Introduction and Objective: Due to the shortcomings of current T2D and obesity therapies, new therapies are required. In previous work, OGAP-Verify (oncology target discovery) has been successfully used to identify new cancer-specific proteins which have been targeted therapeutically and are now in Phase I/II clinical trials. While mRNA can be used to measure gene expression, mRNA only has 40% correlation with protein abundance. This study investigates development of proteomic methods to identify T2D-specific protein pathways which could be exploited therapeutically. Methods: T2D and healthy pancreatic tissues and islets, as well as many normal healthy tissues, were obtained from consenting donors, processed and analysed using quantitative Mass Spectrometry. Proteins were identified and their abundance determined in copies per cell. Up- and down-regulated proteins in T2D islets were identified using bioinformatics. Results: Preliminary results show up- and down-regulation of protein pathways unique to T2D using quantitative proteomics. Conclusion: Results demonstrate the ability of OGAP-Diabetes, a proprietary target discovery platform, to successfully identify new proteins and pathways involved in T2D for therapeutic intervention. This coupled with Artificial Intelligence Machine Learning (AI/ML) based approaches gives new insights into the molecular mechanisms behind T2D pathology. J. Lewis: None. B. Thomas: None. J.C. Allen: None. M. Ameer-Beg: None. M.P. Barnes: None. D.L. Davies: None. M.D. Agarwal: None. A. Houghton: None. C. Rohlff: None.

The ability of transcription factors to discriminate between different classes of binding sites associated with specific biological functions underpins effective gene regulation in development and homeostasis. How this is achieved is poorly understood. The microphthalmia-associated transcription factor MITF is a lineage-survival oncogene that plays a crucial role in melanocyte development and melanoma. MITF suppresses invasion, reprograms metabolism and promotes both proliferation and differentiation. How MITF distinguishes between differentiation and proliferation-associated targets is unknown. Here we show that compared to many transcription factors MITF exhibits a very long residence time which is reduced by p300/CBP-mediated MITF acetylation at K206. While K206 acetylation also decreases genome-wide MITF DNA-binding affinity, it preferentially directs DNA binding away from differentiation-associated CATGTG motifs toward CACGTG elements. The results reveal an acetylation-mediated switch that suppresses differentiation and provides a mechanistic explanation of why a human K206Q MITF mutation is associated with Waardenburg syndrome.

Pain and emotional distress have a reciprocal relation. The amygdala has been implicated in emotional processing. The central nucleus of the amygdala (CeA) receives nociceptive information from the dorsal horn of spinal cord and is responsible for the central plasticity in chronic pain. Neuropathic pain is a type of severe chronic pain and can be strongly influenced by emotional components. Plastic changes in the CeA may play a key role in the development or maintenance or both of neuropathic pain. We studied the expression levels of proteins in the CeA of spinal nerve transection (SNT) model rats. Total tissue lysate proteins were separated by two-dimensional-gel electrophoresis (2D-PAGE). Gels from different time points were compared using Progenesis SameSpot software, and the spots with Fold Change greater than 2 were excised for protein identification by mass spectrometry. We identified more than 50 cytosolic proteins as significantly altered in their expression levels in the CeA of SNT rats, and most of these changes have been validated at mRNA levels by qRT-PCR. We also identified more than 40 membrane proteins as notably up- or down-regulated in the CeA of SNT model rats relative to a control using stable isotope dimethyl labeling nano-LC-MS/MS based proteomics and found that one such protein, doublecortin (DCX), a microtubule-associated protein expressed by neuronal precursor cells during development, is specifically localized in the membrane fraction without changes in total amount of the protein. Immunohistochemistry showed that doublecortin is expressed in processes in the CeA of rats 7 and 21 days after SNT surgery, suggesting that doublecortin is one of the proteins that may contribute to the plastic changes, namely, redevelopment or rewiring of neural networks, in the CeA in the neuropathic pain model. These dysregulated proteins may play roles in reciprocal relationships between pain and psychological distress in the amygdala and contribute to central sensitization. Data are available via ProteomeXchange with identifier PXD017473.

It is widely assumed that decreasing transcription factor DNA-binding affinity reduces transcription initiation by diminishing occupancy of sequence-specific regulatory elements. However, in vivo transcription factors find their binding sites while confronted with a large excess of low-affinity degenerate motifs. Here, using the melanoma lineage survival oncogene MITF as a model, we show that low-affinity binding sites act as a competitive reservoir in vivo from which transcription factors are released by mitogen-activated protein kinase (MAPK)-stimulated acetylation to promote increased occupancy of their regulatory elements. Consequently, a low-DNA-binding-affinity acetylation-mimetic MITF mutation supports melanocyte development and drives tumorigenesis, whereas a high-affinity non-acetylatable mutant does not. The results reveal a paradoxical acetylation-mediated molecular clutch that tunes transcription factor availability via genome-wide redistribution and couples BRAF to tumorigenesis. Our results further suggest that p300/CREB-binding protein-mediated transcription factor acetylation may represent a common mechanism to control transcription factor availability.

Additional file 6: Table S6. Osteoblast microarray candidates involved associated with glucose homeostasis. 21 genes from the WT and Phospho1−/− osteoblast microarray were identified by Ingenuity Pathway Analysis to be associated with glucose homeostasis p = 1.04 × 10− 6.

