Publications by authors named "Arthur Salomon"

52 Publications

Quantitative Interactomics of Lck-TurboID in Living Human T Cells Unveils T Cell Receptor Stimulation-Induced Proximal Lck Interactors.

J Proteome Res 2021 01 13;20(1):715-726. Epub 2020 Nov 13.

Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, Rhode Island, United States.

While Lck has been widely recognized to play a pivotal role in the initiation of the T cell receptor (TCR) signaling pathway, an understanding of the precise regulation of Lck in T cells upon TCR activation remains elusive. Investigation of protein-protein interaction (PPI) using proximity labeling techniques such as TurboID has the potential to provide valuable molecular insights into Lck regulatory networks. By expressing Lck-TurboID in Jurkat T cells, we have uncovered a dynamic, short-range Lck protein interaction network upon 30 min of TCR stimulation. In this novel application of TurboID, we detected 27 early signaling-induced Lck-proximal interactors in living T cells, including known and novel Lck interactors, validating the discovery power of this tool. Our results revealed previously unappreciated Lck PPI which may be associated with cytoskeletal rearrangement, ubiquitination of TCR signaling proteins, activation of the mitogen-activated protein kinase cascade, coalescence of the LAT signalosome, and formation of the immunological synapse. In this study, we demonstrated for the first time in immune cells and for the kinase Lck that TurboID can be utilized to unveil PPI dynamics in living cells at a time scale consistent with early TCR signaling. Data are available ProteomeXchange with identifier PXD020759.
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http://dx.doi.org/10.1021/acs.jproteome.0c00616DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7962135PMC
January 2021

Tandem Mass Tag Approach Utilizing Pervanadate BOOST Channels Delivers Deeper Quantitative Characterization of the Tyrosine Phosphoproteome.

Mol Cell Proteomics 2020 04 18;19(4):730-743. Epub 2020 Feb 18.

Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, Rhode Island. Electronic address:

Dynamic tyrosine phosphorylation is fundamental to a myriad of cellular processes. However, the inherently low abundance of tyrosine phosphorylation in the proteome and the inefficient enrichment of phosphotyrosine(pTyr)-containing peptides has led to poor pTyr peptide identification and quantitation, critically hindering researchers' ability to elucidate signaling pathways regulated by tyrosine phosphorylation in systems where cellular material is limited. The most popular approaches to wide-scale characterization of the tyrosine phosphoproteome use pTyr enrichment with pan-specific, anti-pTyr antibodies from a large amount of starting material. Methods that decrease the amount of starting material and increase the characterization depth of the tyrosine phosphoproteome while maintaining quantitative accuracy and precision would enable the discovery of tyrosine phosphorylation networks in rarer cell populations. To achieve these goals, the BOOST (road-spectrum ptimization f elective riggering) method leveraging the multiplexing capability of tandem mass tags (TMT) and the use of pervanadate (PV) boost channels (cells treated with the broad-spectrum tyrosine phosphatase inhibitor PV) selectively increased the relative abundance of pTyr-containing peptides. After PV boost channels facilitated selective fragmentation of pTyr-containing peptides, TMT reporter ions delivered accurate quantitation of each peptide for the experimental samples while the quantitation from PV boost channels was ignored. This method yielded up to 6.3-fold boost in pTyr quantification depth of statistically significant data derived from contrived ratios, compared with TMT without PV boost channels or intensity-based label-free (LF) quantitation while maintaining quantitative accuracy and precision, allowing quantitation of over 2300 unique pTyr peptides from only 1 mg of T cell receptor-stimulated Jurkat T cells. The BOOST strategy can potentially be applied in analyses of other post-translational modifications where treatments that broadly elevate the levels of those modifications across the proteome are available.
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http://dx.doi.org/10.1074/mcp.TIR119.001865DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7124467PMC
April 2020

Regulation of Respiration and Apoptosis by Cytochrome c Threonine 58 Phosphorylation.

Sci Rep 2019 11 1;9(1):15815. Epub 2019 Nov 1.

Center for Molecular Medicine and Genetics, Wayne State University, Detroit, MI, 48201, USA.

Cytochrome c (Cytc) is a multifunctional protein, acting as an electron carrier in the electron transport chain (ETC), where it shuttles electrons from bc complex to cytochrome c oxidase (COX), and as a trigger of type II apoptosis when released from the mitochondria. We previously showed that Cytc is regulated in a highly tissue-specific manner: Cytc isolated from heart, liver, and kidney is phosphorylated on Y97, Y48, and T28, respectively. Here, we have analyzed the effect of a new Cytc phosphorylation site, threonine 58, which we mapped in rat kidney Cytc by mass spectrometry. We generated and overexpressed wild-type, phosphomimetic T58E, and two controls, T58A and T58I Cytc; the latter replacement is found in human and testis-specific Cytc. In vitro, COX activity, caspase-3 activity, and heme degradation in the presence of HO were decreased with phosphomimetic Cytc compared to wild-type. Cytc-knockout cells expressing T58E or T58I Cytc showed a reduction in intact cell respiration, mitochondrial membrane potential (∆Ψ), ROS production, and apoptotic activity compared to wild-type. We propose that, under physiological conditions, Cytc is phosphorylated, which controls mitochondrial respiration and apoptosis. Under conditions of stress Cytc phosphorylations are lost leading to maximal respiration rates, ∆Ψ hyperpolarization, ROS production, and apoptosis.
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http://dx.doi.org/10.1038/s41598-019-52101-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6825195PMC
November 2019

Serine-47 phosphorylation of cytochrome in the mammalian brain regulates cytochrome oxidase and caspase-3 activity.

FASEB J 2019 12 28;33(12):13503-13514. Epub 2019 Sep 28.

Center for Molecular Medicine and Genetics, Wayne State University, Detroit, Michigan, USA.

