Collagen induces maturation of human monocyte-derived dendritic cells by signaling through osteoclast-associated receptor.

Osteoclast-associated receptor (OSCAR) is widely expressed on human myeloid cells. Collagen types (Col)I, II, and III have been described as OSCAR ligands, and ColII peptides can induce costimulatory signaling in receptor activator for NF-κB-dependent osteoclastogenesis. In this study, we isolated collagen as an OSCAR-interacting protein from the membranes of murine osteoblasts. We have investigated a functional outcome of the OSCAR-collagen interaction in human monocyte-derived dendritic cells (DCs). OSCAR engagement by ColI/II-induced activation/maturation of DCs is characterized by upregulation of cell surface markers and secretion of cytokines. These collagen-matured DCs (Col-DCs) were efficient drivers of allogeneic and autologous naive T cell proliferation. The T cells expanded by Col-DCs secreted cytokines with no clear T cell polarization pattern. Global RNA profiling revealed that multiple proinflammatory mediators, including cytokines and cytokine receptors, components of the stable immune synapse (namely CD40, CD86, CD80, and ICAM-1), as well as components of TNF and TLR signaling, are transcriptional targets of OSCAR in DCs. Our findings indicate the existence of a novel pathway by which extracellular matrix proteins locally drive maturation of DCs during inflammatory conditions, for example, within synovial tissue of rheumatoid arthritis patients, where collagens become exposed during tissue remodeling and are thus accessible for interaction with infiltrating precursors of DCs.

Hemostatic effect of a monoclonal antibody mAb 2021 blocking the interaction between FXa and TFPI in a rabbit hemophilia model.

Hemophilia is treated by IV replacement therapy with Factor VIII (FVIII) or Factor IX (FIX), either on demand to resolve bleeding, or as prophylaxis. Improved treatment may be provided by drugs designed for subcutaneous and less frequent administration with a reduced risk of inhibitor formation. Tissue factor pathway inhibitor (TFPI) down-regulates the initiation of coagulation by inhibition of Factor VIIa (FVIIa)/tissue factor/Factor Xa (FVIIa/TF/FXa). Blockage of TFPI inhibition may facilitate thrombin generation in a hemophilic setting. A high-affinity (K(D) = 25pM) mAb, mAb 2021, against TFPI was investigated. Binding of mAb 2021 to TFPI effectively prevented inhibition of FVIIa/TF/FXa and improved clot formation in hemophilia blood and plasma. The binding epitope on the Kunitz-type protease inhibitor domain 2 of TFPI was mapped by crystallography, and showed an extensive overlap with the FXa contact region highlighting a structural basis for its mechanism of action. In a rabbit hemophilia model, an intravenous or subcutaneous dose significantly reduced cuticle bleeding. mAb 2021 showed an effect comparable with that of rFVIIa. Cuticle bleeding in the model was reduced for at least 7 days by a single intravenous dose of mAb 2021. This study suggests that neutralization of TFPI by mAb 2021 may constitute a novel treatment option in hemophilia.

Rat liver sinusoidal endothelial cells (LSECs) express functional low density lipoprotein receptor-related protein-1 (LRP-1).

BACKGROUND & AIMS: The low density lipoprotein receptor-related protein-1 (LRP-1) is a large, multifunctional endocytic receptor from the LDL receptor family, highly expressed in liver parenchymal cells (PCs), neurons, activated astrocytes, and fibroblasts. The aim of the study was to investigate if liver sinusoidal endothelial cells (LSECs), highly specialized scavenger cells, express LRP-1. METHODS: To address this question, experiments were performed in vivo and in vitro to determine if receptor associated protein (RAP) and trypsin-activated α(2)-macroglobulin (α(2)M∗) were endocytosed in LSECs. RESULTS: Both ligands were cleared from the circulation mainly by the liver. Hepatocellular distribution of intravenously administered ligands, assessed after magnetic bead cell separation using LSEC- and KC-specific antibodies, showed that PCs contained 93% and 82% of liver-associated (125)I-RAP and (125)I-α(2)M∗, whereas 5% and 11% were associated with LSECs. Uptake of RAP and α(2)M∗ in the different liver cell population in vitro was specific and followed by degradation. The uptake of (125)I-RAP was not inhibited by ligands to known endocytosis receptors in LSECs, while uptake of (125)I-α(2)M∗ was significantly inhibited by RAP, suggesting the involvement of LRP-1. Immunofluorescence using LRP-1 antibody showed positive staining in LSECs. Ligand blot analyses using total cell proteins and (125)I-RAP followed by mass spectrometry further confirmed and identified LRP-1 in LSECs. CONCLUSIONS: LSECs express functional LRP-1. An important implication of our findings is that LSECs contribute to the rapid removal of blood borne ligands for LRP-1 and may thus play a role in lipid homeostasis.

A comparative integrated transcript analysis and functional characterization of differential mechanisms for induction of liver hypertrophy in the rat.

