Label-free quantitative phosphoproteomic profiling of cellular response induced by an insect cytokine paralytic peptide

Song, Liang; Wang, Fei; Dong, Zhaoming; Hua, Xiaoting; Xia, Qingyou

doi:10.1016/j.jprot.2016.11.018

Cited by 14 publications

(17 citation statements)

References 49 publications

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“…Subsequent tandem MS analysis identified a phosphorylated peptide, RMS2STDNSPDVIK, in which the Ser-186 residue, which is not located in the BTB domain or the zinc finger domain of silkworm BR-C, was phosphorylated. In addition, in our previous report (12) we extracted total proteins from the fat bodies of silkworm larvae on day 3 of the 5th instar and enriched phosphorylated peptides via TiO 2 enrichment. The resulting phosphopeptides were analyzed by LC-MS/MS.…”

Section: Lc-ms/ms Identification Of Br-c Ser-186 As a Phosphorylationmentioning

confidence: 99%

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Protein kinase A-mediated phosphorylation of the Broad-Complex transcription factor in silkworm suppresses its transcriptional activity

Qian

Gang

Zhang

et al. 2017

Journal of Biological Chemistry

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The insect-specific transcription factor Broad-Complex (BR-C) is transcriptionally activated by the steroid 20-hydroxyecdysone (20E) and regulates the expression of many target genes involved in insect growth and development. However, although the transcriptional regulation of BR-C proteins has been well studied, how BR-C is regulated at post-transcription and -translation levels is poorly understood. To this end, using liquid chromatography-tandem mass spectrometry analysis, we identified residue Ser-186 as a phosphorylation site of BR-C in silkworm. Site-directed mutagenesis and treatment with specific kinase activators and inhibitors indicated that the Ser-186 residue in silkworm BR-C is phosphorylated by protein kinase A (PKA). Immunostaining assays disclosed that PKA-mediated phosphorylation of silkworm BR-C has no effect on its nuclear import. However, luciferase reporter analysis, electrophoretic mobility shift assays, and chromatin immunoprecipitation revealed that the PKA phosphorylation event suppresses the transcriptional activation of silkworm BR-C target genes and that this inhibition was caused by repression of BR-C binding to its DNA targets. Of note, both and experiments disclosed that a continuous 20E signal inhibits the PKA-mediated BR-C phosphorylation and also the cAMP/PKA pathway, indicating that 20E's inhibitory effect on PKA-mediated phosphorylation of silkworm BR-C contributes to maintaining BR-C transcriptional activity. In conclusion, our findings indicate that PKA-mediated phosphorylation inhibits silkworm BR-C activity by interfering with its binding to DNA and that 20E signaling relieves PKA-mediated phosphorylation of BR-C, thereby maintaining its transcriptional activity.

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Section: Lc-ms/ms Identification Of Br-c Ser-186 As a Phosphorylationmentioning

confidence: 99%

“…In addition, in our previous report (12) we extracted total proteins from the fat bodies of five silkworm larvae. The collected fat bodies were first homogenized in RIPA lysis buffer, which contained a mixture of proteinase inhibitors, on ice.…”

Section: Protein Purification and Lc-ms/ms Analysismentioning

confidence: 99%

Protein kinase A-mediated phosphorylation of the Broad-Complex transcription factor in silkworm suppresses its transcriptional activity

Qian

Gang

Zhang

et al. 2017

Journal of Biological Chemistry

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“…To get insight into the functional implication of the proteins found to contain Cys ox -PTMs under basal or oxidative stress conditions, a Label Free Quantification (LFQ) method was applied considering only Mal-PEG 2 -Bio-labeled Cys identified in at least two out of the three independent replicates per condition to eliminate possible "false positive" hits (51,52). The accuracy of our approach was first validated by the identification of Cys ox-PTM sites previously documented to be oxidized in vitro or in vivo ( fig.…”

Section: Identification Of Pathways Related To Virus-host Interactionmentioning

confidence: 99%

“…For label-free quantification (LFQ), only peptides containing at least 1 Cys ox-PTMs and detected in at least two out of the three biological replicates were considered for spectral counting. Scaffold algorithms were used to perform standard cutoffs of significance for peptide ion signal peak intensity-based quantification (52,99). Peptide identifications were accepted if they could be established at greater than 95.0% probability by the Peptide Prophet algorithm (100) with Scaffold delta-mass correction and a false discovery rate (FDR) <1%.…”

Section: Lc-ms/ms Data Analysismentioning

confidence: 99%

Pinpointing of cysteine oxidation sites in vivo by high-resolution proteomics reveals mechanism of redox-dependent inhibition of STING

