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.

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.