Cytochrome (Cyt) is a multifunctional protein that operates as an electron carrier in the mitochondrial electron transport chain and plays a key role in apoptosis. We have previously shown that tissue-specific phosphorylations of Cyt in the heart, liver, and kidney play an important role in the regulation of cellular respiration and cell death. Here, we report that Cyt purified from mammalian brain is phosphorylated on S47 and that this phosphorylation is lost during ischemia. We have characterized the functional effects using phosphorylated Cyt purified from pig brain tissue and a recombinant phosphomimetic mutant (S47E). We crystallized S47E phosphomimetic Cyt at 1.55 Å and suggest that it spatially matches S47-phosphorylated Cyt, making it a good model system. Both S47-phosphorylated and phosphomimetic Cyt showed a lower oxygen consumption rate in reaction with isolated Cyt oxidase, which we propose maintains intermediate mitochondrial membrane potentials under physiologic conditions, thus minimizing production of reactive oxygen species. S47-phosphorylated and phosphomimetic Cyt showed lower caspase-3 activity. Furthermore, phosphomimetic Cyt had decreased cardiolipin peroxidase activity and is more stable in the presence of HO. Our data suggest that S47 phosphorylation of Cyt is tissue protective and promotes cell survival in the brain.-Kalpage, H. A., Vaishnav, A., Liu, J., Varughese, A., Wan, J., Turner, A. A., Ji, Q., Zurek, M. P., Kapralov, A. A., Kagan, V. E., Brunzelle, J. S., Recanati, M.-A., Grossman, L. I., Sanderson, T. H., Lee, I., Salomon, A. R., Edwards, B. F. P, Hüttemann, M. Serine-47 phosphorylation of cytochrome in the mammalian brain regulates cytochrome oxidase and caspase-3 activity.
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http://dx.doi.org/10.1096/fj.201901120RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6894086PMC
December 2019

Fibroblasts Mobilize Tumor Cell Glycogen to Promote Proliferation and Metastasis.

Cell Metab 2019 01 30;29(1):141-155.e9. Epub 2018 Aug 30.

Department of Obstetrics and Gynecology/Section of Gynecologic Oncology, University of Chicago, Chicago, IL 60637, USA. Electronic address:

Successful metastasis requires the co-evolution of stromal and cancer cells. We used stable isotope labeling of amino acids in cell culture coupled with quantitative, label-free phosphoproteomics to study the bidirectional signaling in ovarian cancer cells and human-derived, cancer-associated fibroblasts (CAFs) after co-culture. In cancer cells, the interaction with CAFs supported glycogenolysis under normoxic conditions and induced phosphorylation and activation of phosphoglucomutase 1, an enzyme involved in glycogen metabolism. Glycogen was funneled into glycolysis, leading to increased proliferation, invasion, and metastasis of cancer cells co-cultured with human CAFs. Glycogen mobilization in cancer cells was dependent on p38α MAPK activation in CAFs. In vivo, deletion of p38α in CAFs and glycogen phosphorylase inhibition in cancer cells reduced metastasis, suggesting that glycogen is an energy source used by cancer cells to facilitate metastatic tumor growth.
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http://dx.doi.org/10.1016/j.cmet.2018.08.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6326875PMC
January 2019

Lck promotes Zap70-dependent LAT phosphorylation by bridging Zap70 to LAT.

Nat Immunol 2018 07 18;19(7):733-741. Epub 2018 Jun 18.

Division of Rheumatology, Rosalind Russell and Ephraim P. Engleman Arthritis Research Center, Department of Medicine, University of California, San Francisco, San Francisco, CA, USA.

T cell-antigen receptor (TCR) signaling requires the sequential activities of the kinases Lck and Zap70. Upon TCR stimulation, Lck phosphorylates the TCR, thus leading to the recruitment, phosphorylation, and activation of Zap70. Lck binds and stabilizes phosho-Zap70 by using its SH2 domain, and Zap70 phosphorylates the critical adaptors LAT and SLP76, which coordinate downstream signaling. It is unclear whether phosphorylation of these adaptors occurs through passive diffusion or active recruitment. We report the discovery of a conserved proline-rich motif in LAT that mediates efficient LAT phosphorylation. Lck associates with this motif via its SH3 domain, and with phospho-Zap70 via its SH2 domain, thereby acting as a molecular bridge that facilitates the colocalization of Zap70 and LAT. Elimination of this proline-rich motif compromises TCR signaling and T cell development. These results demonstrate the remarkable multifunctionality of Lck, wherein each of its domains has evolved to orchestrate a distinct step in TCR signaling.
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http://dx.doi.org/10.1038/s41590-018-0131-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6202249PMC
July 2018

Sucrose Nonfermenting-Related Kinase Regulates Both Adipose Inflammation and Energy Homeostasis in Mice and Humans.

Diabetes 2018 03 3;67(3):400-411. Epub 2018 Jan 3.

Department of Epidemiology, Brown University, Providence, RI

Sucrose nonfermenting-related kinase (SNRK) is a member of the AMPK-related kinase family, and its physiological role in adipose energy homeostasis and inflammation remains unknown. We previously reported that SNRK is ubiquitously and abundantly expressed in both white adipose tissue (WAT) and brown adipose tissue (BAT), but SNRK expression diminishes in adipose tissue in obesity. In this study we report novel experimental findings from both animal models and human genetics. SNRK is essential for survival; SNRK globally deficient pups die within 24 h after birth. Heterozygous mice are characterized by inflamed WAT and less BAT. Adipocyte-specific ablation of SNRK causes inflammation in WAT, ectopic lipid deposition in liver and muscle, and impaired adaptive thermogenesis in BAT. These metabolic disorders subsequently lead to decreased energy expenditure, higher body weight, and insulin resistance. We further confirm the significant association of common variants of the gene with obesity risk in humans. Through applying a phosphoproteomic approach, we identified eukaryotic elongation factor 1δ and histone deacetylase 1/2 as potential SNRK substrates. Taking these data together, we conclude that SNRK represses WAT inflammation and is essential to maintain BAT thermogenesis, making it a novel therapeutic target for treating obesity and associated metabolic disorders.
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http://dx.doi.org/10.2337/db17-0745DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5828454PMC
March 2018

A PLC-γ1 Feedback Pathway Regulates Lck Substrate Phosphorylation at the T-Cell Receptor and SLP-76 Complex.

J Proteome Res 2017 08 6;16(8):2729-2742. Epub 2017 Jul 6.

Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University , Providence, Rhode Island 02912, United States.

