Gender-specific changes in energy metabolism and protein degradation as major pathways affected in livers of mice treated with ibuprofen.

Ibuprofen, an inhibitor of prostanoid biosynthesis, is a common pharmacological agent used for the management of pain, inflammation and fever. However, the chronic use of ibuprofen at high doses is associated with increased risk for cardiovascular, renal, gastrointestinal and liver injuries. The underlying mechanisms of ibuprofen-mediated effects on liver remain unclear. To determine the mechanisms and signaling pathways affected by ibuprofen (100 mg/kg/day for seven days), we performed proteomic profiling of male mice liver with quantitative liquid chromatography tandem mass spectrometry (LC-MS/MS) using ten-plex tandem mass tag (TMT) labeling. More than 300 proteins were significantly altered between the control and ibuprofen-treated groups. The data suggests that several major pathways including (1) energy metabolism, (2) protein degradation, (3) fatty acid metabolism and (4) antioxidant system are altered in livers from ibuprofen treated mice. Independent validation of protein changes in energy metabolism and the antioxidant system was carried out by Western blotting and showed sex-related differences. Proteasome and immunoproteasome activity/expression assays showed ibuprofen induced gender-specific proteasome and immunoproteasome dysfunction in liver. The study observed multifactorial gender-specific ibuprofen-mediated effects on mice liver and suggests that males and females are affected differently by ibuprofen

Proteomes of Lactobacillus delbrueckii subsp. bulgaricus LBB.B5 Incubated in Milk at Optimal and Low Temperatures.

We identified the proteins synthesized by Lactobacillus delbrueckii subsp. bulgaricus strain LBB.B5 in laboratory culture medium (MRS) at 37°C and milk at 37 and 4°C. Cell-associated proteins were measured by gel-free, shotgun proteomics using high-performance liquid chromatography coupled with tandem mass spectrophotometry. A total of 635 proteins were recovered from all cultures, among which 72 proteins were milk associated (unique or significantly more abundant in milk). LBB.B5 responded to milk by increasing the production of proteins required for purine biosynthesis, carbohydrate metabolism (LacZ and ManM), energy metabolism (TpiA, PgK, Eno, SdhA, and GapN), amino acid synthesis (MetE, CysK, LBU0412, and AspC) and transport (GlnM and GlnP), and stress response (Trx, MsrA, MecA, and SmpB). The requirement for purines was confirmed by the significantly improved cell yields of L. delbrueckii subsp. bulgaricus when incubated in milk supplemented with adenine and guanine. The L. delbrueckii subsp. bulgaricus-expressed proteome in milk changed upon incubation at 4°C for 5 days and included increased levels of 17 proteins, several of which confer functions in stress tolerance (AddB, UvrC, RecA, and DnaJ). However, even with the activation of stress responses in either milk or MRS, L. delbrueckii subsp. bulgaricus did not survive passage through the murine digestive tract. These findings inform efforts to understand how L. delbrueckii subsp. bulgaricus is adapted to the dairy environment and its implications for its health-benefiting properties in the human digestive tract. IMPORTANCELactobacillus delbrueckii subsp. bulgaricus has a long history of use in yogurt production. Although commonly cocultured with Streptococcus salivarius subsp. thermophilus in milk, fundamental knowledge of the adaptive responses of L. delbrueckii subsp. bulgaricus to the dairy environment and the consequences of those responses on the use of L. delbrueckii subsp. bulgaricus as a probiotic remain to be elucidated. In this study, we identified proteins of L. delbrueckii subsp. bulgaricus LBB.B5 that are synthesized in higher quantities in milk at growth-conducive and non-growth-conductive (refrigeration) temperatures compared to laboratory culture medium and further examined whether those L. delbrueckii subsp. bulgaricus cultures were affected differently in their capacity to survive transit through the murine digestive tract. This work provides novel insight into how a major, food-adapted microbe responds to its primary habitat. Such knowledge can be applied to improve starter culture and yogurt production and to elucidate matrix effects on probiotic performance

Label-free shotgun proteomics and metabolite analysis reveal a significant metabolic shift during citrus fruit development.

