HATRIC-based identification of receptors for orphan ligands

Technologies for identifying receptor-ligand pairs on living cells at physiological conditions remain scarce. Here, the authors develop a mass spectrometry-based ligand receptor capture technology that can identify receptors for a diverse range of ligands at physiological pH with as few as a million cells

Proteome wide purification and identification of O-GlcNAc-modified proteins using click chemistry and mass spectrometry

The posttranslational modification of proteins with N-acetylglucosamine (O-GlcNAc) is involved in the regulation of a wide variety of cellular processes and associated with a number of chronic diseases. Despite its emerging biological significance, the systematic identification of O-GlcNAc proteins is still challenging. In the present study, we demonstrate a significantly improved O-GlcNAc protein enrichment procedure, which exploits metabolic labelling of cells by azide modified GlcNAc and copper mediated Click chemistry for purification of modified proteins on an alkyne-resin. On-resin proteolysis using trypsin followed by LC-MS/MS afforded the identification of around1500 O-GlcNAc proteins from a single cell line. Subsequent elution of covalently resin bound O-GlcNAc peptides using selective β-elimination enabled the identification of 192 O-GlcNAc modification sites on 82 proteins. To demonstrate the practical utility of the developed approach, we studied the global effects of the O-GlcNAcase inhibitor GlcNAcstatin G on the level of O-GlcNAc modification of cellular proteins. About 200 proteins including several key players involved in the hexosamine signalling pathway showed significantly increased O-GlcNAcylation levels in response to the drug which further strengthens the link of O-GlcNAc protein modification to cellular nutrient sensing and response

Leukocyte differentiation by histidine-rich glycoprotein/stanniocalcin-2 complex regulates murine glioma growth through modulation of anti-tumor immunity

The plasma-protein histidine-rich glycoprotein (HRG) is implicated in phenotypic switching of tumor-associated macrophages, regulating cytokine production and phagocytotic activity, thereby promoting vessel normalization and anti-tumor immune responses. To assess the therapeutic effect of HRG gene delivery on CNS tumors, we used adenovirus-encoded HRG to treat mouse intracranial GL261 glioma. Delivery of Ad5-HRG to the tumor site resulted in a significant reduction in glioma growth, associated with increased vessel perfusion and increased CD45+ leukocyte and CD8+ T cell accumulation in the tumor. Antibody-mediated neutralization of colony-stimulating factor-1 suppressed the effects of HRG on CD45+ and CD8+ infiltration. Using a novel protein interaction-decoding technology, TRICEPS-based ligand receptor capture (LRC), we identified Stanniocalcin-2 (STC2) as an interacting partner of HRG on the surface of inflammatory cells in vitro and co-localization of HRG and STC2 in gliomas. HRG reduced the suppressive effects of STC2 on monocyte CD14+ differentiation and STC2-regulated immune response pathways. In consequence, Ad5-HRG treated gliomas displayed decreased numbers of Interleukin-35+ Treg cells, providing a mechanistic rationale for the reduction in GL261 growth in response to Ad5-HRG delivery. We conclude that HRG suppresses glioma growth by modulating tumor inflammation through monocyte infiltration and differentiation. Moreover, HRG acts to balance the regulatory effects of its partner, STC2, on inflammation and innate and/or acquired immunity. HRG gene delivery therefore offers a potential therapeutic strategy to control anti-tumor immunity.I. Pietilä and H. Kaito contributed equally to this article.</p

Sulforaphane preconditioning sensitizes human colon cancer cells towards the bioreductive anticancer prodrug PR-104A

The chemoprotective properties of sulforaphane (SF), derived from cruciferous vegetables, are widely acknowledged to arise from its potent induction of xenobiotic-metabolizing and antioxidant enzymes. However, much less is known about the impact of SF on the efficacy of cancer therapy through the modulation of drug-metabolizing enzymes. To identify proteins modulated by a low concentration of SF, we treated HT29 colon cancer cells with 2.5 μM SF. Protein abundance changes were detected by stable isotope labeling of amino acids in cell culture. Among 18 proteins found to be significantly up-regulated, aldo-keto reductase 1C3 (AKR1C3), bioactivating the DNA cross-linking prodrug PR-104A, was further characterized. Preconditioning HT29 cells with SF reduced the EC50 of PR-104A 3.6-fold. The increase in PR-104A cytotoxicity was linked to AKR1C3 abundance and activity, both induced by SF in a dose-dependent manner. This effect was reproducible in a second colon cancer cell line, SW620, but not in other colon cancer cell lines where AKR1C3 abundance and activity were absent or barely detectable and could not be induced by SF. Interestingly, SF had no significant influence on PR-104A cytotoxicity in non-cancerous, immortalized human colonic epithelial cell lines expressing either low or high levels of AKR1C3. In conclusion, the enhanced response of PR-104A after preconditioning with SF was apparent only in cancer cells provided that AKR1C3 is expressed, while its expression in non-cancerous cells did not elicit such a response. Therefore, a subset of cancers may be susceptible to combined food-derived component and prodrug treatments with no harm to normal tissues

Proteome Wide Purification and Identification of <i>O</i>‑GlcNAc-Modified Proteins Using Click Chemistry and Mass Spectrometry

