Oxidized GAPDH transfers S-glutathionylation to a nuclear protein Sirtuin-1 leading to apoptosis.

AIMS: S-glutathionylation is a reversible oxidative modification of protein cysteines that plays a critical role in redox signaling. Glutaredoxin-1 (Glrx), a glutathione-specific thioltransferase, removes protein S-glutathionylation. Glrx, though a cytosolic protein, can activate a nuclear protein Sirtuin-1 (SirT1) by removing its S-glutathionylation. Glrx ablation causes metabolic abnormalities and promotes controlled cell death and fibrosis in mice. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a key enzyme of glycolysis, is sensitive to oxidative modifications and involved in apoptotic signaling via the SirT1/p53 pathway in the nucleus. We aimed to elucidate the extent to which S-glutathionylation of GAPDH and glutaredoxin-1 contribute to GAPDH/SirT1/p53 apoptosis pathway. RESULTS: Exposure of HEK 293T cells to hydrogen peroxide (H2O2) caused rapid S-glutathionylation and nuclear translocation of GAPDH. Nuclear GAPDH peaked 10-15 min after the addition of H2O2. Overexpression of Glrx or redox dead mutant GAPDH inhibited S-glutathionylation and nuclear translocation. Nuclear GAPDH formed a protein complex with SirT1 and exchanged S-glutathionylation to SirT1 and inhibited its deacetylase activity. Inactivated SirT1 remained stably bound to acetylated-p53 and initiated apoptotic signaling resulting in cleavage of caspase-3. We observed similar effects in human primary aortic endothelial cells suggesting the GAPDH/SirT1/p53 pathway as a common apoptotic mechanism. CONCLUSIONS: Abundant GAPDH with its highly reactive-cysteine thiolate may function as a cytoplasmic rheostat to sense oxidative stress. S-glutathionylation of GAPDH may relay the signal to the nucleus where GAPDH trans-glutathionylates nuclear proteins such as SirT1 to initiate apoptosis. Glrx reverses GAPDH S-glutathionylation and prevents its nuclear translocation and cytoplasmic-nuclear redox signaling leading to apoptosis. Our data suggest that trans-glutathionylation is a critical step in apoptotic signaling and a potential mechanism that cytosolic Glrx controls nuclear transcription factors.

Osteocytes control myeloid cell proliferation and differentiation through Gsα-dependent and -independent mechanisms.

Osteocytes, the bone cells embedded in the mineralized matrix, control bone modeling, and remodeling through direct contact with adjacent cells and via paracrine and endocrine factors that affect cells in the bone marrow microenvironment or distant organs. Osteocytes express numerous G protein-coupled receptors (GPCRs) and thus mice lacking the stimulatory subunit of G-protein (Gsα) in osteocytes (Dmp1-GsαKO mice) have abnormal myelopoiesis, osteopenia, and reduced adipose tissue. We previously reported that the severe osteopenia and the changes in adipose tissue present in these mice were mediated by increased sclerostin, which suppress osteoblast functions and promote browning of white adipocytes. Inversely, the myeloproliferation was driven by granulocyte colony-stimulating factor (G-CSF) and administration of neutralizing antibodies against G-CSF only partially restored the myeloproliferation, suggesting that additional osteocyte-derived factors might be involved. We hypothesized that osteocytes secrete Gsα-dependent factor(s) which regulate the myeloid cells proliferation. To identify osteocyte-secreted proteins, we used the osteocytic cell line Ocy454 expressing or lacking Gsα expression (Ocy454-Gsαcont and Ocy454-GsαKO ) to delineate the osteocyte "secretome" and its regulation by Gsα. Here we reported that factors secreted by osteocytes increased the number of myeloid colonies and promoted macrophage proliferation. The proliferation of myeloid cells was further promoted by osteocytes lacking Gsα expression. Myeloid cells can differentiate into bone-resorbing osteoclasts, therefore, we hypothesized that osteocyte-secreted factors might also regulate osteoclastogenesis in a Gsα-dependent manner. Conditioned medium (CM) from Ocy454 (both Gsαcont and GsαKO ) significanlty increased the proliferation of bone marrow mononuclear cells (BMNC) and, at the same time, inhibited their differentiation into mature osteoclasts via a Gsα-dependent mechanism. Proteomics analysis of CM from Ocy454 Gsαcont and GsαKO cells identified neuropilin-1 (Nrp-1) and granulin (Grn) as osteocytic-secreted proteins upregulated in Ocy454-GsαKO cells compared to Ocy454-Gsαcont , whereas semaphorin3A was significantly suppressed. Treatment of Ocy454-Gsαcont cells with recombinant proteins or knockdown of Nrp-1 and Grn in Ocy454-GsαKO cells partially rescued the inhibition of osteoclasts, demonstrating that osteocytes control osteoclasts differentiation through Nrp-1 and Grn which are regulated by Gsα signaling.