Pelagia noctiluca is the most venomous jellyfish in the Mediterranean Sea where it forms dense blooms. Although there is several published research on this species, until now none of the works has been focused on a complete protein profile of the all body constituents of this organism. Here, we have performed a detailed proteomics characterization of the major protein components expressed by P. noctiluca. With that aim, we have considered the study of jellyfish proteins involved in defense, body constituents and metabolism, and furthered explore the significance and potential application of such bioactive molecules. P. noctiluca body proteins were separated by1D SDS–PAGE and 2DE followed by characterization by nanoLC-MS/MS and MALDI-TOF/TOF techniques. Altogether, both methods revealed 68 different proteins, including a Zinc Metalloproteinase, a Red Fluorescent Protein (RFP) and a Peroxiredoxin. These three proteins were identified for the first time in P. noctiluca. Zinc Metalloproteinase was previously reported in the venom of other jellyfish species. Besides the proteins described above, the other 65 proteins found in P. noctiluca body content were identified and associated with its clinical significance. Among all the proteins identified in this work we highlight: Zinc metalloproteinase, which has a ShK toxin domain and therefore should be implicated in the sting toxicity of P. noctiluca.; the RFP which are a very important family of proteins due to its possible application as molecular markers; and last but not least the discovery of a Peroxiredoxin in this organism makes it a new natural resource of antioxidant and anti-UV radiation agents.

Wolbachia are intracellular maternally inherited bacteria that can spread through insect populations and block virus transmission by mosquitoes, providing an important approach to dengue control. To better understand the mechanisms of virus inhibition, we here perform proteomic quantification of the effects of Wolbachia in Aedes aegypti mosquito cells and midgut. Perturbations are observed in vesicular trafficking, lipid metabolism and in the endoplasmic reticulum that could impact viral entry and replication. Wolbachia- infected cells display a differential cholesterol profile, including elevated levels of esterified cholesterol, that is consistent with perturbed intracellular cholesterol trafficking. Cyclodextrins have been shown to reverse lipid accumulation defects in cells with disrupted cholesterol homeostasis. Treatment of Wolbachia- infected Ae . aegypti cells with 2-hydroxypropyl-β-cyclodextrin restores dengue replication in Wolbachia -carrying cells, suggesting dengue is inhibited in Wolbachia- infected cells by localised cholesterol accumulation. These results demonstrate parallels between the cellular Wolbachia viral inhibition phenotype and lipid storage genetic disorders.

Tregs can adopt a catabolic metabolic program with increased capacity for fatty acid oxidation-fueled oxidative phosphorylation (OXPHOS). It is unclear why this form of metabolism is favored in Tregs and, more specifically, whether this program represents an adaptation to the environment and developmental cues or is "hardwired" by Foxp3. Here we show, using metabolic analysis and an unbiased mass spectroscopy-based proteomics approach, that Foxp3 is both necessary and sufficient to program Treg-increased respiratory capacity and Tregs' increased ability to utilize fatty acids to fuel oxidative phosphorylation. Foxp3 drives upregulation of components of all the electron transport complexes, increasing their activity and ATP generation by oxidative phosphorylation. Increased fatty acid β-oxidation also results in selective protection of Foxp3+ cells from fatty acid-induced cell death. This observation may provide novel targets for modulating Treg function or selection therapeutically.

Profiling of proteome dynamics is crucial for understanding cellular behavior in response to intrinsic and extrinsic stimuli and maintenance of homeostasis. Over the last 20 years, mass spectrometry (MS) has emerged as the most powerful tool for large-scale identification and characterization of proteins. Bottom-up proteomics, the most common MS-based proteomics approach, has always been challenging in terms of data management, processing, analysis and visualization, with modern instruments capable of producing several gigabytes of data out of a single experiment. Here, we present ProteoSign, a freely available web application, dedicated in allowing users to perform proteomics differential expression/abundance analysis in a userfriendly and self-explanatory way. Although several non-commercial standalone tools have been developed for post-quantification statistical analysis of proteomics data, most of them are not end-user appealing as they often require very stringent installation of programming environments, third-party software packages and sometimes further scripting or computer programming. To avoid this bottleneck, we have developed a user-friendly software platform accessible via a web interface in order to enable proteomics laboratories and core facilities to statistically analyse quantitative proteomics data sets in a resource-efficient manner. ProteoSign is available at http://bioinformatics.med.uoc.gr/ProteoSign and the source code at https://github.com/yorgodillo/ProteoSign.

Summary Vacuolar accumulation of acidic metabolites is an important aspect of tomato fruit flavour and nutritional quality. The amino acids Asp and Glu accumulate to high concentrations during ripening, while γ‐aminobutyrate ( GABA ) shows an approximately stoichiometric decline. Given that GABA can be catabolised to form Glu and subsequently Asp, and the requirement for the fruit to maintain osmotic homeostasis during ripening, we hypothesised the existence of a tonoplast transporter that exports GABA from the vacuole in exchange for import of either Asp or Glu. We show here that the tomato vacuolar membrane possesses such a transport property: transport of Glu across isolated tonoplast vesicle membranes was trans ‐stimulated in counterexchange mode by GABA , Glu and Asp. We identified Sl CAT 9 as a candidate protein for this exchanger using quantitative proteomics of a tonoplast‐enriched membrane fraction. Transient expression of a Sl CAT 9‐ YFP fusion in tobacco confirmed a tonoplast localisation. The function of the protein was examined by overexpression of Sl CAT 9 in transgenic tomato plants. Tonoplast vesicles isolated from transgenic plants showed higher rates of Glu and GABA transport than wild‐type ( WT ) only when assayed in counterexchange mode with Glu, Asp, or GABA . Moreover, there were substantial increases in the content of all three cognate amino acids in ripe fruit from the transgenic plants. We conclude that Sl CAT 9 is a tonoplast Glu/Asp/ GABA exchanger that strongly influences the accumulation of these amino acids during fruit development.

