intronic region of the HK2 gene, providing evidence of a direct interaction. Our data support the notion of ΔNp63 as a master regulator in epithelial cells of a combined subset of molecular mechanisms, including cellular energy metabolism and respiration. The ΔNp63-HK2 axis is also present in epithelial cancer cells, suggesting that ΔNp63 could participate in cancer metabolic reprogramming.
Proceedings of the National Academy of Sciences 09/2015; 112(37). DOI:10.1073/pnas.1508871112 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Piedmontese meat tenderness becomes higher by extending the ageing period after slaughter up to 44 days. Classical physical analysis only partially explain this evidence, so in order to discover the reason of the potential beneficial effects of prolonged ageing, we performed omic analysis in the Longissimus thoracis muscle by examining main biochemical changes through mass spectrometry-based metabolomics and proteomics. We observed a progressive decline in myofibrillar structural integrity (underpinning meat tenderness) and impaired energy metabolism. Markers of autophagic responses (e.g. serine and glutathione metabolism) and nitrogen metabolism (urea cycle intermediates) accumulated until the end of the assayed period. Key metabolites such as glutamate, a mediator of the appreciated umami taste of the meat, were found to constantly accumulate until day 44. Finally, statistical analyses revealed that glutamate, serine and arginine could serve as good predictors of ultimate meat quality parameters, even though further studies are mandatory.
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND
Refrigerated storage of red blood cell (RBC) units promotes the progressive accumulation of the so-called storage lesions, a widespread series of alterations to morphology, metabolism, and proteome integrity of stored RBCs. However, while storage lesions targeting the RBC membrane fraction have been widely documented, the cytosolic fraction is as yet an underinvestigated cause of the technical inconveniences related to the high abundance of hemoglobin.STUDY DESIGN AND METHODS
By exploiting a recently ideated preparative two-dimensional clear native electrophoresis, followed by mass spectrometry analysis, we could monitor the changes of soluble multiprotein complexes (MPCs) in RBCs after 0, 21, and 35 days of storage under standard blood banking conditions.RESULTSData indicate a substantial storage-dependent alteration of RBC MPCs, particularly of those involved in energy and redox metabolism, confirming previous evidence about the progressive dysregulation of these pathways in long-stored units.CONCLUSION
The use of native gel–based proteomics to investigate MPCs present in the RBC cytosolic fraction proved to be a powerful tool. Results collected represent a preliminary advance in the knowledge of the key role of native cytosolic MPCs in context of RBC storage lesion. Multiprotein organization and interacting partners of some key enzymes have been found to change during storage duration, suggesting that future studies will be needed to assess whether such alterations could influence their activity and efficiency.
[Show abstract][Hide abstract] ABSTRACT: The yeast Saccharomyces cerevisiae expresses one member of metacaspase Cys protease family, encoded by YCA1 gene. Combination of proteomics and metabolomics data showed YCA1 deletion down-regulated glycolysis, TCA cycle and alcoholic fermentation as compared with WT cells. Δyca1 cells also showed a down-regulation of the pentose phosphate pathway and an accumulation of pyruvate, correlated with higher levels of certain amino acids found in these cells. Accordingly, there is a decrease in protein biosynthesis, and up-regulation of specific stress response protein like Ahp1p, which possibly provides these cells with a better protection against stress. Moreover, in agreement with the down-regulation of protein biosynthesis machinery in Δyca1 cells, we have found that regulation of transcription, co-translational protein folding and protein targeting to different subcellular locations were also down-regulated. Metabolomics analysis of the nucleotide content showed a significant reduction in Δyca1 cells in comparison with the WT, except for GTP content which remained unchanged. Thus, our combined proteome/metabolome approach added a new dimension to the non-apoptotic function of yeast metacaspase, which can specifically affect cell metabolism through as yet unknown mechanisms and possibly stress-response pathways, like HOG and cell wall integrity pathways. Certainly, YCA1 deletion may induce compensatory changes in stress response proteins offering a better protection against apoptosis to Δyca1 cells rather than a loss in a pro-apoptotic YCA1-associated activity.
[Show abstract][Hide abstract] ABSTRACT: One of the hallmarks of blood bank stored red blood cells (RBCs) is the irreversible transition from a discoid to a spherocyte-like
morphology withmembrane perturbation and cytoskeleton disorders. Therefore, identification of the storage-associated modifications
in the protein–protein interactions between the cytoskeleton and the lipid bilayer may contribute to enlighten the molecular
mechanisms involved in the alterations ofmechanical properties of stored RBCs. Here we report the results obtained analyzing RBCs
after 0, 21 and 35 days of storage under standard blood banking conditions by label free mass spectrometry (MS)-based experiments.