Endothelial cell-specific redox gene modulation inhibits angiogenesis but promotes B16F0 tumor growth in mice.

Glutaredoxin-1 (Glrx) is a small cytosolic enzyme that removes S-glutathionylation, glutathione adducts of protein cysteine residues, thus modulating redox signaling and gene transcription. Although Glrx up-regulation prevented endothelial cell (EC) migration and global Glrx transgenic mice had impaired ischemic vascularization, the effects of cell-specific Glrx overexpression remained unknown. Here, we examined the role of EC-specific Glrx up-regulation in distinct models of angiogenesis; namely, hind limb ischemia and tumor angiogenesis. EC-specific Glrx transgenic (EC-Glrx TG) overexpression in mice significantly impaired EC migration in Matrigel implants and hind limb revascularization after femoral artery ligation. Additionally, ECs migrated less into subcutaneously implanted B16F0 melanoma tumors as assessed by decreased staining of EC markers. Despite reduced angiogenesis, EC-Glrx TG mice unexpectedly developed larger tumors compared with control mice. EC-Glrx TG mice showed higher levels of VEGF-A in the tumors, indicating hypoxia, which may stimulate tumor cells to form vascular channels without EC, referred to as vasculogenic mimicry. These data suggest that impaired ischemic vascularization does not necessarily associate with suppression of tumor growth, and that antiangiogenic therapies may be ineffective for melanoma tumors because of their ability to implement vasculogenic mimicry during hypoxia.-Yura, Y., Chong, B. S. H., Johnson, R. D., Watanabe, Y., Tsukahara, Y., Ferran, B., Murdoch, C. E., Behring, J. B., McComb, M. E., Costello, C. E., Janssen-Heininger, Y. M. W., Cohen, R. A., Bachschmid, M. M., Matsui, R. Endothelial cell-specific redox gene modulation inhibits angiogenesis but promotes B16F0 tumor growth in mice.

Altered Domain Structure of the Prion Protein Caused by Cu2+ Binding and Functionally Relevant Mutations: Analysis by Cross-Linking, MS/MS, and NMR.

The cellular isoform of the prion protein (PrPC) serves as precursor to the infectious isoform (PrPSc), and as a cell-surface receptor, which binds misfolded protein oligomers as well as physiological ligands such as Cu2+ ions. PrPC consists of two domains: a flexible N-terminal domain and a structured C-terminal domain. Both the physiological and pathological functions of PrP depend on intramolecular interactions between these two domains, but the specific amino acid residues involved have proven challenging to define. Here, we employ a combination of chemical cross-linking, mass spectrometry, NMR, molecular dynamics simulations, and functional assays to identify residue-level contacts between the N- and C-terminal domains of PrPC. We also determine how these interdomain contacts are altered by binding of Cu2+ ions and by functionally relevant mutations. Our results provide a structural basis for interpreting both the normal and toxic activities of PrP.

Improved mass spectrometry-based activity assay reveals oxidative and metabolic stress as sirtuin-1 regulators.