ADP-ribosylation is a post-translational modification (PTM) of proteins found in organisms from all kingdoms of life which regulates many important biological functions including DNA repair, chromatin structure, unfolded protein response and apoptosis. Several cellular enzymes, such as macrodomain containing proteins PARG [poly(ADP-ribose) glycohydrolase] and TARG1 [terminal ADP-ribose (ADPr) protein glycohydrolase], reverse protein ADP-ribosylation. In the present study, we show that human Nudix (nucleoside diphosphate-linked moiety X)-type motif 16 (hNUDT16) represents a new enzyme class that can process protein ADP-ribosylation in vitro, converting it into ribose-5'-phosphate (R5P) tags covalently attached to the modified proteins. Furthermore, our data show that hNUDT16 enzymatic activity can be used to trim ADP-ribosylation on proteins in order to facilitate analysis of ADP-ribosylation sites on proteins by MS.

The impact of protein arginine methylation on the regulation of immune functions is virtually unknown. Here, we apply a novel method—isomethionine methyl-SILAC—coupled with antibody-mediated arginine-methylated peptide enrichment to identify methylated peptides in human T cells by mass spectrometry. This approach allowed the identification of 2,502 arginine methylation sites from 1,257 tissue-specific and housekeeping proteins. We find that components of T cell antigen receptor signal machinery and several key transcription factors that regulate T cell fate determination are methylated on arginine. Moreover, we demonstrate changes in arginine methylation stoichiometry during cellular stimulation in a subset of proteins critical to T cell differentiation. Our data suggest that protein arginine methyltransferases exert key regulatory roles in T cell activation and differentiation, opening a new field of investigation in T cell biology.

This protocol describes the isolation of tonoplast vesicles from tomato fruit. The vesicles isolated using this procedure are of sufficiently high purity for downstream proteomic analysis whilst remaining transport competent for functional assays. The methodology was used to study the transport of amino acids during tomato fruit ripening (Snowden et al., 2015) and based on the procedure used by Betty and Smith (Bettey and Smith, 1993). Such vesicles may be useful in further studies into the dynamic transfer of metabolites across the tonoplast for storage and metabolism during tomato fruit development.

ABSTRACT Leptospirosis is a life-threatening and emerging zoonotic disease with a worldwide annual occurrence of more than 1 million cases. Leptospirosis is caused by spirochetes belonging to the genus Leptospira. The mechanisms of disease manifestation in the host remain elusive, and the roles of leptospiral exoproteins in these processes have yet to be determined. Our aim in this study was to assess the composition and quantity of exoproteins of pathogenic Leptospira interrogans and to construe how these proteins contribute to disease pathogenesis. Label-free quantitative mass spectrometry of proteins obtained from Leptospira spirochetes cultured in vitro under conditions mimicking infection identified 325 exoproteins. The majority of these proteins are conserved in the nonpathogenic species Leptospira biflexa, and proteins involved in metabolism and energy-generating functions were overrepresented and displayed the highest relative abundance in culture supernatants. Conversely, proteins of unknown function, which represent the majority of pathogen-specific proteins (presumably involved in virulence mechanisms), were underrepresented. Characterization of various L. interrogans exoprotein mutants in the animal infection model revealed host mortality rates similar to those of hosts infected with wild-type L. interrogans. Collectively, these results indicate that pathogenic Leptospira exoproteins primarily function in heterotrophic processes (the processes by which organisms utilize organic substances as nutrient sources) to maintain the saprophytic lifestyle rather than the virulence of the bacteria. The underrepresentation of proteins homologous to known virulence factors, such as toxins and effectors in the exoproteome, also suggests that disease manifesting from Leptospira infection is likely caused by a combination of the primary and potentially moonlight functioning of exoproteins.