Phospholipase C gamma 1 (PLC-γ1) occupies a critically important position in the T-cell signaling pathway. While its functions as a regulator of both Ca signaling and PKC-family kinases are well characterized, PLC-γ1's role in the regulation of early T-cell receptor signaling events is incompletely understood. Activation of the T-cell receptor leads to the formation of a signalosome complex between SLP-76, LAT, PLC-γ1, Itk, and Vav1. Recent studies have revealed the existence of both positive and negative feedback pathways from SLP-76 to the apical kinase in the pathway, Lck. To determine if PLC-γ1 contributes to the regulation of these feedback networks, we performed a quantitative phosphoproteomic analysis of PLC-γ1-deficient T cells. These data revealed a previously unappreciated role for PLC-γ1 in the positive regulation of Zap-70 and T-cell receptor tyrosine phosphorylation. Conversely, PLC-γ1 negatively regulated the phosphorylation of SLP-76-associated proteins, including previously established Lck substrate phosphorylation sites within this complex. While the positive and negative regulatory phosphorylation sites on Lck were largely unchanged, Tyr phosphorylation was elevated in Jgamma1. The data supports a model wherein Lck's targeting, but not its kinase activity, is altered by PLC-γ1, possibly through Lck Tyr phosphorylation and increased association of the kinase with protein scaffolds SLP-76 and TSAd.
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http://dx.doi.org/10.1021/acs.jproteome.6b01026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5549626PMC
August 2017

Highly reproducible improved label-free quantitative analysis of cellular phosphoproteome by optimization of LC-MS/MS gradient and analytical column construction.

J Proteomics 2017 08 17;165:69-74. Epub 2017 Jun 17.

Center for Cancer Research Development, Proteomics Core Facility, Rhode Island Hospital, Providence, RI 02903, USA; Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, RI 02912, USA; Department of Chemistry, Brown University, Providence, RI 02912, USA. Electronic address:

Expanding the sequencing depth of the peptides with a statistically significant quantitative change derived from a biological stimulation is critical. Here we demonstrate that optimization of LC gradient and analytical column construction can reveal over 30,000 unique peptides and 23,000 phosphopeptides at high confidence. The quantitative reproducibility of different analytical workflows was evaluated by comparing the phosphoproteome of CD3/4 stimulated and unstimulated T-cells as a model system. A fritless, 50cm-long column packed with 1.9μm particles operated with a standard pressure HPLC significantly improved the sequencing depth 51% and decreased the selected ion chromatogram peak spreading. Most importantly, under the optimal workflow we observed an improvement of over 300% in detection of significantly changed phosphopeptides in the stimulated cells compared with the other workflows. The discovery power of the optimized column configuration was illustrated by identification of significantly altered phosphopeptides harboring novel sites from proteins previously established as important in T cell signaling including A-Raf, B-Raf, c-Myc, CARMA1, Fyn, ITK, LAT, NFAT1/2/3, PKCα, PLCγ1/2, RAF1, and SOS1. Taken together, our results reveal the analytical power of optimized chromatography using sub 2μm particles for the analysis of the T cell phosphoproteome to reveal a vast landscape of significantly altered phosphorylation changes in response to T cell receptor stimulation.
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http://dx.doi.org/10.1016/j.jprot.2017.06.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5542054PMC
August 2017

CD95/Fas Increases Stemness in Cancer Cells by Inducing a STAT1-Dependent Type I Interferon Response.

Cell Rep 2017 03;18(10):2373-2386

Division of Hematology/Oncology, Department of Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA; Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA. Electronic address:

Stimulation of CD95/Fas drives and maintains cancer stem cells (CSCs). We now report that this involves activation of signal transducer and activator of transcription 1 (STAT1) and induction of STAT1-regulated genes and that this process is inhibited by active caspases. STAT1 is enriched in CSCs in cancer cell lines, patient-derived human breast cancer, and CD95-expressing glioblastoma neurospheres. CD95 stimulation of cancer cells induced secretion of type I interferons (IFNs) that bind to type I IFN receptors, resulting in activation of Janus-activated kinases, activation of STAT1, and induction of a number of STAT1-regulated genes that are part of a gene signature recently linked to therapy resistance in five primary human cancers. Consequently, we identified type I IFNs as drivers of cancer stemness. Knockdown or knockout of STAT1 resulted in a strongly reduced ability of CD95L or type I IFN to increase cancer stemness. This identifies STAT1 as a key regulator of the CSC-inducing activity of CD95.
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http://dx.doi.org/10.1016/j.celrep.2017.02.037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5474321PMC
March 2017

Quantitative Phosphoproteomic Analysis of T-Cell Receptor Signaling.

Methods Mol Biol 2017 ;1584:369-382

Center for Cancer Research and Development, Proteomics Core Facility, Rhode Island Hospital, Providence, RI, 02903, USA.

TCR signaling critically depends on protein phosphorylation across many proteins. Localization of each phosphorylation event relative to the T-cell receptor (TCR) and canonical T-cell signaling proteins will provide clues about the structure of TCR signaling networks. Quantitative phosphoproteomic analysis by mass spectrometry provides a wide-scale view of cellular phosphorylation networks. However, analysis of phosphorylation by mass spectrometry is still challenging due to the relative low abundance of phosphorylated proteins relative to all proteins and the extraordinary diversity of phosphorylation sites across the proteome. Highly selective enrichment of phosphorylated peptides is essential to provide the most comprehensive view of the phosphoproteome. Optimization of phosphopeptide enrichment methods coupled with highly sensitive mass spectrometry workflows significantly improves the sequencing depth of the phosphoproteome to over 10,000 unique phosphorylation sites from complex cell lysates. Here we describe a step-by-step method for phosphoproteomic analysis that has achieved widespread success for identification of serine, threonine, and tyrosine phosphorylation. Reproducible quantification of relative phosphopeptide abundance is provided by intensity-based label-free quantitation. An ideal set of mass spectrometry analysis parameters is also provided that optimize the yield of identified sites. We also provide guidelines for the bioinformatic analysis of this type of data to assess the quality of the data and to comply with proteomic data reporting requirements.
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http://dx.doi.org/10.1007/978-1-4939-6881-7_22DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5573147PMC
February 2018

Proteomic analysis of laser capture microdissected focal lesions in a rat model of progenitor marker-positive hepatocellular carcinoma.

Oncotarget 2017 Apr;8(16):26041-26056

Department of Pediatrics, Rhode Island Hospital and Brown University, Providence, RI, USA.