Sirtuin-1 (SirT1) catalyzes NAD+-dependent protein lysine deacetylation and is a critical regulator of energy and lipid metabolism, mitochondrial biogenesis, apoptosis, and senescence. Activation of SirT1 mitigates metabolic perturbations associated with diabetes and obesity. Pharmacologic molecules, cellular redox, and nutritional states can regulate SirT1 activity. Technical barriers against measuring endogenous SirT1 activity have limited characterization of SirT1 in disease and its activation by small molecules. Herein, we developed a relative quantitative mass spectrometry-based technique for measuring endogenous SirT1 activity (RAMSSAY/RelAtive Mass Spectrometry Sirt1 Activity assaY) in cell and tissue homogenates using a biotin-labeled, acetylated p53-derived peptide as a substrate. We demonstrate that oxidative and metabolic stress diminish SirT1 activity in the hepatic cell line HepG2. Moreover, pharmacologic molecules including nicotinamide and EX-527 attenuate SirT1 activity; purported activators of SirT1, the polyphenol S17834, the polyphenol resveratrol, or the non-polyphenolic Sirtris compound SRT1720, failed to activate endogenous SirT1 significantly. Furthermore, we provide evidence that feeding a high fat high sucrose diet (HFHS) to mice inhibits endogenous SirT1 activity in mouse liver. In summary, we introduce a robust, specific and sensitive mass spectrometry-based assay for detecting and quantifying endogenous SirT1 activity using a biotin-labeled peptide in cell and tissue lysates. With this assay, we determine how pharmacologic molecules and metabolic and oxidative stress regulate endogenous SirT1 activity. The assay may also be adapted for other sirtuin isoforms.

Glycomic and Proteomic Changes in Aging Brain Nigrostriatal Pathway.

Parkinson's disease (PD) is a neurological disorder characterized by the progressive loss of functional dopaminergic neurons in the nigrostriatal pathway in the brain. Although current treatments provide only symptomatic relief, gene therapy has the potential to slow or halt the degeneration of nigrostriatal dopamine neurons in PD patients. Adeno-associated viruses (AAV) are vectors of choice in gene therapy because of their well-characterized safety and efficacy profiles; however, although gene therapy has been successful in preclinical models of the disease, clinical trials in humans have failed to demonstrate efficacy. Significantly, all primary AAV receptors of the virus are glycans. We thus hypothesize that age related changes in glycan receptors of heparan sulfate (HS) proteoglycans (receptor for rAAV2), and/or N-glycans with terminal galactose (receptor for rAAV9) results in poor adeno-associated virus binding in either the striatum or substantia nigra, or both, affecting transduction and gene delivery. To test our hypothesis we analyzed the striatum and substantia nigra for changes in HS, N-glycans and proteomic signatures in young versus aged rat brain striatum and substantia nigra. We observed different brain region-specific HS disaccharide profiles in aged compared with young adult rats for brain region-specific profiles in striatum versus substantia nigra. We observed brain region- and age-specific N-glycan compositional profiles with respect to the terminal galactose units that serve as receptors for AAV9. We also observed brain region-specific changes in protein expression in the aging nigrostriatal pathway. These studies provide insight into age- and brain region-specific changes in glycan receptors and proteome that will inform design of improved viral vectors for Parkinson Disease (PD) gene therapy.

Human Regulatory Protein Ki-1/57 Is a Target of SUMOylation and Affects PML Nuclear Body Formation.