The reasons for the disproportionate contribution of peanuts to prevalent and severe cases of food allergy in the Western world are unclear. Emerging statistics from East Asia generally match the overall common food allergies in the West, with the striking exception of peanuts, which are consumed equivalently in both regions.1Beyer K. Morrow E. Li X.M. Bardina L. Bannon G.A. Burks A.W. et al.Effects of cooking methods on peanut allergenicity.J Allergy Clin Immunol. 2001; 107: 1077-1081Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar, 2Lee A.J. Thalayasingam M. Lee B.W. Food allergy in Asia: how does it compare?.Asia Pac Allergy. 2013; 3: 3-14Crossref PubMed Google Scholar Differences in peanut preparation, roasted and dry roasted (DR) in the West versus raw, boiled, or fried in the East, have been proposed to contribute to this trend1Beyer K. Morrow E. Li X.M. Bardina L. Bannon G.A. Burks A.W. et al.Effects of cooking methods on peanut allergenicity.J Allergy Clin Immunol. 2001; 107: 1077-1081Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar, 2Lee A.J. Thalayasingam M. Lee B.W. Food allergy in Asia: how does it compare?.Asia Pac Allergy. 2013; 3: 3-14Crossref PubMed Google Scholar, 3Prusak A. Schlegel-Zawadzka M. Boulay A. Rowe G. Characteristics of the peanut chain in Europe—implications for peanut allergy.Acta Sci Pol Technol Aliment. 2014; 13: 321-333Crossref PubMed Scopus (10) Google Scholar and are supported by serological studies.1Beyer K. Morrow E. Li X.M. Bardina L. Bannon G.A. Burks A.W. et al.Effects of cooking methods on peanut allergenicity.J Allergy Clin Immunol. 2001; 107: 1077-1081Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar, 4Maleki S.J. Chung S.Y. Champagne E.T. Raufman J.P. The effects of roasting on the allergenic properties of peanut proteins.J Allergy Clin Immunol. 2000; 106: 763-768Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar Results obtained with other proteins have highlighted the potential immunomodulatory properties of advanced glycation end products (AGEs),5Ilchmann A. Burgdorf S. Scheurer S. Waibler Z. Nagai R. Wellner A. et al.Glycation of a food allergen by the Maillard reaction enhances its T-cell immunogenicity: role of macrophage scavenger receptor class A type I and II.J Allergy Clin Immunol. 2010; 125 (e1-11): 175-183Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar which are extensively formed during the high-temperature dry roasting of peanuts.6Hebling C.M. McFarland M.A. Callahan J.H. Ross M.M. Global proteomic screening of protein allergens and advanced glycation end products in thermally processed peanuts.J Agric Food Chem. 2013; 61: 5638-5648Crossref PubMed Scopus (48) Google Scholar However, despite this information, the immunogenicity and allergenicity of raw compared with DR peanuts has not been characterized in vivo.To address this, we first primed BALB/c mice subcutaneously with endotoxin-depleted soluble fractions of peanut protein extract (PPE) from raw or DR peanuts in PBS without adjuvant (see Figs E1, A; E2; and E3, A, and the Methods section in this article's Online Repository at www.jacionline.org). This resulted in enhanced peanut-specific IgG titers in DR-primed groups across a range of doses reactive against both raw and DR peanut extracts and with a dominant IgG1/IgG2a bias (see Fig E4, A-C, in this article's Online Repository at www.jacionline.org). After 3 subsequent intragastric gavages of endotoxin-undetectable DR crude peanut homogenate (CPH; see Figs E2 and E3, A), anti–raw peanut IgG titers increased by an average of 100-fold more in the DR group compared with those in the raw PPE group and were associated with significant titers of anti–raw peanut IgE, which was functional in basophil degranulation (Fig 1, A and B). Mesenteric lymph node cells from DR but not raw PPE–primed mice proliferated robustly in response to raw and DR PPE, and cytokines produced were dominated by IL-4 and IL-5 but not IFN-γ and TNF-α (Fig 1, C and D, and see Fig E4, D and E). This pattern of enhanced DR PPE immunogenicity was maintained in a similar experiment, in which PPE and CPH were kept homologous (raw/raw or DR/DR) during the subcutaneous prime and intragastric exposures (see Fig E4, F). In a further experiment we fed mice previously primed with DR or raw PPE with raw peanut kernels (see Fig E3, B) and observed significantly increased anti–raw and anti–DR PPE IgE only in DR-primed mice (Fig 1, E, and see E4, G). Finally, we excluded subcutaneous priming and directly administered multiple adjuvant-free intragastric CPH gavages (see Fig E3, C, and the Methods section in this article's Online Repository). DR but not raw CPH elicited anti-peanut IgG1 and functional IgE responses (Fig 1, F-H, and see Fig E5 in this article's Online Repository at www.jacionline.org).Atopic dermal sensitization could be highly relevant to peanut allergy.7Lack G. Update on risk factors for food allergy.J Allergy Clin Immunol. 2012; 129: 1187-1197Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar Therefore we adopted a previously described epicutaneous sensitizing strategy (see Fig E3, D, and the Methods section in this article's Online Repository), with raw or DR PPE applied daily to a developing atopic dermatitis–like skin lesion induced by topical administration of the exfoliating vitamin D analogue calcipotriol (MC903). Subsequent intragastric gavage and whole peanut feeding elicited higher peanut-specific IgG and IgE titers in the DR compared with raw PPE–sensitized mice (Fig 1, I and J, and see Fig E6 in this article's Online Repository at www.jacionline.org). Moreover, DR PPE–sensitized mice had higher eosinophilic infiltration of the lamina propria and increased IL-4, IL-5, and IL-13 secretion by mesenteric lymph node cells (Fig 1, K-M). Taken together, these data consolidate an enhanced TH2/allergenic profile of DR compared with raw peanuts that was maintained across the subcutaneous, epicutaneous, and gastrointestinal routes of sensitization.Dry roasting modifies the physicochemical properties of peanut proteins through the Maillard reaction in a manner that might influence protein immunogenicity through cross-linking, AGE adduction, or both.4Maleki S.J. Chung S.Y. Champagne E.T. Raufman J.P. The effects of roasting on the allergenic properties of peanut proteins.J Allergy Clin Immunol. 2000; 106: 763-768Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar, 5Ilchmann A. Burgdorf S. Scheurer S. Waibler Z. Nagai R. Wellner A. et al.Glycation of a food allergen by the Maillard reaction enhances its T-cell immunogenicity: role of macrophage scavenger receptor class A type I and II.J Allergy Clin Immunol. 2010; 125 (e1-11): 175-183Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 6Hebling C.M. McFarland M.A. Callahan J.H. Ross M.M. Global proteomic screening of protein allergens and advanced glycation end products in thermally processed peanuts.J Agric Food Chem. 2013; 61: 5638-5648Crossref PubMed Scopus (48) Google Scholar We excluded cross-linking as a major contributor to immunogenicity because our DR PPE samples underwent multiple rounds of filtration and centrifugation that depleted cross-linked species (see Fig E1). Furthermore, we purified the major peanut allergen Ara h 1 from DR PPE and observed, by using native gel electrophoresis and multiangle light scattering, that it retained fewer multimers compared with raw peanut-derived Ara h 1 (see Fig E1, C and D), yet was more immunogenic in a subcutaneous immunization of BALB/c mice (see Figs E3, E, and E4, H). However, consistent with the presence of AGE-related adducts,6Hebling C.M. McFarland M.A. Callahan J.H. Ross M.M. Global proteomic screening of protein allergens and advanced glycation end products in thermally processed peanuts.J Agric Food Chem. 2013; 61: 5638-5648Crossref PubMed Scopus (48) Google Scholar DR but not raw Ara h 1 bound to 2 well-characterized AGE receptors, CD36 and the receptor for advanced glycation end products (RAGE),8Mueller G.A. Maleki S.J. Johnson K. Hurlburt B.K. Cheng H. Ruan S. et al.Identification of Maillard reaction products on peanut allergens that influence binding to the receptor for advanced glycation end products.Allergy. 