The main goal of the present work was to better understand the molecular mechanisms underlying liver hypertrophy (LH), a recurrent finding observed following acute or repeated drug administration to animals, using transcriptomic technologies together with the results from conventional toxicology methods. Administration of 5 terminated proprietary drug candidates from participating companies involved in the EU Innomed PredTox Project or the reference hepatotoxicant troglitazone to rats for up to a 14-day duration induced LH as the main liver phenotypic toxicity outcome. The integrated analysis of transcriptomic liver expression data across studies turned out to be the most informative approach for the generation of mechanistic models of LH. In response to a xenobiotic stimulus, a marked increase in the expression of xenobiotic metabolizing enzymes (XME) was observed in a subset of 4 studies. Accumulation of these newly-synthesized proteins within the smooth endoplasmic reticulum (SER) would suggest proliferation of this organelle, which most likely is the main molecular process underlying the LH observed in XME studies. In another subset of 2 studies (including troglitazone), a marked up-regulation of genes involved in peroxisomal fatty acid ß-oxidation was noted, associated with induction of genes involved in peroxisome proliferation. Therefore, an increase in peroxisome abundance would be the main mechanism underlying LH noted in this second study subset. Together, the use of transcript profiling provides a means to generate putative mechanistic models underlying the pathogenesis of liver hypertrophy, to distinguish between subtle variations in subcellular organelle proliferation and creates opportunities for improved mechanism-based risk assessment.

The enhanced value of combining conventional and "omics" analyses in early assessment of drug-induced hepatobiliary injury.

The InnoMed PredTox consortium was formed to evaluate whether conventional preclinical safety assessment can be significantly enhanced by incorporation of molecular profiling ("omics") technologies. In short-term toxicological studies in rats, transcriptomics, proteomics and metabolomics data were collected and analyzed in relation to routine clinical chemistry and histopathology. Four of the sixteen hepato- and/or nephrotoxicants given to rats for 1, 3, or 14days at two dose levels induced similar histopathological effects. These were characterized by bile duct necrosis and hyperplasia and/or increased bilirubin and cholestasis, in addition to hepatocyte necrosis and regeneration, hepatocyte hypertrophy, and hepatic inflammation. Combined analysis of liver transcriptomics data from these studies revealed common gene expression changes which allowed the development of a potential sequence of events on a mechanistic level in accordance with classical endpoint observations. This included genes implicated in early stress responses, regenerative processes, inflammation with inflammatory cell immigration, fibrotic processes, and cholestasis encompassing deregulation of certain membrane transporters. Furthermore, a preliminary classification analysis using transcriptomics data suggested that prediction of cholestasis may be possible based on gene expression changes seen at earlier time-points. Targeted bile acid analysis, based on LC-MS metabonomics data demonstrating increased levels of conjugated or unconjugated bile acids in response to individual compounds, did not provide earlier detection of toxicity as compared to conventional parameters, but may allow distinction of different types of hepatobiliary toxicity. Overall, liver transcriptomics data delivered mechanistic and molecular details in addition to the classical endpoint observations which were further enhanced by targeted bile acid analysis using LC/MS metabonomics.

Protocols of two-dimensional difference gel electrophoresis to investigate mechanisms of toxicity.

In recent years, several global omics technologies have been increasingly used to better understand the molecular mechanisms of drug toxicity. Two-dimensional difference gel electrophoresis (2D-DIGE) is a large-scale proteomics high-resolution gel-based quantitative method widely used to detect protein expression alterations after drug treatment. The 2D-DIGE technology is based on the labeling of proteins with different fluorescent dyes, allowing the separation of different samples on the same gel with the use of an internal standard, thus reducing the complexity of spot pattern comparison and providing a reliable method applied to toxicology studies for the detection of modulated proteins in targeted organs.

The sphingolipid long-chain base-Pkh1/2-Ypk1/2 signaling pathway regulates eisosome assembly and turnover.

Eisosomes are recently described fungal structures that play roles in the organization of the plasma membrane and endocytosis. Their major protein components are Pil1 and Lsp1, and previous studies showed that these proteins are phosphorylated by the sphingolipid long-chain base-activated Pkh1 and Pkh2 protein kinases in vitro. We show that Pkh1 and Pkh2 phosphorylate Pil1 and Lsp1 in vivo to produce species B, and that heat stress, which activates Pkh1 and Pkh2, generates a more highly phosphorylated species, C. Cells with low Pkh activity lack species B and C and contain abnormally organized eisosomes. To verify that Pil1 phosphorylation is essential for correct eisosome organization, phosphorylated serine and threonine residues were identified and changed to alanines. A variant Pil1 protein lacking five phosphorylation sites did not form eisosomes during log phase growth, indicating that phosphorylation is critical for eisosome organization. We also found that eisosomes are dynamic structures and disassemble when the Ypk protein kinases, which are activated by the sphingolipid-Pkh signaling pathway, are inactivated or when the sphingolipid signal is pharmacologically blocked with myriocin. We conclude that eisosome formation and turnover are regulated by the sphingolipid-Pkh1/2-Ypk1/2 signaling pathway. These data and previous data showing that endocytosis is regulated by the sphingolipid-Pkh1/2-Ypk1/2 signaling pathway suggest that Pkh1 and -2 respond to changes in membrane sphingolipids and transmit this information to eisosomes via Pil1 phosphorylation. Eisosomes then control endocytosis to align the composition and function of the plasma membrane to match demand.