Label-free LC-MS/MS-based shot-gun proteomics was used to quantify the differential protein synthesis and metabolite profiling in order to assess metabolic changes during the development of citrus fruits. Our results suggested the occurrence of a metabolic change during citrus fruit maturation, where the organic acid and amino acid accumulation seen during the early stages of development shifted into sugar synthesis during the later stage of citrus fruit development. The expression of invertases remained unchanged, while an invertase inhibitor was up-regulated towards maturation. The increased expression of sucrose-phosphate synthase and sucrose-6-phosphate phosphatase and the rapid sugar accumulation suggest that sucrose is also being synthesized in citrus juice sac cells during the later stage of fruit development

Anopheles stephensi p38 MAPK signaling regulates innate immunity and bioenergetics during Plasmodium falciparum infection.

BackgroundFruit flies and mammals protect themselves against infection by mounting immune and metabolic responses that must be balanced against the metabolic needs of the pathogens. In this context, p38 mitogen-activated protein kinase (MAPK)-dependent signaling is critical to regulating both innate immunity and metabolism during infection. Accordingly, we asked to what extent the Asian malaria mosquito Anopheles stephensi utilizes p38 MAPK signaling during infection with the human malaria parasite Plasmodium falciparum.MethodsA. stephensi p38 MAPK (AsP38 MAPK) was identified and patterns of signaling in vitro and in vivo (midgut) were analyzed using phospho-specific antibodies and small molecule inhibitors. Functional effects of AsP38 MAPK inhibition were assessed using P. falciparum infection, quantitative real-time PCR, assays for reactive oxygen species and survivorship under oxidative stress, proteomics, and biochemical analyses.ResultsThe genome of A. stephensi encodes a single p38 MAPK that is activated in the midgut in response to parasite infection. Inhibition of AsP38 MAPK signaling significantly reduced P. falciparum sporogonic development. This phenotype was associated with AsP38 MAPK regulation of mitochondrial physiology and stress responses in the midgut epithelium, a tissue critical for parasite development. Specifically, inhibition of AsP38 MAPK resulted in reduction in mosquito protein synthesis machinery, a shift in glucose metabolism, reduced mitochondrial metabolism, enhanced production of mitochondrial reactive oxygen species, induction of an array of anti-parasite effector genes, and decreased resistance to oxidative stress-mediated damage. Hence, P. falciparum-induced activation of AsP38 MAPK in the midgut facilitates parasite infection through a combination of reduced anti-parasite immune defenses and enhanced host protein synthesis and bioenergetics to minimize the impact of infection on the host and to maximize parasite survival, and ultimately, transmission.ConclusionsThese observations suggest that, as in mammals, innate immunity and mitochondrial responses are integrated in mosquitoes and that AsP38 MAPK-dependent signaling facilitates mosquito survival during parasite infection, a fact that may attest to the relatively longer evolutionary relationship of these parasites with their invertebrate compared to their vertebrate hosts. On a practical level, improved understanding of the balances and trade-offs between resistance and metabolism could be leveraged to generate fit, resistant mosquitoes for malaria control

A label-free differential quantitative mass spectrometry method for the characterization and identification of protein changes during citrus fruit development

