The isoprene-responsive phosphoproteome provides new insights into the putative signalling pathways and novel roles of isoprene.

Many plants, especially trees, emit isoprene in a highly light- and temperature-dependent manner. The advantages for plants that emit, if any, have been difficult to determine. Direct effects on membranes have been disproven. New insights have been obtained by RNA sequencing, proteomic and metabolomic studies. We determined the responses of the phosphoproteome to exposure of Arabidopsis leaves to isoprene in the gas phase for either 1 or 5 h. Isoprene effects that were not apparent from RNA sequencing and other methods but were apparent in the phosphoproteome include effects on chloroplast movement proteins and membrane remodelling proteins. Several receptor kinases were found to have altered phosphorylation levels. To test whether potential isoprene receptors could be identified, we used molecular dynamics simulations to test for proteins that might have strong binding to isoprene and, therefore might act as receptors. Although many Arabidopsis proteins were found to have slightly higher binding affinities than a reference set of Homo sapiens proteins, no specific receptor kinase was found to have a very high binding affinity. The changes in chloroplast movement, photosynthesis capacity and so forth, found in this work, are consistent with isoprene responses being especially useful in the upper canopy of trees.

Interaction of intramembrane metalloprotease SpoIVFB with substrate Pro-σK.

Intramembrane proteases (IPs) cleave membrane-associated substrates in nearly all organisms and regulate diverse processes. A better understanding of how these enzymes interact with their substrates is necessary for rational design of IP modulators. We show that interaction of Bacillus subtilis IP SpoIVFB with its substrate Pro-σK depends on particular residues in the interdomain linker of SpoIVFB. The linker plus either the N-terminal membrane domain or the C-terminal cystathione-ß-synthase (CBS) domain of SpoIVFB was sufficient for the interaction but not for cleavage of Pro-σK Chemical cross-linking and mass spectrometry of purified, inactive SpoIVFB-Pro-σK complex indicated residues of the two proteins in proximity. A structural model of the complex was built via partial homology and by using constraints based on cross-linking data. In the model, the Proregion of Pro-σK loops into the membrane domain of SpoIVFB, and the rest of Pro-σK interacts extensively with the linker and the CBS domain of SpoIVFB. The extensive interaction is proposed to allow coordination between ATP binding by the CBS domain and Pro-σK cleavage by the membrane domain.

Dynamic changes in ribosome-associated proteome and phosphoproteome during deoxynivalenol-induced translation inhibition and ribotoxic stress.

Deoxynivalenol (DON), a trichothecene mycotoxin produced by Fusarium that commonly contaminates cereal-based food, interacts with the ribosome to cause translation inhibition and activate stress kinases in mononuclear phagocytes via the ribotoxic stress response (RSR). The goal of this study was to test the hypothesis that the ribosome functions as a platform for spatiotemporal regulation of translation inhibition and RSR. Specifically, we employed stable isotope labeling of amino acids in cell culture (SILAC)-based proteomics to quantify the early (≤ 30 min) DON-induced changes in ribosome-associated proteins in RAW 264.7 murine macrophage. Changes in the proteome and phosphoproteome were determined using off-gel isoelectric focusing and titanium dioxide chromatography, respectively, in conjunction with LC-MS/MS. Following exposure of RAW 264.7 to a toxicologically relevant concentration of DON (250 ng/ml), we observed an overall decrease in translation-related proteins interacting with the ribosome, concurrently with a compensatory increase in proteins that mediate protein folding, biosynthesis, and cellular organization. Alterations in the ribosome-associated phosphoproteome reflected proteins that modulate translational and transcriptional regulation, and others that converged with signaling pathways known to overlap with phosphorylation changes characterized previously in intact RAW 264.7 cells. These results suggest that the ribosome plays a central role as a hub for association and phosphorylation of proteins involved in the coordination of early translation inhibition as well as recruitment and maintenance of stress-related proteins-both of which enable cells to adapt and respond to ribotoxin exposure. This study provides a template for elucidating the molecular mechanisms of DON and other ribosome-targeting agents.

Early phosphoproteomic changes in the mouse spleen during deoxynivalenol-induced ribotoxic stress.

The trichothecene mycotoxin deoxynivalenol (DON) targets the innate immune system and is of public health significance because of its frequent presence in human and animal food. DON-induced proinflammatory gene expression and apoptosis in the lymphoid tissue have been associated with a ribotoxic stress response (RSR) that involves rapid phosphorylation of mitogen-activated protein kinases (MAPKs). To better understand the relationship between protein phosphorylation and DON's immunotoxic effects, stable isotope dimethyl labeling-based proteomics in conjunction with titanium dioxide chromatography was employed to quantitatively profile the immediate (≤ 30min) phosphoproteome changes in the spleens of mice orally exposed to 5mg/kg body weight DON. A total of 90 phosphoproteins indicative of novel phosphorylation events were significantly modulated by DON. In addition to critical branches and scaffolds of MAPK signaling being affected, DON exposure also altered phosphorylation of proteins that mediate phosphatidylinositol 3-kinase/AKT pathways. Gene ontology analysis revealed that DON exposure affected biological processes such as cytoskeleton organization, regulation of apoptosis, and lymphocyte activation and development, which likely contribute to immune dysregulation associated with DON-induced RSR. Consistent with these findings, DON impacted phosphorylation of proteins within diverse immune cell populations, including monocytes, macrophages, T cells, B cells, dendritic cells, and mast cells. Fuzzy c-means clustering analysis further indicated that DON evoked several distinctive temporal profiles of regulated phosphopeptides. Overall, the findings from this investigation can serve as a template for future focused exploration and modeling of cellular responses associated with the immunotoxicity evoked by DON and other ribotoxins.

eIF4B phosphorylation by pim kinases plays a critical role in cellular transformation by Abl oncogenes.

