Defining TNF-α and IL-1ß induced nascent proteins: combining bio-orthogonal non-canonical amino acid tagging and proteomics.

An impediment in the development of new therapeutic strategies for chronic inflammatory diseases is the limited understanding of underlying molecular mechanisms. The objective of this study was to identify newly synthesized (nascent) proteins induced by critical inflammatory cytokines TNF-α and IL-1ß in human monocytic THP-1 cells. We optimized methods to combine two different approaches, bio-orthogonal non-canonical amino acid tagging (BONCAT) along with proteomics using isobaric tags (iTRAQ). BONCAT employed the incorporation of l-azidohomoalanine (AHA), an analog of methionine, into TNF-α or IL-1ß induced nascent proteins. The AHA-containing nascent proteins were tagged with alkyne-biotin to allow enrichment using avidin affinity purification. The differential expressions of the enriched proteins were further determined using iTRAQ reagents and mass spectrometry (MS). The combination of BONCAT and proteomics represents a unique approach that has uncovered the nascent proteome induced by inflammatory cytokines TNF-α and IL-1ß.

Glia maturation factor gamma regulates the migration and adherence of human T lymphocytes.

BACKGROUND: Lymphocyte migration and chemotaxis are essential for effective immune surveillance. A critical aspect of migration is cell polarization and the extension of pseudopodia in the direction of movement. However, our knowledge of the underlying molecular mechanisms responsible for these events is incomplete. Proteomic analysis of the isolated leading edges of CXCL12 stimulated human T cell lines was used to identify glia maturation factor gamma (GMFG) as a component of the pseudopodia. This protein is predominantly expressed in hematopoietic cells and it has been shown to regulate cytoskeletal branching. The present studies were undertaken to examine the role of GMFG in lymphocyte migration. RESULTS: Microscopic analysis of migrating T-cells demonstrated that GMFG was distributed along the axis of movement with enrichment in the leading edge and behind the nucleus of these cells. Inhibition of GMFG expression in T cell lines and IL-2 dependent human peripheral blood T cells with shRNAmir reduced cellular basal and chemokine induced migration responses. The failure of the cells with reduced GMFG to migrate was associated with an apparent inability to detach from the substrates that they were moving on. It was also noted that these cells had an increased adherence to extracellular matrix proteins such as fibronectin. These changes in adherence were associated with altered patterns of ß1 integrin expression and increased levels of activated integrins as detected with the activation specific antibody HUTS4. GMFG loss was also shown to increase the expression of the ß2 integrin LFA-1 and to increase the adhesion of these cells to ICAM-1. CONCLUSIONS: The present studies demonstrate that GMFG is a component of human T cell pseudopodia required for migration. The reduction in migration and increased adherence properties associated with inhibition of GMFG expression suggest that GMFG activity influences the regulation of integrin mediated adhesion.

Modulation of interleukin-1ß-induced inflammatory responses by a synthetic cationic innate defence regulator peptide, IDR-1002, in synovial fibroblasts.

INTRODUCTION: Innate defence regulator (IDR) peptides are synthetic cationic peptides, variants of naturally occurring innate immune effector molecules known as host defence peptides. IDR peptides were recently demonstrated to limit infection-associated inflammation selectively without compromising host innate immune functions. This study examined the impact of a 12-amino acid IDR peptide, IDR-1002, in pro-inflammatory cytokine interleukin (IL)-1ß-induced responses in synovial fibroblasts, a critical cell type in the pathogenesis of inflammatory arthritis. METHODS: Human fibroblast-like synoviocytes (FLS) were stimulated with IL-1ß in the presence and absence of IDR-1002. Production of enzyme matrix metalloproteinase-3 (MMP-3) and IL-1-receptor antagonist (IL-1RA) was monitored by enzyme-linked immunosorbent assay (ELISA), and various chemokines were evaluated by using multiplex cytometric bead array. Transcriptional responses were analyzed by quantitative real-time PCR. The impact on IL-1ß-induced proteome was investigated by quantitative proteomics by using isobaric tags. IL-1ß-induced pathways altered by IDR-1002 implicated by the proteomics analyses were further investigated by using various immunochemical assays. Cellular uptake of the peptide was monitored by using a biotinylated IDR-1002 peptide followed by microscopy probing with streptavidin-Alexa Fluor. RESULTS: This study demonstrated that IDR-1002 suppressed the production of IL-1ß-induced MMP-3 and monocyte chemotactic protein-1 (MCP-1); in contrast, IDR-1002 enhanced the production of IL-1RA, without neutralizing all chemokine responses. IDR-1002 altered the IL-1ß-induced proteome primarily by altering the expression of members of nuclear factor kappa-B (NF-κB) and c-Jun N-terminal kinase (JNK) pathways. The proteomics data also suggested that IDR-1002 was altering the transcription factor HNF-4α-mediated responses, known to be critical in metabolic regulation. With various immunochemical assays, it was further demonstrated that IL-1ß-induced NF-κB, JNK, and p38 mitogen-activated protein kinase (MAPK) activations were significantly suppressed by IDR-1002. CONCLUSIONS: This study demonstrates the ability of an innate immune-modulatory IDR-peptide to influence the IL-1ß-induced regulatory pathways and selectively to suppress inflammatory responses in synovial fibroblasts. The results of this study provide a rationale for examining the use of IDR-peptides as potential therapeutic candidates for chronic inflammatory diseases such as inflammatory arthritis.