We have shown previously that rapamycin, the canonical inhibitor of the mechanistic target of rapamycin (mTOR) complex 1, markedly inhibits the growth of focal lesions in the resistant hepatocyte (Solt-Farber) model of hepatocellular carcinoma (HCC) in the rat. In the present study, we characterized the proteome of persistent, pre-neoplastic focal lesions in this model. One group was administered rapamycin by subcutaneous pellet for 3 weeks following partial hepatectomy and euthanized 4 weeks after the cessation of rapamycin. A second group received placebo pellets. Results were compared to unmanipulated control animals and to animals that underwent an incomplete Solt-Farber protocol to activate hepatic progenitor cells. Regions of formalin-fixed, paraffin-embedded tissue were obtained by laser capture microdissection (LCM). Proteomic analysis yielded 11,070 unique peptides representing 2,227 proteins. Quantitation of the peptides showed increased abundance of known HCC markers (e.g., glutathione S-transferase-P, epoxide hydrolase, 6 others) and potential markers (e.g., aflatoxin aldehyde reductase, glucose 6-phosphate dehydrogenase, 10 others) in foci from placebo-treated and rapamycin-treated rats. Peptides derived from cytochrome P450 enzymes were generally reduced. Comparisons of the rapamycin samples to normal liver and to the progenitor cell model indicated that rapamycin attenuated a loss of differentiation relative to placebo. We conclude that early administration of rapamycin in the Solt-Farber model not only inhibits the growth of pre-neoplastic foci but also attenuates the loss of differentiated function. In addition, we have demonstrated that the combination of LCM and mass spectrometry-based proteomics is an effective approach to characterize focal liver lesions.
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http://dx.doi.org/10.18632/oncotarget.15219DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5432236PMC
April 2017

Phosphorylation of Cytochrome c Threonine 28 Regulates Electron Transport Chain Activity in Kidney: IMPLICATIONS FOR AMP KINASE.

J Biol Chem 2017 Jan 7;292(1):64-79. Epub 2016 Oct 7.

From the Center for Molecular Medicine and Genetics and

Mammalian cytochrome c (Cytc) plays a key role in cellular life and death decisions, functioning as an electron carrier in the electron transport chain and as a trigger of apoptosis when released from the mitochondria. However, its regulation is not well understood. We show that the major fraction of Cytc isolated from kidneys is phosphorylated on Thr, leading to a partial inhibition of respiration in the reaction with cytochrome c oxidase. To further study the effect of Cytc phosphorylation in vitro, we generated T28E phosphomimetic Cytc, revealing superior behavior regarding protein stability and its ability to degrade reactive oxygen species compared with wild-type unphosphorylated Cytc Introduction of T28E phosphomimetic Cytc into Cytc knock-out cells shows that intact cell respiration, mitochondrial membrane potential (ΔΨ), and ROS levels are reduced compared with wild type. As we show by high resolution crystallography of wild-type and T28E Cytc in combination with molecular dynamics simulations, Thr is located at a central position near the heme crevice, the most flexible epitope of the protein apart from the N and C termini. Finally, in silico prediction and our experimental data suggest that AMP kinase, which phosphorylates Cytc on Thr in vitro and colocalizes with Cytc to the mitochondrial intermembrane space in the kidney, is the most likely candidate to phosphorylate Thr in vivo We conclude that Cytc phosphorylation is mediated in a tissue-specific manner and leads to regulation of electron transport chain flux via "controlled respiration," preventing ΔΨ hyperpolarization, a known cause of ROS and trigger of apoptosis.
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http://dx.doi.org/10.1074/jbc.M116.744664DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5217700PMC
January 2017

Targeted proteomics: Current status and future perspectives for quantification of food allergens.

J Proteomics 2016 06 22;143:15-23. Epub 2016 Apr 22.

Center for Cancer Research and Development, Proteomics Core Facility, Rhode Island Hospital, Providence, RI 02903, USA; Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, RI 02903, USA.

Unlabelled: Allergen levels in fresh and processed foods can vary dynamically. As different sources of foods can cause different types of allergic reactions, the food industry and regulatory bodies urgently require reliable detection and absolute quantitation methods for allergen detection in complex food products to effectively safeguard the food-allergic population. Recent advances of targeted proteomic technologies namely multiple-reaction monitoring (MRM) mass spectrometry (MS) coupled with isotope-labeled internal standard, also known as AQUA peptides offers absolute quantitation of food allergens even at 10ppb level in a multiplex fashion. However, development of successful AQUA-MRM assay relies on a number of pre and post MS criteria. In this review, we briefly describe how allergen levels could potentially change in plant and animal based foods, necessitating the development of a high throughput multiplexed allergen quantification methodology for successful AQUA-MRM assay. We also propose some future strategies that could provide better management of food allergy.

Biological Significance: Given the rapid increases of food allergenicity, it has become imperative to know absolute allergen levels in foods. This essential information could be the most effective means of protecting humans suffering from allergies. In this review, we emphasize the significance of the absolute quantitation of food allergens using AQUA-MRM approach and discuss the likely critical steps for successful assay development.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5082697PMC
http://dx.doi.org/10.1016/j.jprot.2016.04.018DOI Listing
June 2016

Vav1 Regulates T-Cell Activation through a Feedback Mechanism and Crosstalk between the T-Cell Receptor and CD28.

J Proteome Res 2015 Jul 16;14(7):2963-75. Epub 2015 Jun 16.

†Department of Molecular Pharmacology, Physiology, and Biotechnology and ‡Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, Rhode Island 02903, United States.

Vav1, a Rac/Rho guanine nucleotide exchange factor and a critical component of the T-cell receptor (TCR) signaling cascade is tyrosine phosphorylated rapidly in response to T-cell activation. Vav1 has established roles in proliferation, cytokine secretion, Ca(2+) responses, and actin cytoskeleton regulation; however, its function in the regulation of phosphorylation of TCR components, including the ζ chain, the CD3 δ, ε, γ chains, and the associated kinases Lck and ZAP-70, is not well established. To obtain a more comprehensive picture of the role of Vav1 in receptor proximal signaling, we performed a wide-scale characterization of Vav1-dependent tyrosine phosphorylation events using quantitative phosphoproteomic analysis of Vav1-deficient T cells across a time course of TCR stimulation. Importantly, this study revealed a new function for Vav1 in the negative feedback regulation of the phosphorylation of immunoreceptor tyrosine-based activation motifs within the ζ chains, CD3 δ, ε, γ chains, as well as activation sites on the critical T cell tyrosine kinases Itk, Lck, and ZAP-70. Our study also uncovered a previously unappreciated role for Vav1 in crosstalk between the CD28 and TCR signaling pathways.
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http://dx.doi.org/10.1021/acs.jproteome.5b00340DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4490005PMC
July 2015

Adaptation of HepG2 cells to a steady-state reduction in the content of protein phosphatase 6 (PP6) catalytic subunit.