Ki-1/57 is a nuclear and cytoplasmic regulatory protein first identified in malignant cells from Hodgkin's lymphoma. It is involved in gene expression regulation on both transcriptional and mRNA metabolism levels. Ki-1/57 belongs to the family of intrinsically unstructured proteins and undergoes phosphorylation by PKC and methylation by PRMT1. Previous characterization of its protein interaction profile by yeast two-hybrid screening showed that Ki-1/57 interacts with proteins of the SUMOylation machinery, the SUMO E2 conjugating enzyme UBC9 and the SUMO E3 ligase PIAS3, which suggested that Ki-1/57 could be involved with this process. Here we identified seven potential SUMO target sites (lysine residues) on Ki-1/57 sequence and observed that Ki-1/57 is modified by SUMO proteins in vitro and in vivo. We showed that SUMOylation of Ki-1/57 occurred on lysines 213, 276, and 336. In transfected cells expressing FLAG-Ki-1/57 wild-type, its paralog FLAG-CGI-55 wild-type, or their non-SUMOylated triple mutants, the number of PML-nuclear bodies (PML-NBs) is reduced compared with the control cells not expressing the constructs. More interestingly, after treating cells with arsenic trioxide (As2O3), the number of PML-NBs is no longer reduced when the non-SUMOylated triple mutant Ki-1/57 is expressed, suggesting that the SUMOylation of Ki-1/57 has a role in the control of As2O3-induced PML-NB formation. A proteome-wide analysis of Ki-1/57 partners in the presence of either SUMO-1 or SUMO-2 suggests that the involvement of Ki-1/57 with the regulation of gene expression is independent of the presence of either SUMO-1 or SUMO-2; however, the presence of SUMO-1 strongly influences the interaction of Ki-1/57 with proteins associated with cellular metabolism, maintenance, and cell cycle.

Microfluidic Capillary Electrophoresis-Mass Spectrometry for Analysis of Monosaccharides, Oligosaccharides, and Glycopeptides.

Glycomics and glycoproteomics analyses by mass spectrometry require efficient front-end separation methods to enable deep characterization of heterogeneous glycoform populations. Chromatography methods are generally limited in their ability to resolve glycoforms using mobile phases that are compatible with online liquid chromatography-mass spectrometry (LC-MS). The adoption of capillary electrophoresis-mass spectrometry methods (CE-MS) for glycomics and glycoproteomics is limited by the lack of convenient interfaces for coupling the CE devices to mass spectrometers. Here, we describe the application of a microfluidics-based CE-MS system for analysis of released glycans, glycopeptides and monosaccharides. We demonstrate a single CE method for three different modalities, thus contributing to comprehensive glycoproteomics analyses. In addition, we explored compatible sample derivatization methods. We used glycan TMT-labeling to improve electrophoretic migration and enable multiplexed quantitation by tandem MS. We used sialic acid linkage-specific derivatization methods to improve separation and the level of information obtained from a single analytical step. Capillary electrophoresis greatly improved glycoform separation for both released glycans and glycopeptides over that reported for chromatography modes more frequently employed for such analyses. Overall, the CE-MS method described here enables rapid setup and analysis of glycans and glycopeptides using mass spectrometry.

Comparative Proteomics Reveals Dysregulated Mitochondrial O-GlcNAcylation in Diabetic Hearts.

O-linked ß-N-acetylglucosamine (O-GlcNAc), a post-translational modification on serine and threonine residues of many proteins, plays crucial regulatory roles in diverse biological events. As a nutrient sensor, O-GlcNAc modification (O-GlcNAcylation) on nuclear and cytoplasmic proteins underlies the pathology of diabetic complications including cardiomyopathy. However, mitochondrial O-GlcNAcylation, especially in response to chronic hyperglycemia in diabetes, has been poorly explored. We performed a comparative O-GlcNAc profiling of mitochondria from control and streptozotocin (STZ)-induced diabetic rat hearts by using an improved ß-elimination/Michael addition with isotopic DTT reagents (BEMAD) followed by tandem mass spectrometric analysis. In total, 86 mitochondrial proteins, involved in diverse pathways, were O-GlcNAcylated. Among them, many proteins have site-specific alterations in O-GlcNAcylation in response to diabetes, which suggests that protein O-GlcNAcylation is a novel layer of regulation mediating adaptive changes in mitochondrial metabolism during the progression of diabetic cardiomyopathy.

