Analysis of the STAT3 interactome using in-situ biotinylation and SILAC

Blumert, Conny; Kalkhof, Stefan; Brocke-Heidrich, Katja; Kohajda, Tibor; Bergen, Martin M. Von; Horn, Friedemann

doi:10.1016/j.jprot.2013.08.021

Cited by 10 publications

(11 citation statements)

References 75 publications

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“…A total of 149 known or predicted proteins were identified, which were repeatedly quantified in at least two of the three independent experiments in RAW-Bio-USTAT1 cells, but not detected or where at relatively low abundance in RAW-Bio-CT cells (Table S1). Among them was the closely related factor STAT3, which forms heterodimers with STAT1, as previous studies suggested (Buro et al, 2010;Blumert et al, 2013). Interaction of unphosphorylated STAT1 with STAT3, eEF1A, Rps3, Anxa2, Ybx1 and Bag2 were confirmed by a further immunoprecipitation (IP) and immunoblotting experiment, indicating that the interaction between these proteins and unphosphorylated STAT1 occurs in vivo (Fig.…”

Section: Characterization Of the Unphosphorylated Stat1 Protein Intersupporting

confidence: 63%

“…To map the unphosphorylated STAT1 interactome in macrophages, a one-step pull-down procedure, using streptavidin and biotinylated (Bio-tagged) unphosphorylated STAT1, was undertaken in RAW264.7 cells; in this procedure, the Bio tag allows a high affinity binding without affecting protein function (Blumert et al, 2013). We constructed a multi-cistronic lentiviral construct encoding a Bio tag fused to the Cterminus of the unphosphorylated STAT1 protein and Escherichia coli biotin ligase (BirA) separated by a self-cleaving 2A peptide sequence (P2A) derived from porcine teschovirus-1 (Kim et al, 2011) (Fig.…”

Section: Characterization Of the Unphosphorylated Stat1 Protein Intermentioning

confidence: 99%

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Unphosphorylated STAT1 represses apoptosis in macrophages during Mycobacterium t uberculosis infection

Yao

Chen

et al. 2017

Journal of Cell Science

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In murine macrophages infected with Mycobacterium tuberculosis (Mtb), the level of phosphorylated STAT1 (P-STAT1), which drives the expression of many pro-apoptosis genes, increases quickly but then declines over a period of hours. By contrast, infection induces a continued increase in the level of unphosphorylated STAT1 that persists for several days. Here, we found that the level of unphosphorylated STAT1 correlated with the intracellular bacterial burden during the later stages of infection. To investigate the significance of a high level of unphosphorylated STAT1, we increased its concentration exogenously, and found that the apoptosis rate induced by Mtb was sufficiently decreased. Further experiments confirmed that unphosphorylated STAT1 affects the expression of several immune-associated genes and lessens the sensitivity of macrophages to CD95 (FAS)-mediated apoptosis during Mtb infection. Furthermore, we characterized 149 proteins that interacted with unphosphorylated STAT1 and the interactome network. The cooperation between unphosphorylated STAT1 and STAT3 results in downregulation of CD95 expression. Additionally, we verified that unphosphorylated STAT1 and IFIT1 competed for binding to eEF1A. Taken together, our data show that the role of unphosphorylated STAT1 differs from that of P-STAT1, and represses apoptosis in macrophages to promote immune evasion during Mtb infection.

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Section: Characterization Of the Unphosphorylated Stat1 Protein Intersupporting

confidence: 63%

Section: Characterization Of the Unphosphorylated Stat1 Protein Intermentioning

confidence: 99%

Unphosphorylated STAT1 represses apoptosis in macrophages during Mycobacterium t uberculosis infection

Yao

Chen

et al. 2017

Journal of Cell Science

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“…An expression vector of biotinylated STAT1 was prepared as has been descripted recently for STAT3 [ 14 ]. Briefly, Rc/CMV-STAT1-Bio was constructed by cloning the human STAT1 cDNA into the EcoRI and SalI sites of pBluescript II KS (−).…”

Section: Methodsmentioning

confidence: 99%

“…Subsequently, the cDNA was inserted into the KpnI and XbaI sites of the expression vector pcDNA 3.1+ (Invitrogen, Karlsruhe, Germany). The expression vector for GFP-Bio was prepared as descripted [ 14 ].…”

Section: Methodsmentioning

confidence: 99%

PIPINO: A Software Package to Facilitate the Identification of Protein-Protein Interactions from Affinity Purification Mass Spectrometry Data

Kalkhof

Schildbach

Blumert

et al. 2016

BioMed Research International

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The functionality of most proteins is regulated by protein-protein interactions. Hence, the comprehensive characterization of the interactome is the next milestone on the path to understand the biochemistry of the cell. A powerful method to detect protein-protein interactions is a combination of coimmunoprecipitation or affinity purification with quantitative mass spectrometry. Nevertheless, both methods tend to precipitate a high number of background proteins due to nonspecific interactions. To address this challenge the software Protein-Protein-Interaction-Optimizer (PIPINO) was developed to perform an automated data analysis, to facilitate the selection of bona fide binding partners, and to compare the dynamic of interaction networks. In this study we investigated the STAT1 interaction network and its activation dependent dynamics. Stable isotope labeling by amino acids in cell culture (SILAC) was applied to analyze the STAT1 interactome after streptavidin pull-down of biotagged STAT1 from human embryonic kidney 293T cells with and without activation. Starting from more than 2,000 captured proteins 30 potential STAT1 interaction partners were extracted. Interestingly, more than 50% of these were already reported or predicted to bind STAT1. Furthermore, 16 proteins were found to affect the binding behavior depending on STAT1 phosphorylation such as STAT3 or the importin subunits alpha 1 and alpha 6.

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“…Additionally, the quantitative analysis of PTMs, e.g. phosphorylation [69] and the detection of protein-protein interactions can be facilitated by SILAC [70,71]. Finally, pulsed SILAC could be used for the determination of protein synthesis, degradation, and turnover rates of H. pylori [72].…”

Section: Half Of the H Pylori Proteome Was Reproducibly Quantifiedmentioning

confidence: 99%

Stable isotope labeling by amino acids in cell culture based proteomics reveals differences in protein abundances between spiral and coccoid forms of the gastric pathogen Helicobacter pylori

Müller

Pernitzsch

Haange

et al. 2015

Journal of Proteomics

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Analysis of the STAT3 interactome using in-situ biotinylation and SILAC

Cited by 10 publications

References 75 publications

Unphosphorylated STAT1 represses apoptosis in macrophages during Mycobacterium t uberculosis infection

Unphosphorylated STAT1 represses apoptosis in macrophages during Mycobacterium t uberculosis infection

PIPINO: A Software Package to Facilitate the Identification of Protein-Protein Interactions from Affinity Purification Mass Spectrometry Data

Stable isotope labeling by amino acids in cell culture based proteomics reveals differences in protein abundances between spiral and coccoid forms of the gastric pathogen Helicobacter pylori

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