Exp Cell Res 2015 Jul 18;335(2):224-37. Epub 2015 May 18.

Department of Pediatrics, Brown University and Rhode Island Hospital, Providence, RI, USA; Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI, USA. Electronic address:

Protein phosphatase 6 (PP6) is a ubiquitous Ser/Thr phosphatase involved in an array of cellular processes. To assess the potential of PP6 as a therapeutic target in liver disorders, we attenuated expression of the PP6 catalytic subunit in HepG2 cells using lentiviral-transduced shRNA. Two PP6 knock-down (PP6KD) cell lines (90% reduction of PP6-C protein content) were studied in depth. Both proliferated at a rate similar to control cells. However, flow cytometry indicated G2/M cell cycle arrest that was accounted for by a shift of the cells from a diploid to tetraploid state. PP6KD cells did not show an increase in apoptosis, nor did they exhibit reduced viability in the presence of bleomycin or taxol. Gene expression analysis by microarray showed attenuated anti-inflammatory signaling. Genes associated with DNA replication were downregulated. Mass spectrometry-based phosphoproteomic analysis yielded 80 phosphopeptides representing 56 proteins that were significantly affected by a stable reduction in PP6-C. Proteins involved in DNA replication, DNA damage repair and pre-mRNA splicing were overrepresented among these. PP6KD cells showed intact mTOR signaling. Our studies demonstrated involvement of PP6 in a diverse set of biological pathways and an adaptive response that may limit the effectiveness of targeting PP6 in liver disorders.
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http://dx.doi.org/10.1016/j.yexcr.2015.05.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4485549PMC
July 2015

The catalytic activity of the kinase ZAP-70 mediates basal signaling and negative feedback of the T cell receptor pathway.

Sci Signal 2015 May 19;8(377):ra49. Epub 2015 May 19.

Howard Hughes Medical Institute, UCSF, San Francisco, CA 94143, USA.

T cell activation by antigens binding to the T cell receptor (TCR) must be properly regulated to ensure normal T cell development and effective immune responses to pathogens and transformed cells while avoiding autoimmunity. The Src family kinase Lck and the Syk family kinase ZAP-70 (ζ chain-associated protein kinase of 70 kD) are sequentially activated in response to TCR engagement and serve as critical components of the TCR signaling machinery that leads to T cell activation. We performed a mass spectrometry-based phosphoproteomic study comparing the quantitative differences in the temporal dynamics of phosphorylation in stimulated and unstimulated T cells with or without inhibition of ZAP-70 catalytic activity. The data indicated that the kinase activity of ZAP-70 stimulates negative feedback pathways that target Lck and thereby modulate the phosphorylation patterns of the immunoreceptor tyrosine-based activation motifs (ITAMs) of the CD3 and ζ chain components of the TCR and of signaling molecules downstream of Lck, including ZAP-70. We developed a computational model that provides a mechanistic explanation for the experimental findings on ITAM phosphorylation in wild-type cells, ZAP-70-deficient cells, and cells with inhibited ZAP-70 catalytic activity. This model incorporated negative feedback regulation of Lck activity by the kinase activity of ZAP-70 and predicted the order in which tyrosines in the ITAMs of TCR ζ chains must be phosphorylated to be consistent with the experimental data.
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http://dx.doi.org/10.1126/scisignal.2005596DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4445242PMC
May 2015

Wide-scale quantitative phosphoproteomic analysis reveals that cold treatment of T cells closely mimics soluble antibody stimulation.

J Proteome Res 2015 May 3;14(5):2082-9. Epub 2015 Apr 3.

†Department of Chemistry, Brown University, Providence, Rhode Island 02912, United States.

The activation of T lymphocytes through antigen-mediated T cell receptor (TCR) clustering is vital in regulating the adaptive immune response. Although T cell receptor signaling has been extensively studied, the fundamental mechanisms for signal initiation are not fully understood. Reduced temperatures have initiated some of the hallmarks of TCR signaling, such as increased phosphorylation and activation on ERK and calcium release from the endoplasmic reticulum, as well as coalesced the T cell membrane microdomains. The precise mechanism of the TCR signaling initiation due to temperature change remains obscure. One critical question is whether the signaling initiated by the cold treatment of T cells differs from the signaling initiated by the cross-linking of the T cell receptor. To address this uncertainty, we performed a wide-scale, quantitative mass-spectrometry-based phosphoproteomic analysis on T cells stimulated either by temperature shifts or through the cross-linking of the TCR. Careful statistical comparisons between the two stimulations revealed a striking level of identity among the subset of 339 sites that changed significantly with both stimulations. This study demonstrates for the first time, in unprecedented detail, that T cell cold treatment was sufficient to initiate signaling patterns that were nearly identical to those of soluble antibody stimulation, shedding new light on the mechanism of activation of these critically important immune cells.
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http://dx.doi.org/10.1021/pr501172uDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4428545PMC
May 2015

Protein networks and activation of lymphocytes.

Curr Opin Immunol 2015 Apr 14;33:78-85. Epub 2015 Feb 14.

Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, RI 02912, USA. Electronic address:

The signal transduction pathways initiated by lymphocyte activation play a critical role in regulating host immunity. High-resolution mass spectrometry has accelerated the investigation of these complex and dynamic pathways by enabling the qualitative and quantitative investigation of thousands of proteins and phosphoproteins simultaneously. In addition, the unbiased and wide-scale identification of protein-protein interaction networks and protein kinase substrates in lymphocyte signaling pathways can be achieved by mass spectrometry-based approaches. Critically, the integration of these discovery-driven strategies with single-cell analysis using mass cytometry can facilitate the understanding of complex signaling phenotypes in distinct immunophenotypes.
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http://dx.doi.org/10.1016/j.coi.2015.01.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4397151PMC
April 2015

SRC homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) N-terminal tyrosine residues regulate a dynamic signaling equilibrium involving feedback of proximal T-cell receptor (TCR) signaling.

Mol Cell Proteomics 2015 Jan 14;14(1):30-40. Epub 2014 Oct 14.