The post-translational modification of proteins with <i>N</i>-acetylglucosamine (<i>O</i>-GlcNAc) is involved in the regulation of a wide variety of cellular processes and associated with a number of chronic diseases. Despite its emerging biological significance, the systematic identification of <i>O</i>-GlcNAc proteins is still challenging. In the present study, we demonstrate a significantly improved <i>O</i>-GlcNAc protein enrichment procedure, which exploits metabolic labeling of cells by azide-modified GlcNAc and copper-mediated Click chemistry for purification of modified proteins on an alkyne-resin. On-resin proteolysis using trypsin followed by LC–MS/MS afforded the identification of around 1500 <i>O</i>-GlcNAc proteins from a single cell line. Subsequent elution of covalently resin bound <i>O</i>-GlcNAc peptides using selective β-elimination enabled the identification of 185 <i>O</i>-GlcNAc modification sites on 80 proteins. To demonstrate the practical utility of the developed approach, we studied the global effects of the <i>O</i>-GlcNAcase inhibitor GlcNAcstatin G on the level of <i>O</i>-GlcNAc modification of cellular proteins. About 200 proteins including several key players involved in the hexosamine signaling pathway showed significantly increased <i>O</i>-GlcNAcylation levels in response to the drug, which further strengthens the link of <i>O</i>-GlcNAc protein modification to cellular nutrient sensing and response

Proteome Wide Purification and Identification of <i>O</i>‑GlcNAc-Modified Proteins Using Click Chemistry and Mass Spectrometry

The post-translational modification of proteins with <i>N</i>-acetylglucosamine (<i>O</i>-GlcNAc) is involved in the regulation of a wide variety of cellular processes and associated with a number of chronic diseases. Despite its emerging biological significance, the systematic identification of <i>O</i>-GlcNAc proteins is still challenging. In the present study, we demonstrate a significantly improved <i>O</i>-GlcNAc protein enrichment procedure, which exploits metabolic labeling of cells by azide-modified GlcNAc and copper-mediated Click chemistry for purification of modified proteins on an alkyne-resin. On-resin proteolysis using trypsin followed by LC–MS/MS afforded the identification of around 1500 <i>O</i>-GlcNAc proteins from a single cell line. Subsequent elution of covalently resin bound <i>O</i>-GlcNAc peptides using selective β-elimination enabled the identification of 185 <i>O</i>-GlcNAc modification sites on 80 proteins. To demonstrate the practical utility of the developed approach, we studied the global effects of the <i>O</i>-GlcNAcase inhibitor GlcNAcstatin G on the level of <i>O</i>-GlcNAc modification of cellular proteins. About 200 proteins including several key players involved in the hexosamine signaling pathway showed significantly increased <i>O</i>-GlcNAcylation levels in response to the drug, which further strengthens the link of <i>O</i>-GlcNAc protein modification to cellular nutrient sensing and response

Transcriptomic Responses of Cancerous and Noncancerous Human Colon Cells to Sulforaphane and Selenium

Diets enriched with bioactive food components trigger molecular changes in cells that may contribute to either health-promoting or adverse effects. Recent technological advances in high-throughput data generation allow for observing systems-wide molecular responses to cellular perturbations with nontoxic and dietary-relevant doses while considering the intrinsic differences between cancerous and noncancerous cells. In this chemical profile, we compared molecular responses of the colon cancer cell line HT29 and a noncancerous colon epithelial cell line (HCEC) to two widely encountered food components, sulforaphane and selenium. We conducted this comparison by generating new transcriptome data by microarray gene-expression profiling, analyzing them statistically on the single gene, network, and functional pathway levels, and integrating them with protein expression data. Sulforaphane and selenium, at doses that did not inhibit the growth of the tested cells, induced or repressed the transcription of a limited number of genes in a manner distinctly dependent on the chemical and the cell type. The genes that most strongly responded in cancer cells were observed after treatment with sulforaphane and were members of the aldo-keto reductase (AKR) superfamily. These genes were in high agreement in terms of fold change with their corresponding proteins (correlation coefficient <i>r</i>² = 0.98, <i>p</i> = 0.01). Conversely, selenium had little influence on the cancer cells. In contrast, in noncancerous cells, selenium induced numerous genes involved in apoptotic, angiogenic, or tumor proliferation pathways, whereas the influence of sulforaphane was very limited. These findings contribute to defining the significance of cell type in interpreting human cellular transcriptome-level responses to exposures to natural components of the diet

Fold changes in protein abundance for SF-treated HT29 cells compared to untreated counterparts.^a

<p>Fold changes in protein abundance for SF-treated HT29 cells compared to untreated counterparts.<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150219#t001fn001" target="_blank">^a</a></p

Volcano plot showing significant changes in the proteome following exposure of HT29 cells to 2.5 μM SF for 48 h, determined by SILAC.

<p>2653 proteins (black and gray) were quantified and tested for significance. Among the 23 significantly regulated proteins (black), 18 were up-regulated and 5 down-regulated in abundance.</p

PR-104A and CBL cytotoxicity in colon cell lines pretreated with SF.^a

<p>PR-104A and CBL cytotoxicity in colon cell lines pretreated with SF.<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150219#t002fn001" target="_blank">^a</a></p