We could quantitatively measure changes in the phosphorylation level of crucial phosphopeptides belonging
to β-spectrin, ankyrin-1, α-adducin, dematin, glycophorin A and glycophorin C proteins. Data have been validated by
both western blotting and pseudo-Multiple Reaction Monitoring (MRM). Although each phosphopeptide showed a distinctive
trend, a sharp increase in the phosphorylation level during the storage duration was observed. Phosphopeptide
mapping and structural modeling analysis indicated that the phosphorylated residues localize in protein functional domains
fundamental for the maintenance of membrane structural integrity. Along with previous morphological evidence
acquired by electron microscopy, our results seem to indicate that 21-day storage may represent a key point for the
molecular processes leading to the erythrocyte deformability reduction observed during blood storage. These findings
could therefore be helpful in understanding and preventing the morphology-linked mechanisms responsible for the
post-transfusion survival of preserved RBCs.
Journal of Mass Spectrometry 02/2015; 50(2):326-335. DOI:10.1002/jms.3531 · 2.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In the present study, we performed an integrated proteomics (2DE) and metabolomics (HPLC-MS) investigation to determine the molecular mechanisms underlying cadmium (Cd) tolerance in the halophyte Cakile maritima. Recent physiological reports have documented how C. maritima could accumulate high doses of Cd in roots and shoots, while appearing to be naturally equipped to cope with it, since mild or heavy Cd stress did not alter physiological parameters, including mineral uptake, pigment contents, other than transpiration, water use efficiency and variation of net CO2 assimilation. In the present study, metabolomics and proteomics results highlighted the Cd-dependent up-regulation of thiol compound anabolism, including glutathione and phytochelatin homeostasis, especially in response to elevated Cd stress (100 µM), which allows an intracellular chelation of Cd and its compartmentalization into vacuole. Altered energy metabolism at the triose phosphate level was accompanied by altered accumulation of Calvin cycle intermediates and photorespiration byproducts at high (100 µM), albeit not at mild (25 µM), CdCl2 stress, suggesting that elevated doses of Cd might promote photorespiration. Metabolomics results confirmed proteomics and previous physiological evidence, also suggesting that osmoprotectants betaine and proline, together with plant hormones methyl jasmonate and salicylic acid might be involved in mediating responses to Cd-induced stress. Taken together, from the present study we conclude that C. maritima might represent an ideal candidate for phytoremediation interventions in Cd-contaminated soils.
[Show abstract][Hide abstract] ABSTRACT: Nibrin (also named NBN or NBS1) is a component of the MRE11/RAD50/NBN complex, which is involved in early steps of DNA double strand breaks sensing and repair. Mutations within the NBN gene are responsible for the Nijmegen breakage syndrome (NBS). The 90% of NBS patients are homozygous for the 657del5 mutation, which determines the synthesis of two truncated proteins of 26 kDa (p26) and 70 kDa (p70). Here, HEK293 cells have been exploited to transiently express either the full-length NBN protein or the p26 or p70 fragments, followed by affinity chromatography enrichment of the eluates. The application of an unsupervised proteomics approach, based upon SDS-PAGE separation and shotgun digestion of protein bands followed by MS/MS protein identification, indicates the occurrence of previously unreported protein interacting partners of the full-length NBN protein and the p26 fragment containing the FHA/BRCT1 domains, especially after cell irradiation. In particular, results obtained shed light on new possible roles of NBN and of the p26 fragment in ROS scavenging, in the DNA damage response, and in protein folding and degradation. In particular, here we show that p26 interacts with PARP1 after irradiation, and this interaction exerts an inhibitory effect on PARP1 activity as measured by NAD+ levels. Furthermore, the p26-PARP1 interaction seems to be responsible for the persistence of ROS, and in turn of DSBs, at 24 h from IR. Since some of the newly identified interactors of the p26 and p70 fragments have not been found to interact with the full-length NBN, these interactions may somehow contribute to the key biological phenomena underpinning NBS.