Relaxin enhances the collagenolytic activity and in vitro invasiveness by upregulating matrix metalloproteinases in human thyroid carcinoma cells.

In this study, we identified differential expression of immunoreactive matrix metalloproteinase 2 (MMP2)/gelatinase A, membrane-anchored MT1-MMP/MMP14, and human relaxin-2 (RLN2) in human benign and malignant thyroid tissues. MMP2 and MT1-MMP were detected in the majority of thyroid cancer tissues and colocalized with RLN2-positive cells. MMP2 was mostly absent in goiter tissues and, similar to RLN2, may serve as a marker for thyroid cancer. MMP2 and MT1-MMP were identified as novel RLN2 targets. RLN2 caused a significant downregulation of tissue inhibitor of MMP (TIMP) 3 protein levels but did not change the expression levels of MMP13, and TIMP1, TIMP2, and TIMP4 in human thyroid carcinoma cells. RLN2 failed to affect the expression of MMP1, 3, 8, and 9 in the thyroid carcinoma cells investigated. Stable RLN2 transfectants secreted enhanced levels of bioactive MMP2 which contributed to the increased collagenolytic activity and in vitro invasiveness into collagen matrix by human thyroid cancer cells. Three-dimensional reconstitution of confocal fluorescent microscopy images revealed larger-sized invadopodia, with intense MT1-MMP accumulation at the leading migrating edge in RLN2 transfectants when compared with enhanced green fluorescent protein clones. In RLN2 transfectants actin stress fibers contributed to pseudopodia formation. In conclusion, enhanced tumor cell invasion by RLN2 involves the formation of MT1-MMP-enriched invadopodia that lead to increased collagenolytic cell invasion by human thyroid cancer cells.

Immunofluorescence localization of levopimaradiene/abietadiene synthase in methyl jasmonate treated stems of Sitka spruce (Picea sitchensis) shows activation of diterpenoid biosynthesis in cortical and developing traumatic resin ducts.

Conifers produce terpenoid-rich oleoresin in specialized resin ducts as a main line of defence against pests and pathogens. In spruce species (Picea spp.), axial resin ducts are either present constitutively in the cortex tissue (cortical resin ducts, CRDs) or are formed de novo as traumatic resin ducts (TRDs) in the cambial zone upon attack by insects, fungi or treatment with methyl jasmonate (MeJA). Using immunofluorescence localization we tested if previously formed CRDs respond to MeJA treatment with increased capacity for diterpenoid biosynthesis. We also tested the dynamics of diterpene synthase localization in the cambial zone. Immunofluorescence localization was performed using an antibody against a diterpene synthase, levopimaradiene/abietadiene synthase (LAS), in stem cross-sections of untreated and 0.1% MeJA-treated 4-year old Sitka spruce (P. sitchensis) trees. No fluorescence signal was observed in untreated stem cross-sections; however, signal was present 2 days after treatment with MeJA exclusively in the epithelial cells of CRDs. Fluorescence steadily increased in the CRD epithelial cells 4 and 8 days after treatment. At 8days, additional fluorescence was observed in developing epithelial cells of traumatic resin ducts TRDs in the cambial zone. These results confirm that resin duct epithelial cells are the main site of diterpene biosynthesis in Sitka spruce, diterpenoid biosynthesis is induced in CRD epithelial cells early upon treatment with MeJA, and immature developing TRD epithelial cells produce diterpene synthase enzyme. Overall, the results of this work improve our understanding of spatial and temporal patterns of induced diterpene resin acid biosynthesis in conifers.

Defining the membrane proteome of NK cells.

