Improved detection and consistency of RNA-interacting proteomes using DIA SILAC.

The RNA-interacting proteome is commonly characterized by UV-crosslinking followed by RNA purification, with protein recovery quantified using SILAC labeling followed by data-dependent acquisition (DDA) of proteomic data. However, the low efficiency of UV-crosslinking, combined with limited sensitivity of the DDA approach often restricts detection to relatively abundant proteins, necessitating multiple mass spec injections of fractionated peptides for each biological sample. Here we report an application of data-independent acquisition (DIA) with SILAC in a total RNA-associated protein purification (TRAPP) UV-crosslinking experiment. This gave 15% greater protein detection and lower inter-replicate variation relative to the same biological materials analyzed using DDA, while allowing single-shot analysis of the sample. As proof of concept, we determined the effects of arsenite treatment on the RNA-bound proteome of HEK293T cells. The DIA dataset yielded similar GO term enrichment for RNA-binding proteins involved in cellular stress responses to the DDA dataset while detecting extra proteins unseen by DDA. Overall, the DIA SILAC approach improved detection of proteins over conventional DDA SILAC for generating RNA-interactome datasets, at a lower cost due to reduced machine time. Analyses are described for TRAPP data, but the approach is suitable for proteomic analyses following essentially any RNA-binding protein enrichment technique.

The minor chicken class I gene BF1 is deleted between short imperfect direct repeats in the B14 and typical B15 major histocompatibility complex (MHC) haplotypes.

The chicken major histocompatibility complex (MHC, also known as the BF-BL region of the B locus) is notably small and simple with few genes, most of which are involved in antigen processing and presentation. There are two classical class I genes, of which only BF2 is well and systemically expressed as the major ligand for cytotoxic T lymphocytes (CTLs). The other class I gene, BF1, is believed to be primarily a natural killer (NK) cell ligand. Among most standard chicken MHC haplotypes examined in detail, BF1 is expressed tenfold less than BF2 at the RNA level due to defects in the promoter or in a splice site. However, in the B14 and typical B15 haplotypes, BF1 RNA was not detected, and here, we show that a deletion between imperfect 32 nucleotide direct repeats has removed the BF1 gene entirely. The phenotypic effects of not having a BF1 gene (particularly on resistance to infectious pathogens) have not been systematically explored, but such deletions between short direct repeats are also found in some BF1 promoters and in the 5' untranslated region (5'UTR) of some BG genes found in the BG region of the B locus. Despite the opposite transcriptional orientation of homologous genes in the chicken MHC, which might prevent the loss of key genes from a minimal essential MHC, it appears that small direct repeats can still lead to deletion.

Translation factor eIF5a is essential for IFNγ production and cell cycle regulation in primary CD8+ T lymphocytes.

Control of mRNA translation adjusts protein production rapidly and facilitates local cellular responses to environmental conditions. Traditionally initiation of translation is considered to be a major translational control point, however, control of peptide elongation is also important. Here we show that the function of the elongation factor, eIF5a, is regulated dynamically in naïve CD8+ T cells upon activation by post-translational modification, whereupon it facilitates translation of specific subsets of proteins. eIF5a is essential for long-term survival of effector CD8+ T cells and sequencing of nascent polypeptides indicates that the production of proteins which regulate proliferation and key effector functions, particularly the production of IFNγ and less acutely TNF production and cytotoxicity, is dependent on the presence of functional eIF5a. Control of translation in multiple immune cell lineages is required to co-ordinate immune responses and these data illustrate that translational elongation contributes to post-transcriptional regulons important for the control of inflammation.

Suboptimal T-cell receptor signaling compromises protein translation, ribosome biogenesis, and proliferation of mouse CD8 T cells.