O-Linked N-Acetylglucosamine (O-GlcNAc) Transferase and O-GlcNAcase Interact with Mi2ß Protein at the Aγ-Globin Promoter.

One mode of γ-globin gene silencing involves a GATA-1·FOG-1·Mi2ß repressor complex that binds to the -566 GATA site relative to the (A)γ-globin gene cap site. However, the mechanism of how this repressor complex is assembled at the -566 GATA site is unknown. In this study, we demonstrate that the O-linked N-acetylglucosamine (O-GlcNAc) processing enzymes, O-GlcNAc-transferase (OGT) and O-GlcNAcase (OGA), interact with the (A)γ-globin promoter at the -566 GATA repressor site; however, mutation of the GATA site to GAGA significantly reduces OGT and OGA promoter interactions in ß-globin locus yeast artificial chromosome (ß-YAC) bone marrow cells. When WT ß-YAC bone marrow cells are treated with the OGA inhibitor Thiamet-G, the occupancy of OGT, OGA, and Mi2ß at the (A)γ-globin promoter is increased. In addition, OGT and Mi2ß recruitment is increased at the (A)γ-globin promoter when γ-globin becomes repressed in postconception day E18 human ß-YAC transgenic mouse fetal liver. Furthermore, we show that Mi2ß is modified with O-GlcNAc, and both OGT and OGA interact with Mi2ß, GATA-1, and FOG-1. Taken together, our data suggest that O-GlcNAcylation is a novel mechanism of γ-globin gene regulation mediated by modulating the assembly of the GATA-1·FOG-1·Mi2ß repressor complex at the -566 GATA motif within the promoter.

Epithelial Mesenchymal Transition Induces Aberrant Glycosylation through Hexosamine Biosynthetic Pathway Activation.

Deregulated cellular metabolism is a hallmark of tumors. Cancer cells increase glucose and glutamine flux to provide energy needs and macromolecular synthesis demands. Several studies have been focused on the importance of glycolysis and pentose phosphate pathway. However, a neglected but very important branch of glucose metabolism is the hexosamine biosynthesis pathway (HBP). The HBP is a branch of the glucose metabolic pathway that consumes ∼2-5% of the total glucose, generating UDP-GlcNAc as the end product. UDP-GlcNAc is the donor substrate used in multiple glycosylation reactions. Thus, HBP links the altered metabolism with aberrant glycosylation providing a mechanism for cancer cells to sense and respond to microenvironment changes. Here, we investigate the changes of glucose metabolism during epithelial mesenchymal transition (EMT) and the role of O-GlcNAcylation in this process. We show that A549 cells increase glucose uptake during EMT, but instead of increasing the glycolysis and pentose phosphate pathway, the glucose is shunted through the HBP. The activation of HBP induces an aberrant cell surface glycosylation and O-GlcNAcylation. The cell surface glycans display an increase of sialylation α2-6, poly-LacNAc, and fucosylation, all known epitopes found in different tumor models. In addition, modulation of O-GlcNAc levels was demonstrated to be important during the EMT process. Taken together, our results indicate that EMT is an applicable model to study metabolic and glycophenotype changes during carcinogenesis, suggesting that cell glycosylation senses metabolic changes and modulates cell plasticity.

Overexpression of Catalase Diminishes Oxidative Cysteine Modifications of Cardiac Proteins.