Cuervo

Fortin

Caron

et al. 2020

Preprint

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Brief summary of the main results.The function of proteins is regulated by post-translational modifications, among which reversible oxidation of Cys recently emerged as a key component. Comprehension of redox regulation of cellular responses requires identification of specific oxidation sites in vivo. Using a bioswitch method to specifically label Cys subjected to reversible oxidation coupled to mass spectrometry, we identified thousands of novel oxidation sites. Many are relevant to virus -host interaction pathways. Here, we focused on the oxid ation of STING, an adaptor critical for activating the innate immune type I interferon pathway engaged upon cytosolic DNA sensing. Molecular studies led us to establish a new model in which STING Cys 148 is oxidized at basal levels, while Cys 206 oxidation is induced by oxidative stress and ligand binding. We show that oxidation of Cys 206 has an inhibitory function to prevent STING hyperactivation. This study provides ground for novel research avenues aimed at designing therapeutics that target this pathway. AbstractProtein function is regulated by post-translational modifications, among which reversible oxidation of Cys (Cys ox-PTM) emerged as a key regulatory mechanism of cellular responses. The redox regulation of virus-host interactions is well documented, but in most cases, proteins subjected to Cys ox-PTM remain unknown. The identification of Cys ox-PTM sites in vivo is essential to underpin our understanding of the mechanisms of the redox regulation. In this study, we present a proteome-wide identification of reversible Cys ox-PTM sites in vivo during stimulation by oxidants using a maleimide-based bioswitch method coupled to mass spectrometry. We identified 2720 unique Cys ox-PTM sites encompassing 1473 proteins with distinct abundance, location and functions. Label-free quantification (LFQ)-based analysis revealed the enrichment of ox-PTM in numerous pathways, many relevant to virus-host interaction. Here, we focused on the oxidation of STING, the central adaptor of the innate immune type I interferon 2 pathway induced upon detection of cytosolic DNA. We provide the first in vivo demonstration of reversible oxidation of Cys 148 and Cys 206 of STING. Molecular analyses led us to establish a new model in which Cys 148 oxidation is constitutive, while Cys 206 oxidation is inducible by oxidative stress or by the natural ligand 2'3'-cGAMP. We show that oxidation of Cys 206 has an inhibitory function to prevent STING hyperactivation through the constraint of a conformational change associated with the formation of inactive polymers containing intermolecular disulfide bonds. This provides new ground for the design of therapies targeting STING relevant to autoinflammatory disorders, immunotherapies and vaccines.

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“…/www.reactome.org/), IPA (IPA, QIAGEN, Redwood City, CA, USA, www.qiagen.com/ingenuity), Cytoscape (http://www. cytoscape.org/) ve KEGG (http://www.genome.jp/kegg/) gibi programlar kullanılabilmektedir(75)(76)(77)(78).…”

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confidence: 99%

An Overview of The Phosphoproteomic Workflow

Sürmen¹,

Sürmen²,

Pençe³

2018

Experimed

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İnsan genom projesinin tamamlanmasıyla birlikte-protein kodlayan yaklaşık 20.000 gen ortaya çıkarılmış, bu genlere ait mutasyonlar ve ekspresyon farklılıkları ortaya konulmuştur (1). İnsan genomunun haritalanması hastalıkların moleküler mekanizmasının anlaşılmasında, erken tespitinde ve tedavisinde büyük umut kaynağı olmuştur. Kapsamlı genomik çalışmalarla tespit edilen mutasyonlar ve ekspresyon farklılıkları çeşitli hastalıklarda rol oynayan sinyal yolaklarına olan bakış açımızın gelişmesini sağlarken kanser gibi moleküler mekanizması kompleks olan heterojen hastalıkların araştırılmasında sadece genomik analizlerin yeterli olmadığı da anlaşılmıştır. Bununla birlikte yaşanan genomik devrim; protein, transkript, metabolit veya lipid profillerinin biyoniformatik destekle topluca değerlendirildiği genom sonrası "omik" platformlarının gelişmesine zemin hazırlamıştır (2, 3). Hücrenin fonksiyonel makromolekülleri olan proteinlerin; fizyolojik ve patolojik süreçlerdeki rolünü daha iyi anlamaya yönelik yapılan proteomik çalışmalar son onbeş yıl içerisinde özellikle kanser araştırmalarında önem kazanmıştır (4, 5). Post translasyonel değişiklikler (PTM'ler) ve izoform varyasyon nedeniyle genomun aksine çok daha çeşitli olan proteom aynı zamanda dinamik yapıdadır. Genellikle geri dönüşümlü modifikasyonlar olan fosforilasyonlar, proteinleri aktif ya da inaktif formlarına dönüştürürler. Buna bağlı olarak sinyal iletimi, büyüme ve apoptoz gibi hücresel süreçlerin düzenlenmesinde rol oynayan fosforilasyonlar,

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Label-free quantitative phosphoproteomic profiling of cellular response induced by an insect cytokine paralytic peptide

Cited by 14 publications

References 49 publications

Protein kinase A-mediated phosphorylation of the Broad-Complex transcription factor in silkworm suppresses its transcriptional activity

Protein kinase A-mediated phosphorylation of the Broad-Complex transcription factor in silkworm suppresses its transcriptional activity

Pinpointing of cysteine oxidation sites in vivo by high-resolution proteomics reveals mechanism of redox-dependent inhibition of STING

An Overview of The Phosphoproteomic Workflow

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