The present study was initiated to define the composition of the membrane proteome of the Natural Killer (NK) like cell line YTS. Isolated membranes were treated with reagents that have been reported to remove peripheral membrane proteins. Additional steps involving trifluoroethanol (TFE) were introduced in an effort to remove remaining nonintegral membrane proteins. This treatment resulted in the release of a subset of proteins without any apparent disruption of membrane integrity. The membranes were solubilized and digested with trypsin in 25% TFE. The resulting peptides were separated using an off-line two-dimensional reversed phase LC technique at alkaline and acidic pHs. Mass spectrometric analysis identified 1843 proteins with high confidence scores. On the basis of the presence of transmembrane regions or evidence of posttranslational modifications and prediction algorithms, approximately 40% of the identified proteins were predicted as plausible membrane proteins. The remaining species were largely involved in cellular processes and molecular functions that could be predicted to be transiently associated with membranes. The analytical approaches presented in this study offer robust generic methods for the identification and characterization of membrane proteins. These observations highlight the fact that the membrane is a dynamic entity that is composed of integral and stably associated proteins.

Targeted proteomics using selected reaction monitoring reveals the induction of specific terpene synthases in a multi-level study of methyl jasmonate-treated Norway spruce (Picea abies).

Induction of terpene synthase (TPS) gene expression and enzyme activity is known to occur in response to various chemical and biological stimuli in several species of spruce (genus Picea). However, high sequence identity between TPS family members has made it difficult to determine the induction patterns of individual TPS at the protein and transcript levels and whether specific TPS enzymes respond differentially to treatment. In the present study we used a multi-level approach to measure the induction and activity of TPS enzymes in protein extracts of Norway spruce (Picea abies) bark tissue following treatment with methyl jasmonate (MeJA). Measurements were made on the transcript, protein, enzyme activity and metabolite levels. Using a relatively new proteomics application, selective reaction monitoring (SRM), it was possible to differentiate and quantitatively measure the abundance of several known TPS proteins and three 1-deoxy-D-xylulose 5-phosphate synthase (DXS) isoforms in Norway spruce. Protein levels of individual TPS and DXS enzymes were differentially induced upon MeJA treatment and good correlation was generally observed between induction of transcripts, proteins, and enzyme activities. Most of the mono- and diterpenoid metabolites accumulated with similar temporal patterns of induction as part of the coordinated multi-compound chemical defense response. Protein and enzyme activity levels of the monoTPS (+)-3-carene synthase and the corresponding accumulation of (+)-3-carene was induced to a higher fold change than any other TPS or metabolite measured, indicating an important role in the induced terpenoid defense response in Norway spruce.

Intracellular receptor for human host defense peptide LL-37 in monocytes.

The human cationic host defense peptide LL-37 has a broad range of immunomodulatory, anti-infective functions. A synthetic innate defense regulator peptide, innate defense regulator 1 (IDR-1), based conceptually on LL-37, was recently shown to selectively modulate innate immunity to protect against a wide range of bacterial infections. Using advanced proteomic techniques, ELISA, and Western blotting procedures, GAPDH was identified as a direct binding partner for LL-37 in monocytes. Enzyme kinetics and mobility shift studies also indicated LL-37 and IDR-1 binding to GAPDH. The functional relevance of GAPDH in peptide-induced responses was demonstrated by using gene silencing of GAPDH with small interfering RNA (siRNA). Previous studies have established that the induction of chemokines and the anti-inflammatory cytokine IL-10 are critical immunomodulatory functions in the anti-infective properties of LL-37 and IDR-1, and these functions are modulated by the MAPK p38 pathway. Consistent with that, this study demonstrated the importance of the GAPDH interactions with these peptides since gene silencing of GAPDH resulted in impaired p38 MAPK signaling, downstream chemokine and cytokine transcriptional responses induced by LL-37 and IDR-1, and LL-37-induced cytokine production. Bioinformatic analysis, using InnateDB, of the major interacting partners of GAPDH indicated the likelihood that this protein can impact on innate immune pathways including p38 MAPK. Thus, this study has demonstrated a novel function for GAPDH as a mononuclear cell receptor for human cathelicidin LL-37 and immunomodulatory IDR-1 and conclusively demonstrated its relevance in the functioning of cationic host defense peptides.

Thermodynamic effects of noncoded and coded methionine substitutions in calmodulin.

The methionine residues in the calcium (Ca2+) regulatory protein calmodulin (CaM) are structurally and functionally important. They are buried within the N- and C-domains of apo-CaM but become solvent-exposed in Ca2+-CaM, where they interact with numerous target proteins. Previous structural studies have shown that methionine substitutions to the noncoded amino acids selenomethionine, ethionine, or norleucine, or mutation to leucine do not impact the main chain structure of CaM. Here we used differential scanning calorimetry to show that these substitutions enhance the stability of both domains, with the largest increase in melting temperature (19-26 degrees C) achieved with leucine or norleucine in the apo-C-domain. Nuclear magnetic resonance spectroscopy experiments also revealed the loss of a slow conformational exchange process in the Leu-substituted apo-C-domain. In addition, isothermal titration calorimetry experiments revealed considerable changes in the enthalpy and entropy of target binding to apo-CaM and Ca2+-CaM, but the free energy of binding was largely unaffected due to enthalpy-entropy compensation. Collectively, these results demonstrate that noncoded and coded methionine substitutions can be accommodated in CaM because of the structural plasticity of the protein. However, adjustments in side-chain packing and dynamics lead to significant differences in protein stability and the thermodynamics of target binding.