PLoS ONE 12/2014; 9(12):e114651. DOI:10.1371/journal.pone.0114651 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nelumbo nucifera (Gaertn.) or lotus, is an aquatic plant native to India, and presently consumed as food mainly in China and Japan. Lotus is also widely used in Indian and Chinese traditional medicine. Extracts from different parts of the lotus plant have been reported to show diverse biological activities – antioxidant, free radical scavenging, anti-inflammatory and immunomodulatory. Despite this, little work has been done in isolating and identifying proteins responsible for these activities, or yet importantly to establish a lotus proteome. The aim of our group is to develop a proteome catalogue of the lotus plant, starting with its seed, the nutrient rich food source. In this present study, the seed endosperm – most abundant and nutrient storage tissue – was targeted for protein extraction by testing five different extraction protocols, followed by their proteomic analyses using complementary 1DE and 2DE approaches in conjunction with tandem mass spectrometry. The inventory of 66 non-redundant proteins obtained by 1DE-MS and the 30 obtained by 2DE-MS provides the first catalogue of the lotus seed endosperm, where the most abundant protein functions were in categories of metabolic activities related to carbohydrate metabolism and nutrient storage.This article is protected by copyright. All rights reserved
[Show abstract][Hide abstract] ABSTRACT: Heat-shock protein (Hsp)10 is the co-chaperone for Hsp60 inside mitochondria, but it also resides outside the organelle. Variations in its levels and intracellular distribution have been documented in pathological conditions, e.g. cancer and chronic obstructive pulmonary disease (COPD). Here, we show that Hsp10 in COPD undergoes changes at the molecular and subcellular levels in bronchial cells from human specimens and derived cell lines, intact or subjected to stress induced by cigarette smoke extract (CSE). Noteworthy findings are: (i) Hsp10 occurred in nuclei of epithelial and lamina propria cells of bronchial mucosa from non-smokers and smokers; (ii) human bronchial epithelial (16HBE) and lung fibroblast (HFL-1) cells, in vitro, showed Hsp10 in the nucleus, before and after CSE exposure; (iii) CSE stimulation did not increase the levels of Hsp10 but did elicit qualitative changes as indicated by molecular weight and isoelectric point shifts; and (iv) Hsp10 nuclear levels increased after CSE stimulation in HFL-1, indicating cytosol to nucleus migration, and although Hsp10 did not bind DNA, it bound a DNA-associated protein.
Open Biology 10/2014; 4(10). DOI:10.1098/rsob.140125 · 5.78 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In this work we evaluated Cd-phytoextraction ability of the halophyte Cakile maritima comparatively to the glycophyte Brassica juncea commonly recommended for phytoextraction. Seedlings were grown in nutrient solution added with 0–100 M Cd for 21 days. Cd impaired growth in B. juncea but had no sig-nificant impact on C. maritima. The halophyte C. maritima maintained also higher photosynthetic activity than the glycophyte B. juncea. Cd decreased leaf chlorophyll (Chl) and carotenoids concentrations as well as PSII efficiency (F v /F m , F v /F 0 an PSII) in B. juncea while it increased intercellular CO 2 concentration in this species. Shoot Cd content was higher in the halophyte C. maritima reaching 1365 g g −1 dw at 100 M while it was 548 g g −1 dw in B. juncea at the same dose. The translocation factor (TF) was higher for C. maritima than for B. juncea at all external Cd doses. It is concluded that the halophyte C. maritima could be considered as a promising plant material for Cd-phytoextraction.
[Show abstract][Hide abstract] ABSTRACT: Background
Red blood cell (RBC) glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a glycolytic enzyme normally inhibited upon binding to the membrane-spanning protein Band 3, but active when free in the cytosol. Accumulating evidence in other cells indicates that oxidative thiol modifications in cytosolic GAPDH drive this molecule into functional avenues that deviate from glycolysis. This study aimed to investigate the role of GAPDH in oxidative stress–dependent metabolic modulations occurring in SAGM-stored RBCs, to increase the knowledge of the molecular mechanisms affecting RBC survival and viability under blood banking conditions.Study Design and Methods
Membranes and cytosol from CPD SAGM-stored RBCs were subjected to Western blotting with anti-GAPDH at 0, 7, 14, 21, 28, 35, and 42 days of preservation. Immunoreactive bands were excised, digested with trypsin, and analyzed by mass spectrometry for the presence of oxidative posttranslational modifications. GAPDH enzymatic activity was also measured in the cytosolic fraction during storage.ResultsAt 21 days of storage, we demonstrated that cytosolic GAPDH undergoes temporary inactivation due to the formation of an intramolecular disulfide bond between the active-site Cys-152 and nearby Cys-156, a mechanism to rerouting glucose flux toward the pentose phosphate pathway. In addition, an increase in the membrane-bound GAPDH was detected in long-stored RBCs.Conclusion
Reversible inhibition or activation of cytosolic GAPDH may represent a protective strategy against oxidative stress to favor NADPH production in stored RBCs.
[Show abstract][Hide abstract] ABSTRACT: The "New discipline for blood transfusion activities and national production of blood derivatives" (Italian Parliament, October 21st, 2005) brought about a significant update of all regulatory aspects concerning transfusion medicine activities in Italy, including the establishment of a nationally co-ordinated blood system consistent with the autonomy of regional authorities1. The aim was to identify homogeneous standards of quality and safety nationwide1. In this setting, the newly born Italian National Blood Centre was to be responsible for all technical and scientific aspects related to transfusion medicine issues, including (i) blood and blood product self-sufficiency; (ii) blood quality and safety; (iii) appropriate utilisation of blood resources; (iv) accreditation and funding of transfusion medicine activities; (v) a national blood information system; (vi) technology nnnassessment; (vii) external quality assessment; (viii) a national haemovigilance network; (ix) control of medical products deriving from human plasma; (x) inspections and controls of the plasma-derivative industry; (xi) education and scientific research in transfusion medicine, and (xii) promotion of voluntary, non-remunerated, responsible and periodic blood donation1.