Reactive protein cysteine thiolates are instrumental in redox regulation. Oxidants, such as hydrogen peroxide (H2O2), react with thiolates to form oxidative post-translational modifications, enabling physiological redox signaling. Cardiac disease and aging are associated with oxidative stress which can impair redox signaling by altering essential cysteine thiolates. We previously found that cardiac-specific overexpression of catalase (Cat), an enzyme that detoxifies excess H2O2, protected from oxidative stress and delayed cardiac aging in mice. Using redox proteomics and systems biology, we sought to identify the cysteines that could play a key role in cardiac disease and aging. With a 'Tandem Mass Tag' (TMT) labeling strategy and mass spectrometry, we investigated differential reversible cysteine oxidation in the cardiac proteome of wild type and Cat transgenic (Tg) mice. Reversible cysteine oxidation was measured as thiol occupancy, the ratio of total available versus reversibly oxidized cysteine thiols. Catalase overexpression globally decreased thiol occupancy by ≥1.3 fold in 82 proteins, including numerous mitochondrial and contractile proteins. Systems biology analysis assigned the majority of proteins with differentially modified thiols in Cat Tg mice to pathways of aging and cardiac disease, including cellular stress response, proteostasis, and apoptosis. In addition, Cat Tg mice exhibited diminished protein glutathione adducts and decreased H2O2 production from mitochondrial complex I and II, suggesting improved function of cardiac mitochondria. In conclusion, our data suggest that catalase may alleviate cardiac disease and aging by moderating global protein cysteine thiol oxidation.

MALDI-ISD Mass Spectrometry Analysis of Hemoglobin Variants: a Top-Down Approach to the Characterization of Hemoglobinopathies.

Hemoglobinopathies are the most common inherited disorders in humans and are thus the target of screening programs worldwide. Over the past decade, mass spectrometry (MS) has gained a more important role as a clinical means to diagnose variants, and a number of approaches have been proposed for characterization. Here we investigate the use of matrix-assisted laser desorption/ionization time-of-flight MS (MALDI-TOF MS) with sequencing using in-source decay (MALDI-ISD) for the characterization of Hb variants. We explored the effect of matrix selection using super DHB or 1,5-diaminonaphthalene on ISD fragment ion yield and distribution. MALDI-ISD MS of whole blood using super DHB simultaneously provided molecular weights for the alpha and beta chains, as well as extensive fragmentation in the form of sequence defining c-, (z + 2)-, and y-ion series. We observed sequence coverage on the first 70 amino acids positions from the N- and C-termini of the alpha and beta chains in a single experiment. An abundant beta chain N-terminal fragment ion corresponding to ßc34 was determined to be a diagnostic marker ion for Hb S (ß6 Glu→Val, sickle cell), Hb C (ß6 Glu→Lys), and potentially for Hb E (ß26 Glu→Lys). The MALDI-ISD analysis of Hb S and HbSC yielded mass shifts corresponding to the variants, demonstrating the potential for high-throughput screening. Characterization of an alpha chain variant, Hb Westmead (α122 His→Gln), generated fragments that established the location of the variant. This study is the first clinical application of MALDI-ISD MS for the determination and characterization of hemoglobin variants.

Protein Expression by Human Pulmonary Artery Smooth Muscle Cells Containing a BMPR2 Mutation and the Action of ET-1 as Determined by Proteomic Mass Spectrometry.

Pulmonary arterial hypertension (PAH) is a disease characterized by increased pulmonary vascular resistance and remodeling. Increase in the population of vascular smooth muscle cells is among the key events contributing to the remodeling. Endothelin-1 (ET-1), a potent vasoconstrictor, is linked to the etiology and progression of PAH. Here we analyze changes in protein expressions in response to ET-1 in pulmonary arterial smooth muscle cells (PASMC) from a healthy Control (non-PAH) and a PAH subject presenting a bone morphogenetic protein type II receptor (BMPR2) mutation with exon 1-8 deletion. Protein expressions were analyzed by proteomic mass spectrometry using label-free quantitation and the correlations were subjected to Ingenuity™ Pathway Analysis. The results point to eIF2/mTOR/p70S6K, RhoA/actin cytoskeleton/integrin and protein unbiquitination as canonical pathways whose protein expressions increase with the development of PAH. These pathways have an intimal function in the PAH-related physiology of smooth muscle proliferation, apoptosis, contraction and cellular stress. Exposure of the cells to ET-1 further increases protein expression within these pathways. Thus our results show changes in signaling pathways as a consequence of PAH and the effect of ET-1 interference on Control and PAH-affected cells.