Alterations in translation occur in cancer cells, but the precise pathogenic processes and mechanistic underpinnings are not well understood. In this study, we report that interactions between Pim family kinases and the translation initiation factor eIF4B are critical for Abl oncogenicity. Pim kinases, Pim-1 and Pim-2, both directly phosphorylated eIF4B on Ser406 and Ser422. Phosphorylation of eIF4B on Ser422 was highly sensitive to pharmacologic or RNA interference-mediated inhibition of Pim kinases. Expression and phosphorylation of eIF4B relied upon Abl kinase activity in both v-Abl- and Bcr-Abl-expressing leukemic cells based on their blockade by the Abl kinase inhibitor imatinib. Ectopic expression of phosphomimetic mutants of eIF4B conferred resistance to apoptosis by the Pim kinase inhibitor SMI-4a in Abl-transformed cells. In contrast, silencing eIF4B sensitized Abl-transformed cells to imatinib-induced apoptosis and also inhibited their growth as engrafted tumors in nude mice. Extending these observations, we found that primary bone marrow cells derived from eIF4B-knockdown transgenic mice were less susceptible to Abl transformation, relative to cells from wild-type mice. Taken together, our results identify eIF4B as a critical substrate of Pim kinases in mediating the activity of Abl oncogenes, and they highlight eIF4B as a candidate therapeutic target for treatment of Abl-induced cancers.

Global protein phosphorylation dynamics during deoxynivalenol-induced ribotoxic stress response in the macrophage.

Deoxynivalenol (DON), a trichothecene mycotoxin produced by Fusarium that commonly contaminates food, is capable of activating mononuclear phagocytes of the innate immune system via a process termed the ribotoxic stress response (RSR). To encapture global signaling events mediating RSR, we quantified the early temporal (≤30min) phosphoproteome changes that occurred in RAW 264.7 murine macrophage during exposure to a toxicologically relevant concentration of DON (250ng/mL). Large-scale phosphoproteomic analysis employing stable isotope labeling of amino acids in cell culture (SILAC) in conjunction with titanium dioxide chromatography revealed that DON significantly upregulated or downregulated phosphorylation of 188 proteins at both known and yet-to-be functionally characterized phosphosites. DON-induced RSR is extremely complex and goes far beyond its prior known capacity to inhibit translation and activate MAPKs. Transcriptional regulation was the main target during early DON-induced RSR, covering over 20% of the altered phosphoproteins as indicated by Gene Ontology annotation and including transcription factors/cofactors and epigenetic modulators. Other biological processes impacted included cell cycle, RNA processing, translation, ribosome biogenesis, monocyte differentiation and cytoskeleton organization. Some of these processes could be mediated by signaling networks involving MAPK-, NFκB-, AKT- and AMPK-linked pathways. Fuzzy c-means clustering revealed that DON-regulated phosphosites could be discretely classified with regard to the kinetics of phosphorylation/dephosphorylation. The cellular response networks identified provide a template for further exploration of the mechanisms of trichothecenemycotoxins and other ribotoxins, and ultimately, could contribute to improved mechanism-based human health risk assessment.

Proteomic and biochemical evidence support a role for transport vesicles and endosomes in stress response and secondary metabolism in Aspergillus parasiticus.

Aflatoxin is among the most potent naturally occurring carcinogens known. Previous studies demonstrated that endosomes in the filamentous fungus Aspergillus parasiticus carry enzymes that catalyze the final two steps in aflatoxin synthesis, and these structures also play a role in aflatoxin storage and export. We hypothesized that endosomes house a complete and functional aflatoxin biosynthetic pathway. To address this hypothesis, we purified a cellular fraction containing endosomes, transport vesicles, and vacuoles (V fraction) from A. parasiticus grown under aflatoxin inducing and noninducing conditions. We also added (fed) aflatoxin pathway intermediates to V fraction to test the functional status of aflatoxin pathway enzymes. High throughput LC-MS/MS analysis of proteins in V fraction detected 8 aflatoxin enzymes with high reliability and 8 additional enzymes at lower reliability, suggesting that most aflatoxin pathway enzymes are present. Purified V fraction synthesized aflatoxin and addition of the pathway intermediate versicolorin A increased aflatoxin synthesis, confirming that middle and late aflatoxin enzymes in V fraction are functional. Of particular significance, proteomic and biochemical analysis strongly suggested that additional secondary metabolic pathways as well as proteins involved in response to heat, osmotic, and oxidative stress are housed in V fraction.