Quantitation with virtual expert mass spectrometrist.

Quantitative analysis of proteins and peptides by mass spectrometry has been greatly advanced by the development of proteomic technologies within recent years. Particularly, labeling of peptides and proteins with stable isotopes such as 2H, 15N, and 13C facilitated the unbiased comparison of protein amounts in distinct samples in a single mass spectrometric experiment. These methods can be applied to detect quantitative changes in protein amounts and posttranslational modifications such as phosphorylation. Quantitation of mass spectra requires accurate and efficient bioinformatics tools, which can match corresponding peptides, determine peak intensities, and calculate relative amounts. In this chapter, we describe the use of virtual expert mass spectrometrist for the quantitation of mass spectra from samples with peptides and proteins encoded with stable isotopes.

Stable isotope labeling of Arabidopsis thaliana cells and quantitative proteomics by mass spectrometry.

Quantitative analysis of protein expression is an important tool for the examination of complex biological systems. Albeit its importance, quantitative proteomics is still a challenging task because of the high dynamic range of protein amounts in the cell and the variation in the physical properties of proteins. Stable isotope labeling by amino acids in cell culture (SILAC) has been successfully used in yeast and mammalian cells to measure relative protein abundance by mass spectrometry. Here we show for the first time that proteins from Arabidopsis thaliana cell cultures can be selectively isotope-labeled in vivo by growing cells in the presence of a single stable isotope-labeled amino acid. Among the tested amino acids ([2H3]-leucine, [13C6]arginine, and [2H4]lysine), [13C6]arginine proved to be the most suitable. Incorporation of [13C6]arginine into the proteome was homogeneous and reached efficiencies of about 80%. [13C6]Arginine-labeled A. thaliana suspension cells were used to study the regulation of glutathione S-transferase expression in response to abiotic stress caused by salicylic acid and to identify proteins that bind specifically to phosphorylated 14-3-3 binding motifs on synthesized bait peptides in affinity purification experiments. In conclusion, the combination of stable isotope labeling of plant cells and mass spectrometry is a powerful technology that can be applied to study complex biological processes that involve changes in protein expression such as cellular responses to various kinds of stress or activation of cell signaling.

Quantitative phosphoproteomics applied to the yeast pheromone signaling pathway.

Cellular processes such as proliferation, differentiation, and adaptation to environmental changes are regulated by protein phosphorylation. Development of sensitive and comprehensive analytical methods for determination of protein phosphorylation is therefore a necessity in the pursuit of a detailed molecular view of complex biological processes. We present a quantitative modification-specific proteomic approach that combines stable isotope labeling by amino acids in cell culture (SILAC) for quantitation with IMAC for phosphopeptide enrichment and three stages of mass spectrometry (MS/MS/MS) for identification. This integrated phosphoproteomic technology identified and quantified phosphorylation in key regulator and effector proteins of a prototypical G-protein-coupled receptor signaling pathway, the yeast pheromone response. SILAC encoding of yeast proteomes was achieved by incorporation of [(13)C(6)]arginine and [(13)C(6)]lysine in a double auxotroph yeast strain. Pheromone-treated yeast cells were mixed with SILAC-encoded cells as the control and lysed, and extracted proteins were digested with trypsin. Phosphopeptides were enriched by a combination of strong cation exchange chromatography and IMAC. Phosphopeptide fractions were analyzed by LC-MS using a linear ion trap-Fourier transform ion cyclotron resonance mass spectrometer. MS/MS and neutral loss-directed MS/MS/MS analysis allowed detection and sequencing of phosphopeptides with exceptional accuracy and specificity. Of more than 700 identified phosphopeptides, 139 were differentially regulated at least 2-fold in response to mating pheromone. Among these regulated proteins were components belonging to the mitogen-activated protein kinase signaling pathway and to downstream processes including transcriptional regulation, the establishment of polarized growth, and the regulation of the cell cycle.

Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spectrometry.

The recent abundance of genome sequence data has brought an urgent need for systematic proteomics to decipher the encoded protein networks that dictate cellular function. To date, generation of large-scale protein-protein interaction maps has relied on the yeast two-hybrid system, which detects binary interactions through activation of reporter gene expression. With the advent of ultrasensitive mass spectrometric protein identification methods, it is feasible to identify directly protein complexes on a proteome-wide scale. Here we report, using the budding yeast Saccharomyces cerevisiae as a test case, an example of this approach, which we term high-throughput mass spectrometric protein complex identification (HMS-PCI). Beginning with 10% of predicted yeast proteins as baits, we detected 3,617 associated proteins covering 25% of the yeast proteome. Numerous protein complexes were identified, including many new interactions in various signalling pathways and in the DNA damage response. Comparison of the HMS-PCI data set with interactions reported in the literature revealed an average threefold higher success rate in detection of known complexes compared with large-scale two-hybrid studies. Given the high degree of connectivity observed in this study, even partial HMS-PCI coverage of complex proteomes, including that of humans, should allow comprehensive identification of cellular networks.