Characterization of the human NEK7 interactome suggests catalytic and regulatory properties distinct from those of NEK6.

Human NEK7 is a regulator of cell division and plays an important role in growth and survival of mammalian cells. Human NEK6 and NEK7 are closely related, consisting of a conserved C-terminal catalytic domain and a nonconserved and disordered N-terminal regulatory domain, crucial to mediate the interactions with their respective proteins. Here, in order to better understand NEK7 cellular functions, we characterize the NEK7 interactome by two screening approaches: one using a yeast two-hybrid system and the other based on immunoprecipitation followed by mass spectrometry analysis. These approaches led to the identification of 61 NEK7 interactors that contribute to a variety of biological processes, including cell division. Combining additional interaction and phosphorylation assays from yeast two-hybrid screens, we validated CC2D1A, TUBB2B, MNAT1, and NEK9 proteins as potential NEK7 interactors and substrates. Notably, endogenous RGS2, TUBB, MNAT1, NEK9, and PLEKHA8 localized with NEK7 at key sites throughout the cell cycle, especially during mitosis and cytokinesis. Furthermore, we obtained evidence that the closely related kinases NEK6 and NEK7 do not share common interactors, with the exception of NEK9, and display different modes of protein interaction, depending on their N- and C-terminal regions, in distinct fashions. In summary, our work shows for the first time a comprehensive NEK7 interactome that, combined with functional in vitro and in vivo assays, suggests that NEK7 is a multifunctional kinase acting in different cellular processes in concert with cell division signaling and independently of NEK6.

Does reversible cysteine oxidation link the Western diet to cardiac dysfunction?

Using a novel cysteine thiol labeling strategy coupled with mass spectrometric analysis, we identified and quantified the changes in global reversible cysteine oxidation of proteins in the left ventricle of hearts from mice with metabolic syndrome-associated diastolic dysfunction. This phenotype was induced by feeding a high-fat, high-sucrose, type-2 diabetogenic diet to C57BL/6J mice for 8 mo. The extent of reversible thiol oxidation in relationship to the total available (free and reducible) level of each cysteine could be confidently determined for 173 proteins, of which 98 contained cysteines differentially modified ≥1.5-fold by the diet. Our findings suggest that the metabolic syndrome leads to potentially deleterious changes in the oxidative modification of metabolically active proteins. These alterations may adversely regulate energy substrate flux through glycolysis, ß-oxidation, citric acid (TCA) cycle, and oxidative phosphorylation (oxphos), thereby contributing to maladaptive tissue remodeling that is associated with, and possibly contributing to, diastolic left ventricular dysfunction.

Epithelial wounds induce differential phosphorylation changes in response to purinergic and EGF receptor activation.

Protein phosphorylation is a dynamic post-translational modification. Mass spectrometry-based quantitation was performed to determine the phosphoproteome profile of epithelial cells in response to injury, nucleotide, or epidermal growth factor. Phosphotyrosine enrichment used immunoprecipitation and immobilized metal affinity chromatography. Nucleotides released after scratch wounding activate purinergic receptors, leading to a distinct phosphorylation profile on epidermal growth factor receptor (EGFR) compared with its natural ligand. ATP induced a 2- to 15-fold phosphorylation increase over control on EGFR Y974, Y1086, and Y1148, with minimal phosphorylation intensity on EGFR Y1173 compared with the level measured in response to epidermal growth factor. Differential phosphorylation induced by epidermal growth factor or ATP was site specific on Src, Shc, phospholipase Cγ, protein kinase C, focal adhesion kinase, paxillin, and mitogen-activated protein kinases 1, 12, and 13. After wounding, the P2Y2 receptor mRNA expression increased, and after knockdown, migration and Ca(2+) mobilization were impaired. To examine phosphorylation mediated by P2Y2, cells were cultured in media containing stable isotope-labeled amino acids, the receptor was knocked down, and the cells were stimulated. Mass spectrometry-based comparison of the phosphorylation profiles of control versus transfected cells revealed a 50-fold decrease in phosphorylation of EGFR Y974 and 1086, with no decrease in Y1173 phosphorylation. A similarfold decrease in Src Y421 and Y446 and paxillin Y118 was detected, indicating the far-reaching importance of the P2Y2 receptor in mediating migration.