From the ‡Department of Chemistry, Brown University Providence, RI 02903; §Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University Providence, RI 02903

SRC homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (SLP-76) is a cytosolic adaptor protein that plays an important role in the T-cell receptor-mediated T-cell signaling pathway. SLP-76 links proximal receptor stimulation to downstream effectors through interaction with many signaling proteins. Previous studies showed that mutation of three tyrosine residues, Tyr(112), Tyr(128), and Tyr(145), in the N terminus of SLP-76 results in severely impaired phosphorylation and activation of Itk and PLCγ1, which leads to defective calcium mobilization, Erk activation, and NFAT activation. To expand our knowledge of the role of N-terminal phosphorylation of SLP-76 from these three tyrosine sites, we characterized nearly 1000 tyrosine phosphorylation sites via mass spectrometry in SLP-76 reconstituted wild-type cells and SLP-76 mutant cells in which three tyrosine residues were replaced with phenylalanines (Y3F mutant). Mutation of the three N-terminal tyrosine residues of SLP-76 phenocopied SLP-76-deficient cells for the majority of tyrosine phosphorylation sites observed, including feedback on proximal T-cell receptor signaling proteins. Meanwhile, reversed phosphorylation changes were observed on Tyr(192) of Lck when we compared mutants to the complete removal of SLP-76. In addition, N-terminal tyrosine sites of SLP-76 also perturbed phosphorylation of Tyr(440) of Fyn, Tyr(702) of PLCγ1, Tyr(204), Tyr(397), and Tyr(69) of ZAP-70, revealing new modes of regulation on these sites. All these findings confirmed the central role of N-terminal tyrosine sites of SLP-76 in the pathway and also shed light on novel signaling events that are uniquely regulated by SLP-76 N-terminal tyrosine residues.
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http://dx.doi.org/10.1074/mcp.M114.037861DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4288261PMC
January 2015

Novel Cul3 binding proteins function to remodel E3 ligase complexes.

BMC Cell Biol 2014 Jul 10;15:28. Epub 2014 Jul 10.

Department of Biology, Portland State University, Portland, Oregon, USA.

Background: Cullins belong to a family of scaffold proteins that assemble multi-subunit ubiquitin ligase complexes to recruit protein substrates for ubiquitination via unique sets of substrate adaptor, such as Skp1 or Elongin B, and a substrate-binding protein with a conserved protein-protein interacting domain, such as leucine-rich repeats (LRR), a WD40 domain, or a zinc-finger domain. In the case of the Cullin3 (Cul3), it forms a BTB-Cul3-Rbx1 (BCR) ubiquitin ligase complex where it is believed that a BTB domain-containing protein performs dual functions where it serves as both the substrate adaptor and the substrate recognition protein.

Results: Tandem affinity purification and LC/MS-MS analysis of the BCR complex led to the identification of 10,225 peptides. After the SEQUEST algorithm and CDART program were used for protein identification and domain prediction, we discovered a group of Cul3-bound proteins that contain either the LRR or WD40 domain (CLWs). Further biochemical analysis revealed that the LRR domain-containing CLWs could bind both Cul3 and BTB domain-containing proteins. The dual binding role for the LRR domain-containing CLWs results in causing the BTB-domain protein to become a substrate instead of an adaptor.To further distinguish potential substrates from other components that are part of the BCR ubiquitin ligase complex, we altered the parameters in the SEQUEST algorithm to select for peptide fragments with a modified lysine residue. This method not only identifies the potential substrates of the BCR ubiquitin ligase complex, but it also pinpoints the lysine residue in which the post-translational modification occurs. Interestingly, none of the CLWs were identified by this method, supporting our hypothesis that CLWs were not potential substrates but rather additional components of the BCR ubiquitin ligase complex.

Conclusion: Our study identified a new set of Cul3-binding proteins known as CLWs via tandem affinity purification and LC/MS-MS analysis. Subsequently, our biochemical analysis revealed that some CLWs modify binding of BTB domain-containing proteins to the complex, causing degradation of the BTB domain-containing protein. As these CLWs were excluded from our list of substrates, we propose that CLWs serve as unique Cul3 binding proteins that provide an alternative regulatory mechanism for the complex.
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http://dx.doi.org/10.1186/1471-2121-15-28DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4107866PMC
July 2014

Mitogen-activated protein kinase phosphatase 3 (MKP-3)-deficient mice are resistant to diet-induced obesity.

Diabetes 2014 Sep 10;63(9):2924-34. Epub 2014 Apr 10.

Hallett Center for Diabetes and Endocrinology, Rhode Island Hospital, Brown University, Warren Alpert Medical School, Providence, RI Pathobiology Program, Brown University, Providence, RI

Mitogen-activated protein kinase phosphatase 3 (MKP-3) is a negative regulator of extracellular signal-related kinase signaling. Our laboratory recently demonstrated that MKP-3 plays an important role in obesity-related hyperglycemia by promoting hepatic glucose output. This study shows that MKP-3 deficiency attenuates body weight gain induced by a high-fat diet (HFD) and protects mice from developing obesity-related hepatosteatosis. Triglyceride (TG) contents are dramatically decreased in the liver of MKP-3(-/-) mice fed an HFD compared with wild-type (WT) controls. The absence of MKP-3 also reduces adiposity, possibly by repressing adipocyte differentiation. In addition, MKP-3(-/-) mice display increased energy expenditure, enhanced peripheral glucose disposal, and improved systemic insulin sensitivity. We performed global phosphoproteomic studies to search for downstream mediators of MKP-3 action in liver lipid metabolism. Our results revealed that MKP-3 deficiency increases the phosphorylation of histone deacetylase (HDAC) 1 on serine 393 by 3.3-fold and HDAC2 on serine 394 by 2.33-fold. Activities of HDAC1 and 2 are increased in the livers of MKP-3(-/-) mice fed an HFD. Reduction of HDAC1/2 activities is sufficient to restore TG content of MKP-3(-/-) primary hepatocytes to a level similar to that in WT cells.
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http://dx.doi.org/10.2337/db14-0066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4141371PMC
September 2014

Quantitative phosphoproteomic analysis identifies activation of the RET and IGF-1R/IR signaling pathways in neuroblastoma.

PLoS One 2013 11;8(12):e82513. Epub 2013 Dec 11.

Division of Pediatric Hematology-Oncology, Department of Pediatrics, The Warren Albert School of Medicine at Brown University, Providence, Rhode Island, United States of America.