<p>Abstract</p> <p>Background</p> <p>Citrus is one of the most important and widely grown commodity fruit crops. In this study a label-free LC-MS/MS based shot-gun proteomics approach was taken to explore three main stages of citrus fruit development. These approaches were used to identify and evaluate changes occurring in juice sac cells in various metabolic pathways affecting citrus fruit development and quality.</p> <p>Results</p> <p>Protein changes in citrus juice sac cells were identified and quantified using label-free shotgun methodologies. Two alternative methods, differential mass-spectrometry (dMS) and spectral counting (SC) were used to analyze protein changes occurring during earlier and late stages of fruit development. Both methods were compared in order to develop a proteomics workflow that could be used in a non-model plant lacking a sequenced genome. In order to resolve the bioinformatics limitations of EST databases from species that lack a full sequenced genome, we established iCitrus. iCitrus is a comprehensive sequence database created by merging three major sources of sequences (HarvEST:citrus, NCBI/citrus/unigenes, NCBI/citrus/proteins) and improving the annotation of existing unigenes. iCitrus provided a useful bioinformatics tool for the high-throughput identification of citrus proteins. We have identified approximately 1500 citrus proteins expressed in fruit juice sac cells and quantified the changes of their expression during fruit development. Our results showed that both dMS and SC provided significant information on protein changes, with dMS providing a higher accuracy.</p> <p>Conclusion</p> <p>Our data supports the notion of the complementary use of dMS and SC for label-free comparative proteomics, broadening the identification spectrum and strengthening the identification of trends in protein expression changes during the particular processes being compared.</p

A comparison of proteomic, genomic, and osteological methods of archaeological sex estimation

Sex estimation of skeletons is fundamental to many archaeological studies. Currently, three approaches are available to estimate sex–osteology, genomics, or proteomics, but little is known about the relative reliability of these methods in applied settings. We present matching osteological, shotgun-genomic, and proteomic data to estimate the sex of 55 individuals, each with an independent radiocarbon date between 2,440 and 100 cal BP, from two ancestral Ohlone sites in Central California. Sex estimation was possible in 100% of this burial sample using proteomics, in 91% using genomics, and in 51% using osteology. Agreement between the methods was high, however conflicts did occur. Genomic sex estimates were 100% consistent with proteomic and osteological estimates when DNA reads were above 100,000 total sequences. However, more than half the samples had DNA read numbers below this threshold, producing high rates of conflict with osteological and proteomic data where nine out of twenty conditional DNA sex estimates conflicted with proteomics. While the DNA signal decreased by an order of magnitude in the older burial samples, there was no decrease in proteomic signal. We conclude that proteomics provides an important complement to osteological and shotgun-genomic sex estimation

Phosphorylation and Activation of the Plasma Membrane Na+/H+ Exchanger (NHE1) during Osmotic Cell Shrinkage

The Na+/H+ Exchanger isoform 1 (NHE1) is a highly versatile, broadly distributed and precisely controlled transport protein that mediates volume and pH regulation in most cell types. NHE1 phosphorylation contributes to Na+/H+ exchange activity in response to phorbol esters, growth factors or protein phosphatase inhibitors, but has not been observed during activation by osmotic cell shrinkage (OCS). We examined the role of NHE1 phosphorylation during activation by OCS, using an ideal model system, the Amphiuma tridactylum red blood cell (atRBC). Na+/H+ exchange in atRBCs is mediated by an NHE1 homolog (atNHE1) that is 79% identical to human NHE1 at the amino acid level. NHE1 activity in atRBCs is exceptionally robust in that transport activity can increase more than 2 orders of magnitude from rest to full activation. Michaelis-Menten transport kinetics indicates that either OCS or treatment with the phosphatase inhibitor calyculin-A (CLA) increase Na+ transport capacity without affecting transport affinity (Km = 44 mM) in atRBCs. CLA and OCS act non-additively to activate atNHE1, indicating convergent, phosphorylation-dependent signaling in atNHE1 activation. In situ 32P labeling and immunoprecipitation demonstrates that the net phosphorylation of atNHE1 is increased 4-fold during OCS coinciding with a more than 2-order increase in Na+ transport activity. This is the first reported evidence of increased NHE1 phosphorylation during OCS in any vertebrate cell type. Finally, liquid chromatography and mass spectrometry (LC-MS/MS) analysis of atNHE1 immunoprecipitated from atRBC membranes reveals 9 phosphorylated serine/threonine residues, suggesting that activation of atNHE1 involves multiple phosphorylation and/or dephosphorylation events