No abstract

NF-κB is constitutively activated in primary human thyroid tumors, particularly in those of anaplastic type. The inhibition of NF-κB activity in the human anaplastic thyroid carcinoma cell line, FRO, leads to an increased susceptibility to chemotherapeutic drug-induced apoptosis and to the blockage of their ability to form tumors in nude mice. To identify NF-κB target genes involved in thyroid cancer, we analyzed the secretome of conditioned media from parental and NF-κB-null FRO cells. Proteomic analysis revealed that the neutrophil gelatinase-associated lipocalin (NGAL), a protein involved in inflammatory and immune responses, is secreted by FRO cells whereas its expression is strongly reduced in the NF-κB-null FRO cells. NGAL is highly expressed in human thyroid carcinomas, and knocking down its expression blocks the ability of FRO cells to grow in soft agar and form tumors in nude mice. These effects are reverted by the addition of either recombinant NGAL or FRO conditioned medium. In addition, we show that the prosurvival activity of NGAL is mediated by its ability to bind and transport iron inside the cells. Our data suggest that NF-κB contributes to thyroid tumor cell survival by controlling iron uptake via NGAL.

In adult vertebrates, most cells are not in the cell cycle at any one time. Physiological nonproliferation states encompass reversible quiescence and permanent postmitotic conditions such as terminal differentiation and replicative senescence. Although these states appear to be attained and maintained quite differently, they might share a core proliferation-restricting mechanism. Unexpectedly, we found that all sorts of nonproliferating cells can be mitotically reactivated by the sole suppression of histotype-specific cyclin-dependent kinase (cdk) inhibitors (CKIs) in the absence of exogenous mitogens. RNA interference–mediated suppression of appropriate CKIs efficiently triggered DNA synthesis and mitosis in established and primary terminally differentiated skeletal muscle cells (myotubes), quiescent human fibroblasts, and senescent human embryo kidney cells. In serum-starved fibroblasts and myotubes alike, cell cycle reactivation was critically mediated by the derepression of cyclin D–cdk4/6 complexes. Thus, both temporary and permanent growth arrest must be actively maintained by the constant expression of CKIs, whereas the cell cycle–driving cyclins are always present or can be readily elicited. In principle, our findings could find wide application in biotechnology and tissue repair whenever cell proliferation is limiting.

The flagellated protozoan Giardia duodenalis (syn. lamblia or intestinalis) has been chosen as a model parasite to further investigate the multifunctional 14-3-3s, a family of highly conserved eukaryotic proteins involved in many cellular processes, such as cell cycle, differentiation, apoptosis, and signal transduction pathways. We confirmed the presence of a single 14-3-3 homolog gene (g14-3-3) by an in silico screening of the complete genome of Giardia, and we demonstrated its constitutive transcription throughout the life stages of the parasite. We cloned and expressed the g14-3-3 in bacteria, and by protein-protein interaction assays we demonstrated that it is a functional 14-3-3. Using an anti-peptide antibody raised against a unique 18-amino acid sequence at the N terminus, we observed variations both in the intracellular localization and in the molecular size of the native g14-3-3 during the conversion of Giardia from trophozoites to the cyst stage. An affinity chromatography, based on the 14-3-3 binding to the polypeptide difopein, was set to purify the native g14-3-3. By matrix-assisted laser desorption ionization mass spectroscopy analysis, we showed that polyglycylation, an unusual post-translational modification described only for tubulin, occurred at the extreme C terminus of the native g14-3-3 on Glu 246 , Glu 247 , or both and that the Thr 214 , located in the loop between helices 8 and 9, is phosphorylated. We propose that the addition of the polyglycine chain can promote the binding of g14-3-3 to alternative ligands and that the differential rate of polyglycylation/deglycylation during the encystation process can act as a novel mechanism to regulate the intracellular localization of g14-3-3.

The worldwide prevalence of obesity-associated pathologies, including type 2 diabetes, requires thorough investigation of mechanisms and interventions. Recent studies have highlighted thyroid hormone analogs and derivatives as potential agents able to counteract such pathologies. In this study, in rats receiving a high-fat diet (HFD), we analyzed the effects of a 4-wk daily administration of a naturally occurring iodothyronine, 3,5-diiodo-L-thyronine (T2), on the gastrocnemius muscle metabolic/structural phenotype and insulin signaling. The HFD-induced increases in muscle levels of fatty acid translocase (3-fold; P<0.05) and TGs (2-fold, P<0.05) were prevented by T2 (each; P<0.05 vs. HFD). T2 increased insulin-stimulated Akt phosphorylation levels (∼2.5-fold; P<0.05 vs. HFD). T2 induced these effects while sparing muscle mass and without cardiac hypertrophy. T2 increased the muscle contents of fast/glycolytic fibers (2-fold; P<0.05 vs. HFD) and sarcolemmal glucose transporter 4 (3-fold; P<0.05 vs. HFD). Adipocyte differentiation-related protein was predominantly present within the slow/oxidative fibers in HFD-T2. In T2-treated rats (vs. HFD), glycolytic enzymes and associated components were up-regulated (proteomic analysis, significance limit: 2-fold; P<0.05), as was phosphofructokinase activity (by 1.3-fold; P<0.05), supporting the metabolic shift toward a more glycolytic phenotype. These results highlight T2 as a potential therapeutic approach to the treatment of diet-induced metabolic dysfunctions.