Neuroblastoma is an embryonal tumor of childhood with a heterogenous clinical presentation that reflects differences in activation of complex biological signaling pathways. Protein phosphorylation is a key component of cellular signal transduction and plays a critical role in processes that control cancer cell growth and survival. We used shotgun LC/MS to compare phosphorylation between a human MYCN amplified neuroblastoma cell line (NB10), modeling a resistant tumor, and a human neural precursor cell line (NPC), modeling a normal baseline neural crest cell. 2181 unique phosphorylation sites representing 1171 proteins and 2598 phosphopeptides were found. Protein kinases accounted for 6% of the proteome, with a predominance of tyrosine kinases, supporting their prominent role in oncogenic signaling pathways. Highly abundant receptor tyrosine kinase (RTK) phosphopeptides in the NB10 cell line relative to the NPC cell line included RET, insulin-like growth factor 1 receptor/insulin receptor (IGF-1R/IR), and fibroblast growth factor receptor 1 (FGFR1). Multiple phosphorylated peptides from downstream mediators of the PI3K/AKT/mTOR and RAS pathways were also highly abundant in NB10 relative to NPC. Our analysis highlights the importance of RET, IGF-1R/IR and FGFR1 as RTKs in neuroblastoma and suggests a methodology that can be used to identify potential novel biological therapeutic targets. Furthermore, application of this previously unexploited technology in the clinic opens the possibility of providing a new wide-scale molecular signature to assess disease progression and prognosis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0082513PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3859635PMC
October 2014

Cytochrome C is tyrosine 97 phosphorylated by neuroprotective insulin treatment.

PLoS One 2013 5;8(11):e78627. Epub 2013 Nov 5.

Department of Emergency Medicine, Wayne State University School of Medicine, Detroit, Michigan, United States of America ; Cardiovascular Research Institute, Wayne State University School of Medicine, Detroit, Michigan, United States of America.

Recent advancements in isolation techniques for cytochrome c (Cytc) have allowed us to discover post-translational modifications of this protein. We previously identified two distinct tyrosine phosphorylated residues on Cytc in mammalian liver and heart that alter its electron transfer kinetics and the ability to induce apoptosis. Here we investigated the phosphorylation status of Cytc in ischemic brain and sought to determine if insulin-induced neuroprotection and inhibition of Cytc release was associated with phosphorylation of Cytc. Using an animal model of global brain ischemia, we found a ∼50% decrease in neuronal death in the CA1 hippocampal region with post-ischemic insulin administration. This insulin-mediated increase in neuronal survival was associated with inhibition of Cytc release at 24 hours of reperfusion. To investigate possible changes in the phosphorylation state of Cytc we first isolated the protein from ischemic pig brain and brain that was treated with insulin. Ischemic brains demonstrated no detectable tyrosine phosphorylation. In contrast Cytc isolated from brains treated with insulin showed robust phosphorylation of Cytc, and the phosphorylation site was unambiguously identified as Tyr97 by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry. We next confirmed these results in rats by in vivo application of insulin in the absence or presence of global brain ischemia and determined that Cytc Tyr97-phosphorylation is strongly induced under both conditions but cannot be detected in untreated controls. These data suggest a mechanism whereby Cytc is targeted for phosphorylation by insulin signaling, which may prevent its release from the mitochondria and the induction of apoptosis.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0078627PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3818486PMC
August 2014

Hepatic signaling by the mechanistic target of rapamycin complex 2 (mTORC2).

FASEB J 2014 Jan 26;28(1):300-15. Epub 2013 Sep 26.

3Division of Pediatric Endocrinology, Rhode Island Hospital, 593 Eddy Street, Providence, RI 02903, USA.

The mechanistic target of rapamycin (mTOR) exists in two complexes that regulate diverse cellular processes. mTOR complex 1 (mTORC1), the canonical target of rapamycin, has been well studied, whereas the physiological role of mTORC2 remains relatively uncharacterized. In mice in which the mTORC2 component Rictor is deleted in liver [Rictor-knockout (RKO) mice], we used genomic and phosphoproteomic analyses to characterize the role of hepatic mTORC2 in vivo. Overnight food withdrawal followed by refeeding was used to activate mTOR signaling. Rapamycin was administered before refeeding to specify mTORC2-mediated events. Hepatic mTORC2 regulated a complex gene expression and post-translational network that affects intermediary metabolism, ribosomal biogenesis, and proteasomal biogenesis. Nearly all changes in genes related to intermediary metabolic regulation were replicated in cultured fetal hepatocytes, indicating a cell-autonomous effect of mTORC2 signaling. Phosphoproteomic profiling identified mTORC2-related signaling to 144 proteins, among which were metabolic enzymes and regulators. A reduction of p38 MAPK signaling in the RKO mice represents a link between our phosphoproteomic and gene expression results. We conclude that hepatic mTORC2 exerts a broad spectrum of biological effects under physiological conditions. Our findings provide a context for the development of targeted therapies to modulate mTORC2 signaling.
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http://dx.doi.org/10.1096/fj.13-237743DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3868844PMC
January 2014

ERK positive feedback regulates a widespread network of tyrosine phosphorylation sites across canonical T cell signaling and actin cytoskeletal proteins in Jurkat T cells.

PLoS One 2013 17;8(7):e69641. Epub 2013 Jul 17.

Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown University, Providence, Rhode Island, USA.

Competing positive and negative signaling feedback pathways play a critical role in tuning the sensitivity of T cell receptor activation by creating an ultrasensitive, bistable switch to selectively enhance responses to foreign ligands while suppressing signals from self peptides. In response to T cell receptor agonist engagement, ERK is activated to positively regulate T cell receptor signaling through phosphorylation of Ser(59) Lck. To obtain a wide-scale view of the role of ERK in propagating T cell receptor signaling, a quantitative phosphoproteomic analysis of 322 tyrosine phosphorylation sites by mass spectrometry was performed on the human Jurkat T cell line in the presence of U0126, an inhibitor of ERK activation. Relative to controls, U0126-treated cells showed constitutive decreases in phosphorylation through a T cell receptor stimulation time course on tyrosine residues found on upstream signaling proteins (CD3 chains, Lck, ZAP-70), as well as downstream signaling proteins (VAV1, PLCγ1, Itk, NCK1). Additional constitutive decreases in phosphorylation were found on the majority of identified proteins implicated in the regulation of actin cytoskeleton pathway. Although the majority of identified sites on T cell receptor signaling proteins showed decreases in phosphorylation, Tyr(598) of ZAP-70 showed elevated phosphorylation in response to U0126 treatment, suggesting differential regulation of this site via ERK feedback. These findings shed new light on ERK's role in positive feedback in T cell receptor signaling and reveal novel signaling events that are regulated by this kinase, which may fine tune T cell receptor activation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0069641PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3714263PMC
March 2014

Identification of sucrose non-fermenting-related kinase (SNRK) as a suppressor of adipocyte inflammation.