2013; 68: 1546-1554Crossref PubMed Scopus (57) Google Scholar, 9Ohgami N. Nagai R. Ikemoto M. Arai H. Kuniyasu A. Horiuchi S. et al.Cd36, a member of the class b scavenger receptor family, as a receptor for advanced glycation end products.J Biol Chem. 2001; 276: 3195-3202Crossref PubMed Scopus (201) Google Scholar and DR PPE binding to bone marrow–derived dendritic cells (BMDCs) was partially inhibited by scavenger receptor blockers (Fig 2, A and B). Consistent with this, DR PPE was more efficient than raw PPE in inhibiting the in vitro presentation of maleylated or AGE-modified hen egg lysozyme (HEL) by BMDCs to a HEL-specific T-cell hybridoma (Fig 2, C, and see the Methods section in this article's Online Repository).Fig 2AGE adducts in DR peanuts. A, Raw and DR Ara h 1 recombinant receptor binding by means of ELISA. RAGE, Receptor for advanced glycation end products; TLR3, Toll-like receptor 3. B, BMDC binding of fluoresceinated Ara h 1 and its inhibition by means of flow cytometry. MSRA, Macrophage scavenger receptor A. C, DR or raw PPE competition for maleylated or AGE-HEL BMDC presentation to 3A9 hybridoma. D and E, PPE-pulsed BMDC surface marker and cytokine analysis by means of flow cytometry and Luminex. MCP1, Monocyte chemoattractant protein 1; MIP1β, macrophage inflammatory protein 1β; OX40L, OX40 ligand. F, PPE coculture with HEL in BMDC presentation to 3A9 hybridoma. G, Nuclear factor κB (NF-κB)/activator protein 1 (AP1) activation in RAW-Blue cells pulsed with HEL, PPE, or LPS with or without polymixin B. *P ≤ .05, **P ≤ .01, and ***P ≤ .001.View Large Image Figure ViewerDownload Hi-res image Download (PPT)These results confirm the presence of biologically active AGE adducts in DR peanut proteins and their ability to target these proteins to antigen-presenting cells. However, phenotypic and cytokine profile analyses of peanut-pulsed BMDCs revealed no classical activation by raw or DR peanut preparations compared with LPS (Fig 2, D and E, and see Fig E7 in this article's Online Repository at www.jacionline.org). Moreover, by contrast with direct AGE-relevant modification of HEL that showed increased presentation to an HEL-specific T-cell hybridoma, DR PPE–pulsed dendritic cells (DCs) did not enhance the presentation of unmodified HEL, reinforcing the absence of DR PPE–elicited DC costimulation (Fig 2, F, and see Fig E8 in this article's Online Repository at www.jacionline.org). Finally, we confirmed the lack of canonical proinflammatory activation by raw or DR PPE and CPH preparations using the sensitive nuclear factor κB/activator protein 1 murine and human monocyte/macrophage reporter lines RAW-Blue and THP1 Blue, respectively (Fig 2, G, and see Fig E9 in this article's Online Repository at www.jacionline.org). These data suggest that, in accordance with other reports,5Ilchmann A. Burgdorf S. Scheurer S. Waibler Z. Nagai R. Wellner A. et al.Glycation of a food allergen by the Maillard reaction enhances its T-cell immunogenicity: role of macrophage scavenger receptor class A type I and II.J Allergy Clin Immunol. 2010; 125 (e1-11): 175-183Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 10Moghaddam A.E. Gartlan K.H. Kong L. Sattentau Q.J. Reactive carbonyls are a major th2-inducing damage-associated molecular pattern generated by oxidative stress.J Immunol. 2011; 187: 1626-1633Crossref PubMed Scopus (47) Google Scholar enhanced targeting and presentation through AGE receptors rather than conventional DC maturation might be implicated in the increased immunogenicity of DR peanut antigens in vivo. The complex variety of AGE adducts and the diversity and redundancy of their receptors means that more study is required to elucidate the precise receptors and pathways implicated in DC uptake and presentation of peanut antigens to T cells.This report provides the first in vivo support for the enhanced immunogenicity of DR peanuts, which is probably mediated by oxidation-driven generation of AGE-related adducts in peanut allergens. Two other major findings with clinical implications are as follows: (1) the high reactivity of DR peanut–derived responses to raw peanut antigens, suggesting that roasting might have a more important effect on the immunogenicity rather than antigenicity of peanuts at the priming step of sensitization, which could be further aggravated by subsequent exposure to raw antigens, and (2) the fact that cutaneous sensitization provokes further hypersensitivity in the gut on oral antigen exposure. A better understanding of how high-temperature antigen modification, such as peanut dry roasting, leads to allergic sensitization should inform future preventive strategies, including those concerning early age exposure, and therapeutic measures, such as the choice and route of antigen delivery in desensitization strategies. The reasons for the disproportionate contribution of peanuts to prevalent and severe cases of food allergy in the Western world are unclear. Emerging statistics from East Asia generally match the overall common food allergies in the West, with the striking exception of peanuts, which are consumed equivalently in both regions.1Beyer K. Morrow E. Li X.M. Bardina L. Bannon G.A. Burks A.W. et al.Effects of cooking methods on peanut allergenicity.J Allergy Clin Immunol. 2001; 107: 1077-1081Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar, 2Lee A.J. Thalayasingam M. Lee B.W. Food allergy in Asia: how does it compare?.Asia Pac Allergy. 2013; 3: 3-14Crossref PubMed Google Scholar Differences in peanut preparation, roasted and dry roasted (DR) in the West versus raw, boiled, or fried in the East, have been proposed to contribute to this trend1Beyer K. Morrow E. Li X.M. Bardina L. Bannon G.A. Burks A.W. et al.Effects of cooking methods on peanut allergenicity.J Allergy Clin Immunol. 2001; 107: 1077-1081Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar, 2Lee A.J. Thalayasingam M. Lee B.W. Food allergy in Asia: how does it compare?.Asia Pac Allergy. 2013; 3: 3-14Crossref PubMed Google Scholar, 3Prusak A. Schlegel-Zawadzka M. Boulay A. Rowe G. Characteristics of the peanut chain in Europe—implications for peanut allergy.Acta Sci Pol Technol Aliment. 2014; 13: 321-333Crossref PubMed Scopus (10) Google Scholar and are supported by serological studies.1Beyer K. Morrow E. Li X.M. Bardina L. Bannon G.A. Burks A.W. et al.Effects of cooking methods on peanut allergenicity.J Allergy Clin Immunol. 2001; 107: 1077-1081Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar, 4Maleki S.J. Chung S.Y. Champagne E.T. Raufman J.P. The effects of roasting on the allergenic properties of peanut proteins.J Allergy Clin Immunol. 2000; 106: 763-768Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar Results obtained with other proteins have highlighted the potential immunomodulatory properties of advanced glycation end products (AGEs),5Ilchmann A. Burgdorf S. Scheurer S. Waibler Z. Nagai R. Wellner A. et al.Glycation of a food allergen by the Maillard reaction enhances its T-cell immunogenicity: role of macrophage scavenger receptor class A type I and II.J Allergy Clin Immunol. 2010; 125 (e1-11): 175-183Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar which are extensively formed during the high-temperature dry roasting of peanuts.6Hebling C.M. McFarland M.A. Callahan J.H. Ross M.M. Global proteomic screening of protein allergens and advanced glycation end products in thermally processed peanuts.J Agric Food Chem. 2013; 61: 5638-5648Crossref PubMed Scopus (48) Google Scholar However, despite this information, the immunogenicity and allergenicity of raw compared with DR peanuts has not been characterized in vivo. To address this, we first primed BALB/c mice subcutaneously with endotoxin-depleted soluble fractions of peanut protein extract (PPE) from raw or DR peanuts in PBS without adjuvant (see Figs E1, A; E2; and E3, A, and the Methods section in this article's Online Repository at www.jacionline.org). This resulted in enhanced peanut-specific IgG titers in DR-primed groups across a range of doses reactive against both raw and DR peanut extracts and with a dominant IgG1/IgG2a bias (see Fig E4, A-C, in this article's Online Repository at www.jacionline.org). After 3 subsequent intragastric gavages of endotoxin-undetectable DR crude peanut homogenate (CPH; see Figs E2 and E3, A), anti–raw peanut IgG titers increased by an average of 100-fold more in the DR group compared with those in the raw PPE group and were associated with significant titers of anti–raw peanut IgE, which was functional in basophil degranulation (Fig 1, A and B). Mesenteric lymph node cells from DR but not raw PPE–primed mice proliferated robustly in response to raw and DR PPE, and cytokines produced were dominated by IL-4 and IL-5 but not IFN-γ and TNF-α (Fig 1, C and D, and see Fig E4, D and E). This pattern of enhanced DR PPE immunogenicity was maintained in a similar experiment, in which PPE and CPH were kept homologous (raw/raw or DR/DR) during the subcutaneous prime and intragastric exposures (see Fig E4, F). In a further experiment we fed mice previously primed with DR or raw PPE with raw peanut kernels (see Fig E3, B) and observed significantly increased anti–raw and anti–DR PPE IgE only in DR-primed mice (Fig 1, E, and see E4, G). Finally, we excluded subcutaneous priming and directly administered multiple adjuvant-free intragastric CPH gavages (see Fig E3, C, and the Methods section in this article's Online Repository). DR but not raw CPH elicited anti-peanut IgG1 and functional IgE responses (Fig 1, F-H, and see Fig E5 in this article's Online Repository at www.jacionline.org). Atopic dermal sensitization could be highly relevant to peanut allergy.7Lack G. Update on risk factors for food allergy.J Allergy Clin Immunol. 