An antioxidant response phenotype shared between hereditary and sporadic type 2 papillary renal cell carcinoma.

Fumarate hydratase (FH) mutation causes hereditary type 2 papillary renal cell carcinoma (PRCC2). The main effect of FH mutation is fumarate accumulation. The current paradigm posits that the main consequence of fumarate accumulation is HIF-α stabilization. Paradoxically, FH mutation differs from other HIF-α stabilizing mutations, such as VHL and SDH mutations, in its associated tumor types. We identified that fumarate can directly up-regulate antioxidant response element (ARE)-controlled genes. We demonstrated that aldo-keto reductase family 1 member B10 (AKR1B10) is an ARE-controlled gene and is up-regulated upon FH knockdown as well as in FH null cell lines. AKR1B10 overexpression is also a prominent feature in both hereditary and sporadic PRCC2. This phenotype better explains the similarities between hereditary and sporadic PRCC2.

HOIL-1L interacting protein (HOIP) as an NF-kappaB regulating component of the CD40 signaling complex.

The tumor necrosis factor receptor (TNFR) superfamily mediates signals critical for regulation of the immune system. One family member, CD40, is important for the efficient activation of antibody-producing B cells and other antigen-presenting cells. The molecules and mechanisms that mediate CD40 signaling are only partially characterized. Proteins known to interact with the cytoplasmic domain of CD40 include members of the TNF receptor-associated factor (TRAF) family, which regulate signaling and serve as links to other signaling molecules. To identify additional proteins important for CD40 signaling, we used a combined stimulation/immunoprecipitation procedure to isolate CD40 signaling complexes from B cells and characterized the associated proteins by mass spectrometry. In addition to known CD40-interacting proteins, we detected SMAC/DIABLO, HTRA2/Omi, and HOIP/RNF31/PAUL/ZIBRA. We found that these previously unknown CD40-interacting partners were recruited in a TRAF2-dependent manner. HOIP is a ubiquitin ligase capable of mediating NF-kappaB activation through the ubiquitin-dependent activation of IKKgamma. We found that a mutant HOIP molecule engineered to lack ubiquitin ligase activity inhibited the CD40-mediated activation of NF-kappaB. Together, our results demonstrate a powerful approach for the identification of signaling molecules associated with cell surface receptors and indicate an important role for the ubiquitin ligase activity of HOIP in proximal CD40 signaling.

Protein variations in Listeria monocytogenes exposed to sodium lactate, sodium diacetate, and their combination.

Most studies of the effect of adverse conditions on survival of Listeria monocytogenes have focused on stress caused by acid or sodium chloride. However, no information is available on resistance of this pathogen to stress caused by salts of organic acids. Sodium lactate and sodium diacetate are generally recognized as safe substances and are approved as ingredients for use in foods. We evaluated antilisterial properties of each of these salts and the enhanced inhibition effected by their combination in ready-to-eat meat products at pH 6.3. Changes in proteins found in this pathogen were studied in the presence of the salts in a chemically defined medium at the same pH using a proteomic approach. The total numbers of protein spots obtained from two-dimensional electrophoresis were 198, 150, and 131 for sodium diacetate, sodium lactate, and the control, respectively. Sodium diacetate treatment produced the highest number of unmatched proteins (124 versus 53 in lactate), the greatest increase in expression (20 versus 5 in lactate), and the highest number of novel proteins (90 versus 45 in lactate). The number of repressed proteins was highest in the combination treatment (41 versus -30 in the single salt treatment). Six proteins that increased or decreased by > or = 10-fold were further investigated; oxidoreductase and lipoprotein were upregulated, and DNA-binding protein, alpha amylase, and two SecA proteins were downregulated or completely suppressed by the salt treatment. Identification of all protein spots is essential for comparison with proteins induced or suppressed under other stress conditions.

An experimental strategy for quantitative analysis of the humoral immune response to prostate cancer antigens using natural protein microarrays.

The identification of human tumor antigens has potential utility in the diagnosis and treatment of cancers. We demonstrate here a complete strategy to profile immunoreactivity and identify tumor antigens from proteins derived from tumor cell lines. Microarrays of proteins produced from 2-D LC fractionation of prostate tumor cell-line lysates were used to profile immunoreactivity in the sera of prostate cancer patients and control subjects. Cancer-associated immunoreactivity to distinct groups of chromatography fractions was present in about 50% of the patients, with greater immunoreactivity present in patients with non-organ-confined cancer than in patients with organ-confined cancer. We grouped the immunoreactive fractions by similarities in elution order and patterns of immunoreactivity to guide and interpret the MS analysis of selected fractions, which was used to identify the proteins that may be responsible for the immunoreactivity. As a complementary method to further characterize and validate the immunoreactivity of the proteins identified by mass spectrometry, we demonstrate the use of focused microarrays of recombinant proteins. Disease-associated immunoreactivity was confirmed for one of the identified proteins, human Kallikrein 11. These results demonstrate a practical approach to screening, identifying, and validating immunoreactive proteins that could be applied to diverse studies on humoral immune responses.