The aryl hydrocarbon receptor directs hematopoietic progenitor cell expansion and differentiation.

The evolutionarily conserved aryl hydrocarbon receptor (AhR) has been studied for its role in environmental chemical-induced toxicity. However, recent studies have demonstrated that the AhR may regulate the hematopoietic and immune systems during development in a cell-specific manner. These results, together with the absence of an in vitro model system enabling production of large numbers of primary human hematopoietic progenitor cells (HPs) capable of differentiating into megakaryocyte- and erythroid-lineage cells, motivated us to determine if AhR modulation could facilitate both progenitor cell expansion and megakaryocyte and erythroid cell differentiation. Using a novel, pluripotent stem cell-based, chemically-defined, serum and feeder cell-free culture system, we show that the AhR is expressed in HPs and that, remarkably, AhR activation drives an unprecedented expansion of HPs, megakaryocyte-lineage cells, and erythroid-lineage cells. Further AhR modulation within rapidly expanding progenitor cell populations directs cell fate, with chronic AhR agonism permissive to erythroid differentiation and acute antagonism favoring megakaryocyte specification. These results highlight the development of a new Good Manufacturing Practice-compliant platform for generating virtually unlimited numbers of human HPs with which to scrutinize red blood cell and platelet development, including the assessment of the role of the AhR critical cell fate decisions during hematopoiesis.

STRAP PTM: Software Tool for Rapid Annotation and Differential Comparison of Protein Post-Translational Modifications.

The identification of protein post-translational modifications (PTMs) is an increasingly important component of proteomics and biomarker discovery, but very few tools exist for performing fast and easy characterization of global PTM changes and differential comparison of PTMs across groups of data obtained from liquid chromatography-tandem mass spectrometry experiments. STRAP PTM (Software Tool for Rapid Annotation of Proteins: Post-Translational Modification edition) is a program that was developed to facilitate the characterization of PTMs using spectral counting and a novel scoring algorithm to accelerate the identification of differential PTMs from complex data sets. The software facilitates multi-sample comparison by collating, scoring, and ranking PTMs and by summarizing data visually. The freely available software (beta release) installs on a PC and processes data in protXML format obtained from files parsed through the Trans-Proteomic Pipeline. The easy-to-use interface allows examination of results at protein, peptide, and PTM levels, and the overall design offers tremendous flexibility that provides proteomics insight beyond simple assignment and counting.

Differential Characterization and Classification of Tissue Specific Glycosaminoglycans by Tandem Mass Spectrometry and Statistical Methods.

The biological functions of glycoconjugate glycans arise in the context of structural heterogeneity resulting from non-template driven biosynthetic reactions. Such heterogeneity is particularly apparent for the glycosaminoglycan (GAG) classes, of which heparan sulfate (HS) is of particular interest for its properties in binding to many classes of growth factors and growth factor receptors. The structures of HS chains vary according to spatial and temporal factors in biological systems as a mechanism where by the functions of the relatively limited number of associated proteoglycan core proteins is elaborated. Thus, there is a strong driver for the development of methods to discover functionally relevant structures in HS preparations for different sources. In the present work, a set of targeted tandem mass spectra were acquired in automated mode on HS oligosaccharides deriving from two different tissue sources. Statistical methods were used to determine the precursor and product ions, the abundances of which differentiate between the tissue sources. The results demonstrate considerable potential for using this approach to constrain the number of positional glycoform isomers present in different biological preparations toward the end of discovery of functionally relevant structures.