2012; 129: 1187-1197Abstract Full Text Full Text PDF PubMed Scopus (302) Google Scholar Therefore we adopted a previously described epicutaneous sensitizing strategy (see Fig E3, D, and the Methods section in this article's Online Repository), with raw or DR PPE applied daily to a developing atopic dermatitis–like skin lesion induced by topical administration of the exfoliating vitamin D analogue calcipotriol (MC903). Subsequent intragastric gavage and whole peanut feeding elicited higher peanut-specific IgG and IgE titers in the DR compared with raw PPE–sensitized mice (Fig 1, I and J, and see Fig E6 in this article's Online Repository at www.jacionline.org). Moreover, DR PPE–sensitized mice had higher eosinophilic infiltration of the lamina propria and increased IL-4, IL-5, and IL-13 secretion by mesenteric lymph node cells (Fig 1, K-M). Taken together, these data consolidate an enhanced TH2/allergenic profile of DR compared with raw peanuts that was maintained across the subcutaneous, epicutaneous, and gastrointestinal routes of sensitization. Dry roasting modifies the physicochemical properties of peanut proteins through the Maillard reaction in a manner that might influence protein immunogenicity through cross-linking, AGE adduction, or both.4Maleki S.J. Chung S.Y. Champagne E.T. Raufman J.P. The effects of roasting on the allergenic properties of peanut proteins.J Allergy Clin Immunol. 2000; 106: 763-768Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar, 5Ilchmann A. Burgdorf S. Scheurer S. Waibler Z. Nagai R. Wellner A. et al.Glycation of a food allergen by the Maillard reaction enhances its T-cell immunogenicity: role of macrophage scavenger receptor class A type I and II.J Allergy Clin Immunol. 2010; 125 (e1-11): 175-183Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 6Hebling C.M. McFarland M.A. Callahan J.H. Ross M.M. Global proteomic screening of protein allergens and advanced glycation end products in thermally processed peanuts.J Agric Food Chem. 2013; 61: 5638-5648Crossref PubMed Scopus (48) Google Scholar We excluded cross-linking as a major contributor to immunogenicity because our DR PPE samples underwent multiple rounds of filtration and centrifugation that depleted cross-linked species (see Fig E1). Furthermore, we purified the major peanut allergen Ara h 1 from DR PPE and observed, by using native gel electrophoresis and multiangle light scattering, that it retained fewer multimers compared with raw peanut-derived Ara h 1 (see Fig E1, C and D), yet was more immunogenic in a subcutaneous immunization of BALB/c mice (see Figs E3, E, and E4, H). However, consistent with the presence of AGE-related adducts,6Hebling C.M. McFarland M.A. Callahan J.H. Ross M.M. Global proteomic screening of protein allergens and advanced glycation end products in thermally processed peanuts.J Agric Food Chem. 2013; 61: 5638-5648Crossref PubMed Scopus (48) Google Scholar DR but not raw Ara h 1 bound to 2 well-characterized AGE receptors, CD36 and the receptor for advanced glycation end products (RAGE),8Mueller G.A. Maleki S.J. Johnson K. Hurlburt B.K. Cheng H. Ruan S. et al.Identification of Maillard reaction products on peanut allergens that influence binding to the receptor for advanced glycation end products.Allergy. 2013; 68: 1546-1554Crossref PubMed Scopus (57) Google Scholar, 9Ohgami N. Nagai R. Ikemoto M. Arai H. Kuniyasu A. Horiuchi S. et al.Cd36, a member of the class b scavenger receptor family, as a receptor for advanced glycation end products.J Biol Chem. 2001; 276: 3195-3202Crossref PubMed Scopus (201) Google Scholar and DR PPE binding to bone marrow–derived dendritic cells (BMDCs) was partially inhibited by scavenger receptor blockers (Fig 2, A and B). Consistent with this, DR PPE was more efficient than raw PPE in inhibiting the in vitro presentation of maleylated or AGE-modified hen egg lysozyme (HEL) by BMDCs to a HEL-specific T-cell hybridoma (Fig 2, C, and see the Methods section in this article's Online Repository). These results confirm the presence of biologically active AGE adducts in DR peanut proteins and their ability to target these proteins to antigen-presenting cells. However, phenotypic and cytokine profile analyses of peanut-pulsed BMDCs revealed no classical activation by raw or DR peanut preparations compared with LPS (Fig 2, D and E, and see Fig E7 in this article's Online Repository at www.jacionline.org). Moreover, by contrast with direct AGE-relevant modification of HEL that showed increased presentation to an HEL-specific T-cell hybridoma, DR PPE–pulsed dendritic cells (DCs) did not enhance the presentation of unmodified HEL, reinforcing the absence of DR PPE–elicited DC costimulation (Fig 2, F, and see Fig E8 in this article's Online Repository at www.jacionline.org). Finally, we confirmed the lack of canonical proinflammatory activation by raw or DR PPE and CPH preparations using the sensitive nuclear factor κB/activator protein 1 murine and human monocyte/macrophage reporter lines RAW-Blue and THP1 Blue, respectively (Fig 2, G, and see Fig E9 in this article's Online Repository at www.jacionline.org). These data suggest that, in accordance with other reports,5Ilchmann A. Burgdorf S. Scheurer S. Waibler Z. Nagai R. Wellner A. et al.Glycation of a food allergen by the Maillard reaction enhances its T-cell immunogenicity: role of macrophage scavenger receptor class A type I and II.J Allergy Clin Immunol. 2010; 125 (e1-11): 175-183Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 10Moghaddam A.E. Gartlan K.H. Kong L. Sattentau Q.J. Reactive carbonyls are a major th2-inducing damage-associated molecular pattern generated by oxidative stress.J Immunol. 2011; 187: 1626-1633Crossref PubMed Scopus (47) Google Scholar enhanced targeting and presentation through AGE receptors rather than conventional DC maturation might be implicated in the increased immunogenicity of DR peanut antigens in vivo. The complex variety of AGE adducts and the diversity and redundancy of their receptors means that more study is required to elucidate the precise receptors and pathways implicated in DC uptake and presentation of peanut antigens to T cells. This report provides the first in vivo support for the enhanced immunogenicity of DR peanuts, which is probably mediated by oxidation-driven generation of AGE-related adducts in peanut allergens. Two other major findings with clinical implications are as follows: (1) the high reactivity of DR peanut–derived responses to raw peanut antigens, suggesting that roasting might have a more important effect on the immunogenicity rather than antigenicity of peanuts at the priming step of sensitization, which could be further aggravated by subsequent exposure to raw antigens, and (2) the fact that cutaneous sensitization provokes further hypersensitivity in the gut on oral antigen exposure. A better understanding of how high-temperature antigen modification, such as peanut dry roasting, leads to allergic sensitization should inform future preventive strategies, including those concerning early age exposure, and therapeutic measures, such as the choice and route of antigen delivery in desensitization strategies. Supplementary data Download .docx (.07 MB) Help with docx files Online Repository Data Download .pdf (.55 MB) Help with pdf files Fig E1 Download .pdf (.03 MB) Help with pdf files Fig E2 Download .pdf (.04 MB) Help with pdf files Fig E3 Download .pdf (.2 MB) Help with pdf files Fig E4 Download .pdf (.03 MB) Help with pdf files Fig E5 Download .pdf (.02 MB) Help with pdf files Fig E6 Download .pdf (.1 MB) Help with pdf files Fig E7 Download .pdf (.04 MB) Help with pdf files Fig E8 Download .pdf (.12 MB) Help with pdf files Fig E9 Download .docx (.07 MB) Help with docx files Online Repository Data Download .pdf (.55 MB) Help with pdf files Fig E1 Download .pdf (.03 MB) Help with pdf files Fig E2 Download .pdf (.04 MB) Help with pdf files Fig E3 Download .pdf (.2 MB) Help with pdf files Fig E4 Download .pdf (.03 MB) Help with pdf files Fig E5 Download .pdf (.02 MB) Help with pdf files Fig E6 Download .pdf (.1 MB) Help with pdf files Fig E7 Download .pdf (.04 MB) Help with pdf files Fig E8 Download .pdf (.12 MB) Help with pdf files Fig E9