Global transcriptomic and proteomic analyses of T cells have been rich sources of unbiased data for understanding T-cell activation. Lack of full concordance of these datasets has illustrated that important facets of T-cell activation are controlled at the level of translation. We undertook translatome analysis of CD8 T-cell activation, combining polysome profiling and microarray analysis. We revealed that altering T-cell receptor stimulation influenced recruitment of mRNAs to heavy polysomes and translation of subsets of genes. A major pathway that was compromised, when TCR signaling was suboptimal, was linked to ribosome biogenesis, a rate-limiting factor in both cell growth and proliferation. Defective TCR signaling affected transcription and processing of ribosomal RNA precursors, as well as the translation of specific ribosomal proteins and translation factors. Mechanistically, IL-2 production was compromised in weakly stimulated T cells, affecting the abundance of Myc protein, a known regulator of ribosome biogenesis. Consequently, weakly activated T cells showed impaired production of ribosomes and a failure to maintain proliferative capacity after stimulation. We demonstrate that primary T cells respond to various environmental cues by regulating ribosome biogenesis and mRNA translation at multiple levels to sustain proliferation and differentiation.

Telomere maintenance and telomerase activity are differentially regulated in asexual and sexual worms.

In most sexually reproducing animals, replication and maintenance of telomeres occurs in the germ line and during early development in embryogenesis through the use of telomerase. Somatic cells generally do not maintain telomere sequences, and these cells become senescent in adults as telomeres shorten to a critical length. Some animals reproduce clonally and must therefore require adult somatic mechanisms for maintaining their chromosome ends. Here we study the telomere biology of planarian flatworms with apparently limitless regenerative capacity fueled by a population of highly proliferative adult stem cells. We show that somatic telomere maintenance is different in asexual and sexual animals. Asexual animals maintain telomere length somatically during reproduction by fission or when regeneration is induced by amputation, whereas sexual animals only achieve telomere elongation through sexual reproduction. We demonstrate that this difference is reflected in the expression and alternate splicing of the protein subunit of the telomerase enzyme. Asexual adult planarian stem cells appear to maintain telomere length over evolutionary timescales without passage through a germ-line stage. The adaptations we observe demonstrate indefinite somatic telomerase activity in proliferating stem cells during regeneration or reproduction by fission, and establish planarians as a pertinent model for studying telomere structure, function, and maintenance.

Fractalkine has anti-apoptotic and proliferative effects on human vascular smooth muscle cells via epidermal growth factor receptor signalling.

AIMS: Fractalkine (CX3CL1) is a membrane-bound chemokine that signals through the G protein-coupled receptor CX3CR1 that is implicated in the development of atherosclerosis. We have previously reported that CX3CR1 is expressed by primary human coronary artery smooth muscle cells (CASMC), where it mediates chemotaxis towards CX3CL1. We sought to determine the effect of CX3CL1 on CASMC survival and proliferation and elucidate the signalling mechanisms involved. METHODS AND RESULTS: CX3CL1 significantly reduces staurosporine-induced apoptosis of CASMC, as quantified by caspase 3 immunostaining and Annexin-V flow cytometry. Furthermore, CX3CL1 is a potent mitogen for primary CASMC and induces phosphorylation of extracellular signal-regulated kinase (ERK) and Akt, measured by western blotting. Inhibition of either ERK or phosphoinositide 3-kinase (PI3K) signalling abrogates proliferation, while only PI3K signalling is involved in the anti-apoptotic effects of CX3CL1. We describe a novel and specific small molecule antagonist of CX3CR1 (AZ12201182) which abrogates the mitogenic and anti-apoptotic effects of CX3CL1 on CASMC. Pharmacological inhibition of the epidermal growth factor receptor (EGFR) blocks CASMC survival and DNA synthesis, indicating a previously undocumented role for EGFR signalling in response to CX3CL1 involving release of a soluble EGFR ligand. Specifically, CX3CL1 induces shedding of epiregulin and increases epiregulin mRNA expression 20-fold within 2 h. Finally, antibody neutralization of epiregulin abrogates the mitogenic effect of CX3CL1. CONCLUSION: We have demonstrated two novel and important functions of CX3CL1 on primary human SMCs: anti-apoptosis and proliferation, both mediated via epiregulin-induced EGFR signalling. Our data have important implications in vascular pathologies including atherosclerosis, restenosis, and transplant accelerated arteriosclerosis, where the balance of SMC proliferation and apoptosis critically determines both plaque stability and vessel stenosis.