Diabetes 2013 Jul 21;62(7):2396-409. Epub 2013 Mar 21.

Hallett Center for Diabetes and Endocrinology, Rhode Island Hospital, Warren Alpert Medical School of Brown University, Providence, Rhode Island, USA.

In this study, the role of sucrose non-fermenting-related kinase (SNRK) in white adipocyte biology was investigated. SNRK is abundantly expressed in adipose tissue, and the expression level is decreased in obese mice. SNRK expression is repressed by inflammatory signals but increased by insulin sensitizer in cultured adipocytes. In vivo, adipose tissue SNRK expression can be decreased by lipid injection but enhanced by macrophage ablation. Knocking down SNRK in cultured adipocytes activates both JNK and IKKβ pathways as well as promotes lipolysis. Insulin-stimulated Akt phosphorylation and glucose uptake are impaired in SNRK knockdown adipocytes. Phosphoproteomic analysis with SNRK knockdown adipocytes revealed significantly decreased phosphorylation of 49 proteins by 25% or more, which are involved in various aspects of adipocyte function with a clear indication of attenuated mTORC1 signaling. Phosphorylation of 43 proteins is significantly increased by onefold or higher, among which several proteins are known to be involved in inflammatory pathways. The inflammatory responses in SNRK knockdown adipocytes can be partially attributable to defective mTORC1 signaling, since rapamycin treatment activates IKKβ and induces lipolysis in adipocytes. In summary, SNRK may act as a suppressor of adipocyte inflammation and its presence is necessary for maintaining normal adipocyte function.
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http://dx.doi.org/10.2337/db12-1081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3712026PMC
July 2013

Quantitative phosphoproteomics reveals SLP-76 dependent regulation of PAG and Src family kinases in T cells.

PLoS One 2012 11;7(10):e46725. Epub 2012 Oct 11.

Department of Chemistry, Brown University, Providence, Rhode Island, United States of America.

The SH2-domain-containing leukocyte protein of 76 kDa (SLP-76) plays a critical scaffolding role in T cell receptor (TCR) signaling. As an adaptor protein that contains multiple protein-binding domains, SLP-76 interacts with many signaling molecules and links proximal receptor stimulation to downstream effectors. The function of SLP-76 in TCR signaling has been widely studied using the Jurkat human leukaemic T cell line through protein disruption or site-directed mutagenesis. However, a wide-scale characterization of SLP-76-dependant phosphorylation events is still lacking. Quantitative profiling of over a hundred tyrosine phosphorylation sites revealed new modes of regulation of phosphorylation of PAG, PI3K, and WASP while reconfirming previously established regulation of Itk, PLCγ, and Erk phosphorylation by SLP-76. The absence of SLP-76 also perturbed the phosphorylation of Src family kinases (SFKs) Lck and Fyn, and subsequently a large number of SFK-regulated signaling molecules. Altogether our data suggests unique modes of regulation of positive and negative feedback pathways in T cells by SLP-76, reconfirming its central role in the pathway.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0046725PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3469622PMC
April 2013

Phosphoproteomic analysis of liver homogenates.

Methods Mol Biol 2012 ;909:151-63

Department of Pediatrics, Brown University and Rhode Island Hospital, Providence, RI, USA.

Regulation of protein function via reversible phosphorylation is an essential component of cell signaling. Our ability to understand complex phosphorylation networks in the physiological context of a whole organism or tissue remains limited. This is largely due to the technical challenge of isolating serine/threonine phosphorylated peptides from a tissue sample. In the present study, we developed a phosphoproteomic strategy to purify and identify phosphopeptides from a tissue sample by employing protein gel filtration, protein strong anion exchange and strong cation exchange (SCX) chromatography, peptide SCX chromatography, and TiO(2) affinity purification. By applying this strategy to the mass spectrometry-based analysis of rat liver homogenates, we were able to identify with high confidence and quantify over 4,000 unique phosphopeptides. Finally, the reproducibility of our methodology was demonstrated by its application to analysis of the mammalian Target of Rapamycin (mTOR) signaling pathways in liver samples obtained from rats in which hepatic mTOR was activated by refeeding following a period of fasting.
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http://dx.doi.org/10.1007/978-1-61779-959-4_11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3581044PMC
December 2012

Lectin site ligation of CR3 induces conformational changes and signaling.

J Biol Chem 2012 Jan 9;287(5):3337-48. Epub 2011 Dec 9.

Division of Surgical Research, Department of Surgery, Rhode Island Hospital and the Warren Alpert Medical School of Brown University, Providence, Rhode Island 02903, USA.

Neutrophils provide an innate immune response to tissues infected with fungal pathogens such as Candida albicans. This response is tightly regulated in part through the interaction of integrins with extracellular matrix ligands that are distributed within infected tissues. The β(2) integrin, CR3 (CD11b/CD18), is unique among integrins in containing a lectin-like domain that binds the fungal pathogen-associated molecular pattern β-glucan and serves as the dominant receptor for recognition of fungal pathogens by human granulocytes. β-Glucan, when isolated in soluble form, has been shown to be a safe and effective immune potentiator when administered therapeutically. Currently a pharmaceutical grade preparation of β-glucan is in several clinical trials with an anti-cancer indication. CR3 binding of extracellular matrix, carbohydrate, or both ligands simultaneously differentially regulates neutrophil function through a mechanism not clearly understood. Using FRET reporters, we interrogated the effects of soluble β-glucan on intracellular and extracellular CR3 structure. Although the canonical CR3 ligand fibrinogen induced full activation, β-glucan alone or in conjunction with fibrinogen stabilized an intermediate conformation with moderate headpiece extension and full cytoplasmic tail separation. A set of phosphopeptides differentially regulated by β-glucan in a CR3-dependent manner were identified using functional proteomics and found to be enriched for signaling molecules and proteins involved in transcriptional regulation, mRNA processing, and alternative splicing. These data confirm that CR3 is a signaling pattern recognition receptor for β-glucan and represent the first direct evidence of soluble β-glucan binding and affecting a signaling-competent intermediate CR3 conformation on living cells.
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http://dx.doi.org/10.1074/jbc.M111.298307DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3270988PMC
January 2012