Viruses use virions to spread between hosts, and virion composition is therefore the primary determinant of viral transmissibility and immunogenicity. However, the virions of many viruses are complex and pleomorphic, making them difficult to analyse in detail. Here we address this by identifying and quantifying virion proteins with mass spectrometry, producing a complete and quantified model of the hundreds of host-encoded and viral proteins that make up the pleomorphic virions of influenza viruses. We show that a conserved influenza virion architecture is maintained across diverse combinations of virus and host. This 'core' architecture, which includes substantial quantities of host proteins as well as the viral protein NS1, is elaborated with abundant host-dependent features. As a result, influenza virions produced by mammalian and avian hosts have distinct protein compositions. Finally, we note that influenza virions share an underlying protein composition with exosomes, suggesting that influenza virions form by subverting microvesicle production.

Here we demonstrate quantitation of stimuli-induced proteome dynamics in primary cells by combining the power of bio-orthogonal noncanonical amino acid tagging (BONCAT) and stable-isotope labeling of amino acids in cell culture (SILAC). In conjunction with nanoscale liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS), quantitative noncanonical amino acid tagging (QuaNCAT) allowed us to monitor the early expression changes of >600 proteins in primary resting T cells subjected to activation stimuli.

The lack of methods for proteome-scale detection of arginine methylation restricts our knowledge of its relevance in physiological and pathological processes. Here we show that most tryptic peptides containing methylated arginine(s) are highly basic and hydrophilic. Consequently, they could be considerably enriched from total cell extracts by simple protocols using either one of strong cation exchange chromatography, isoelectric focusing, or hydrophilic interaction liquid chromatography, the latter being by far the most effective of all. These methods, coupled with heavy methyl-stable isotope labeling by amino acids in cell culture and mass spectrometry, enabled in T cells the identification of 249 arginine methylation sites in 131 proteins, including 190 new sites and 93 proteins not previously known to be arginine methylated. By extending considerably the number of known arginine methylation sites, our data reveal a novel proline-rich consensus motif and identify for the first time arginine methylation in proteins involved in cytoskeleton rearrangement at the immunological synapse and in endosomal trafficking.