Protective effect of sanguinarine on ultraviolet B-mediated damages in SKH-1 hairless mouse skin: implications for prevention of skin cancer.

Excessive exposure of solar ultraviolet (UV) radiation, particularly its UVB component (280-320 nm), to human skin is the major cause of skin cancers. UV exposure also leads to the development of precancerous conditions such as actinic keratosis and elicits a variety of other adverse effects such as sunburn, inflammation, hyperplasia, immunosuppression and skin aging. Therefore, there is a need to intensify our efforts towards the development of novel mechanism-based approaches/agents for the protection of UVB-mediated damages. Chemoprevention is being investigated as a potential approach for the management of UV damages including skin cancer. We have earlier shown that sanguinarine, a benzophenanthridine alkaloid, inhibits UVB exposure-mediated damages in HaCaT keratinocytes. In this study, to determine the relevance of our in vitro findings to in vivo situations, we assessed the effects of sanguinarine on UVB-mediated damages in SKH-1 hairless mice. Our data demonstrated that a topical application of sanguinarine (5 micromol 0.3 mL(-1) ethanol per mouse), either as a pretreatment (30 min prior to UVB) or posttreatment (5 min after UVB), resulted in a significant decrease in UVB-mediated increases in skin edema, skin hyperplasia and infiltration of leukocytes. Further, sanguinarine treatments (pre and post) also resulted in a significant decrease in UVB mediated (1) generation of H2O2 and (2) increases in the protein levels of markers of tumor promotion/proliferation viz. ornithine decarboxylase (ODC), proliferating cell nuclear antigen (PCNA) and Kiel antigen-67. Based on this data, we suggest that sanguinarine could be developed as an agent for the management of conditions elicited by UV exposure including skin cancer. However, further detailed studies are needed to support this suggestion.

Patch-test reactions to formaldehydes, bioban, and other formaldehyde releasers.

BACKGROUND: Contact allergy to formaldehyde, Bioban, and other formaldehyde releasers and cross-reactivity between them have been reported in the literature; however, not many studies have data on this cross-reactivity. OBJECTIVE: To study (1) the rates of allergy to formaldehyde and to Bioban and other formaldehyde releasers and (2) the rates of cross-reactivity between them. METHODS: We present a retrospective chart analysis of patch-test results for all patients referred for allergic contact dermatitis testing at the Milton S. Hershey Medical Center from June 2004 to September 2005. Anyone allergic to formaldehyde, Bioban, or other formaldehyde releasers was included. Cross-reactivity between the agents was then analyzed. RESULTS: The charts of 210 patients were analyzed. Of these patients, 24 (11%) were allergic to formaldehyde, Bioban, or other formaldehyde-releasing agents. Seventeen (8.1%) of the patients were allergic to formaldehyde, 15 (7.1%) were allergic to Bioban, and 20 (9.5%) were allergic to other formaldehyde-releasing agents. Eleven (65%) of the 17 formaldehyde-allergic patients were also allergic to Bioban. Of the 20 patients allergic to formaldehyde-releasing agents, 14 (70%) were also allergic to one of the three Bioban products tested. Of the 15 patients allergic to Bioban, 11 (73%) were allergic to formaldehyde, 14 (93%) were allergic to formaldehyde-releasing agents, and 11 (73%) were allergic to both formaldehyde and formaldehyde-releasing agents. CONCLUSION: A high cross-reactivity rate between formaldehyde, Bioban, and other formaldehyde-releasing agents was found.