Analysis of the poplar phloem proteome and its response to leaf wounding.

Phloem exudate collected from hybrid poplar (Populus trichocarpa x Populus deltoides) was estimated to have more than 100 proteins, of which 48 were identified using LC-MS/MS. Comparative two-dimensional gel electrophoresis demonstrated that two phloem exudate proteins were significantly (P<0.05) upregulated 24 h after leaf wounding. These were identified as pop3/SP1 and a thaumatin-like protein. This is the first characterization of a phloem proteome from a tree species.

Quantitative iTRAQ proteome and comparative transcriptome analysis of elicitor-induced Norway spruce (Picea abies) cells reveals elements of calcium signaling in the early conifer defense response.

Long-lived conifer trees depend on both constitutive and induced defenses for resistance against a myriad of potential pathogens and herbivores. In species of spruce (Picea spp.), several of the late events of pathogen-, insect-, or elicitor-induced defense responses have previously been characterized at the anatomical, biochemical, transcriptome, and proteome levels in stems and needles. However, accurately measuring the early events of induced cellular responses in a conifer is technically challenging due to limitations in the precise timing of induction and tissue sampling from intact trees following insect or fungal treatment. In the present study, we used the advantages of Norway spruce (Picea abies) cell suspensions combined with chitosan elicitation to investigate the early proteome response in a conifer. A combination of iTRAQ labeling and a new design of iterative sample analysis employing data-dependent exclusion lists were used for proteome analysis. This approach improved the coverage of the spruce proteome beyond that achieved in any prior study in a conifer system. Comparison of elicitor-induced proteome and transcriptome responses in Norway spruce cells consistently identified features associated with calcium-mediated signaling and response to oxidative stress that have not previously been observed in the response of intact trees to fungal attack.

Conifer defense against insects: proteome analysis of Sitka spruce (Picea sitchensis) bark induced by mechanical wounding or feeding by white pine weevils (Pissodes strobi).

Feeding insects can have major ecological and economic impacts on both natural and planted forests. Understanding the molecular and biochemical mechanisms by which conifers defend themselves from insect pests is a major goal of ongoing research in forest health genomics. In previous work, we demonstrated a complex system of anatomical, chemical, and transcriptome responses in Sitka spruce (Picea sitchensis) upon feeding by the economically significant insect pest, the white pine weevil (Pissodes strobi). In this study, changes to the proteome of Sitka spruce bark tissue were examined subsequent to feeding by white pine weevils or mechanical wounding. 2-D PAGE and high-throughput MS/MS were used to examine induced changes in protein abundance and protein modification. Significant changes were observed as early as 2 h following the onset of insect feeding. Among the insect-induced proteins are a series of related small heat shock proteins, other stress response proteins, proteins involved in secondary metabolism, oxidoreductases, and a novel spruce protein. Comparison of protein expression and cDNA microarray profiles of induced spruce stem tissues reveals the complementary nature of transcriptome and proteome analyses and the need to apply a multifaceted approach to the large-scale analysis of plant defense systems.

Proteome analysis of early somatic embryogenesis in Picea glauca.

Forestry is a valuable natural resource for many countries. Rapid production of large quantities of genetically improved and uniform seedlings for restocking harvested lands is a key component of sustainable forest management programs. Clonal propagation through somatic embryogenesis has the potential to meet this need in conifers and can offer the added benefit of ensuring consistent seedling quality. Although in commercial use, mass production of conifers through somatic embryogenesis is relatively new and there are numerous biological unknowns regarding this complex developmental pathway. To aid in unravelling the embryo developmental process, two-dimensional electrophoresis was employed to quantitatively assess the expression levels of proteins across four stages of somatic embryo maturation in white spruce (0, 7, 21 and 35 days post abscisic acid treatment). Forty-eight differentially expressed proteins have been identified, which display a significant change in abundance as early as day 7 of embryo development. These proteins are involved in a variety of cellular processes, many of which have not previously been associated with embryo development. The identification of these proteins was greatly assisted by the availability of a substantial expressed sequence tag (EST) resource developed for white, sitka and interior spruce. The combined use of these spruce ESTs in conjunction with GenBank accessions for other plants improved the rate of protein identification from 38% to 62% when compared with GenBank alone using automated, high-throughput techniques. This underscores the utility of EST resources in a proteomic study of any species for which a genome sequence is unavailable.