Protein phosphorylation is a common post-translational modification in eukaryotic cells and has a wide range of functional effects. Here, we used mass spectrometry to search for phosphorylated residues in all the proteins of influenza A and B viruses - to the best of our knowledge, the first time such a comprehensive approach has been applied to a virus. We identified 36 novel phosphorylation sites, as well as confirming 3 previously-identified sites. N-terminal processing and ubiquitination of viral proteins was also detected. Phosphorylation was detected in the polymerase proteins (PB2, PB1 and PA), glycoproteins (HA and NA), nucleoprotein (NP), matrix protein (M1), ion channel (M2), non-structural protein (NS1) and nuclear export protein (NEP). Many of the phosphorylation sites detected were conserved between influenza virus genera, indicating the fundamental importance of phosphorylation for all influenza viruses. Their structural context indicates roles for phosphorylation in regulating viral entry and exit (HA and NA); nuclear localisation (PB2, M1, NP, NS1 and, through NP and NEP, of the viral RNA genome); and protein multimerisation (NS1 dimers, M2 tetramers and NP oligomers). Using reverse genetics we show that for NP of influenza A viruses phosphorylation sites in the N-terminal NLS are important for viral growth, whereas mutating sites in the C-terminus has little or no effect. Mutating phosphorylation sites in the oligomerisation domains of NP inhibits viral growth and in some cases transcription and replication of the viral RNA genome. However, constitutive phosphorylation of these sites is not optimal. Taken together, the conservation, structural context and functional significance of phosphorylation sites implies a key role for phosphorylation in influenza biology. By identifying phosphorylation sites throughout the proteomes of influenza A and B viruses we provide a framework for further study of phosphorylation events in the viral life cycle and suggest a range of potential antiviral targets.