Water buffalo has been studied in relation to the exclusive use of its milk for the manufacture of high-quality dairy products. Buffalo milk presents physicochemical features different from that of other ruminant species, such as a higher content of fatty acids and proteins. We report here a detailed proteomic analysis of buffalo skim milk, whey and milk fat globule membrane fractions. Notwithstanding the poor information available on buffalo genome, identification of protein isoforms corresponding to 72 genes was achieved by a combined approach based on 2-DE/ MALDI-TOF PMF and 1-DE/mLC-ESI-IT-MS-MS. Major protein components, i.e. a Sl -, a S2 -, b-, kcaseins, a-lactalbumin and b-lactoglobulin, were characterized for PTM, providing a scientific basis to coagulation/cheese making processes used in dairy productions. Minor proteins detected emphasized the multiple functions of milk, which besides affording nutrition to the newborn through its major components, also promotes development and digestive tract protection in the neonate, and ensures optimal mammary gland function in the mother. Defense against pathogens is guaranteed by an arsenal of antimicrobial/immunomodulatory proteins, which are directly released in milk or occur on the surface of secreted milk-lipid droplets. Proteins associated with cell signaling or membrane/protein trafficking functions were also identified, providing putative insights into major secretory pathways in mammary epithelial cells.

The Maillard reaction includes a complex network of processes affecting food and biopharmaceutical products; it also occurs in living organisms and has been strictly related to cell aging, to the pathogenesis of several (chronic) diseases, such as diabetes, uremia, cataract, liver cirrhosis and various neurodegenerative pathologies, as well as to peritoneal dialysis treatment. Dozens of compounds are involved in this process, among which a number of protein-adducted derivatives that have been simplistically defined as early, intermediate and advanced glycation end-products. In the last decade, various bottom-up proteomic approaches have been successfully used for the identification of glycation/glycoxidation protein targets as well as for the characterization of the corresponding adducts, including assignment of the modified amino acids. This article provides an updated overview of the mass spectrometry-based procedures developed to this purpose, emphasizing their partial limits with respect to current proteomic approaches for the analysis of other post-translational modifications. These limitations are mainly related to the concomitant sheer diversity, chemical complexity, and variable abundance of the various derivatives to be characterized. Some challenges to scientists are finally proposed for future proteomic investigations to solve main drawbacks in this research field.

Peroxynitrite is a strong oxidant involved in cell injury. In tissues, most of peroxynitrite reacts preferentially with CO2 or hemoproteins, and these reactions affect its fate and toxicity. CO2 promotes tyrosine nitration but reduces the lifetime of peroxynitrite, preventing, at least in part, membrane crossing. The role of hemoproteins is not easily predictable, because the heme intercepts peroxynitrite, but its oxidation to ferryl species and tyrosyl radical(s) may catalyze tyrosine nitration. The modifications induced by peroxynitrite/CO2 on oxyhemoglobin were determined by mass spectrometry, and we found that αTyr42, βTyr130, and, to a lesser extent, αTyr24 were nitrated. The suggested nitration mechanism is tyrosyl radical formation by long-range electron transfer to ferrylhemoglobin followed by a reaction with •NO2. Dityrosine (α24−α42) and disulfides (β93−β93 and α104−α104) were also detected, but these cross-linkings were largely due to modifications occurring under the denaturing conditions employed for mass spectrometry. Moreover, immunoelectrophoretic techniques showed that the 3-nitrotyrosine content of oxyhemoglobin sharply increased only in molar excess of peroxynitrite, thus suggesting that this hemoprotein is not a catalyst of nitration. The noncatalytic role may be due to the formation of the nitrating species •NO2 mainly in molar excess of peroxynitrite. In agreement with this hypothesis, oxyhemoglobin strongly inhibited tyrosine nitration of a target dipeptide (Ala−Tyr) and of membrane proteins from ghosts resealed with oxyhemoglobin. Erythrocytes were poor inhibitors of Ala−Tyr nitration on account of the membrane barrier. However, at the physiologic hematocrit, Ala−Tyr nitration was reduced by 65%. This “sink” function was facilitated by the huge amount of band 3 anion exchanger on the cell membrane. We conclude that in blood oxyhemoglobin is a peroxynitrite scavenger of physiologic relevance.