Functional analysis of the nuclear proteome of human A549 alveolar epithelial cells by HPLC-high resolution 2-D gel electrophoresis

Forbus, Jeffery; Spratt, Heidi; Wiktorowicz, John E.; Wu, Ziyan; Boldogh, István; Denner, Larry; Kurosky, Alexander; Brasier, Robert C.; Luxon, Bruce A.; Brasier, Allan R.

doi:10.1002/pmic.200500652

Cited by 35 publications

(55 citation statements)

References 55 publications

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“…Proteins were identified by peptide mass fingerprint analysis using the National Center for Biology Information protein database and Mascot algorithm. Positive protein identifications were accepted for proteins having expectation scores of 1 ϫ 10 Ϫ3 or smaller as we previously reported (28).…”

Section: Protein Composition Of Gmr␤ Immunoprecipitatesmentioning

confidence: 99%

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Cross-Talk between ICAM-1 and GM-CSF Receptor Signaling Modulates Eosinophil Survival and Activation

Pazdrak

Young

Stafford

et al. 2008

The Journal of Immunology

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Reversal of eosinophilic inflammation has been an elusive therapeutic goal in the management of asthma pathogenesis. In this regard, GM-CSF is a primary candidate cytokine regulating eosinophil activation and survival in the lung; however, its molecular mechanism of propagation and maintenance of stimulated eosinophil activation is not well understood. In this study, we elucidate those late interactions occurring between the GM-CSF receptor and activated eosinophil signaling molecules. Using coimmunoprecipitation with GM-CSF-stimulated eosinophils, we have identified that the GM-CSF receptor β-chain (GMRβ) interacted with ICAM-1 and Shp2 phosphatase, as well as Slp76 and ADAP adaptor proteins. Separate experiments using affinity binding with a tyrosine-phosphorylated peptide containing an ITIM (ICAM-1 residues 480–488) showed binding to Shp2 phosphatase and GMRβ. However, the interaction of GMRβ with the phosphorylated ICAM-1-derived peptide was observed only with stimulated eosinophil lysates, suggesting that the interaction of GMRβ with ICAM-1 required phosphorylated Shp2 and/or phosphorylated GMRβ. Importantly, we found that inhibition of ICAM-1 in activated eosinophils blocked GM-CSF-induced expression of c-fos, c-myc, IL-8, and TNF-α. Moreover, inhibition of ICAM-1 expression with either antisense oligonucleotide or an ICAM-1-blocking Ab effectively inhibited ERK activation and eosinophil survival. We concluded that the interaction between ICAM-1 and the GM-CSF receptor was essential for GM-CSF-induced eosinophil activation and survival. Taken together, these results provide novel mechanistic insights defining the interaction between ICAM-1 and the GM-CSF receptor and highlight the importance of targeting ICAM-1 and GM-CSF/IL-5/IL-3 receptor systems as a therapeutic strategy to counter eosinophilia in asthma.

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Section: Protein Composition Of Gmr␤ Immunoprecipitatesmentioning

confidence: 99%

“…After staining with Sypro Ruby fluorescent stain (Bio-Rad), UV-visible bands were excised and subjected to mass fingerprinting after trypsin digestion (28). Mass spectra of peptide digests were obtained using a model 4800, MALDI-TOF-TOF-MS (Applied Biosystems).…”

Section: Protein Composition Of Gmr␤ Immunoprecipitatesmentioning

confidence: 99%

Cross-Talk between ICAM-1 and GM-CSF Receptor Signaling Modulates Eosinophil Survival and Activation

Pazdrak

Young

Stafford

et al. 2008

The Journal of Immunology

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“…To isolate nuclear and cytoplasmic proteins, cells were harvested in PBS and centrifuged. The pellets were resuspended sequentially in low salt, sucrose, and high salt solutions to obtain cytosolic and highly purified nuclear extracts, as previously described (Forbus et al, 2006). Protein concentration of the supernatant was measured by the Bradford method.…”

Section: Western Blot Analysismentioning

confidence: 99%

LECT2 association with macrophage-mediated killing of Helicobacter pylori by activating NF-κB and nitric oxide production

Shen

Chen

et al. 2016

Genet. Mol. Res.

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ABSTRACT. Helicobacter pylori employs unique methods to colonize the stomach, which induces chronic inflammation. It is also able to avoid eradication by macrophages and other immune cells. Leukocyte cell-derived chemotaxin 2 (LECT2), a multi-functional cytokine involved in many pathological conditions, has recently been shown to activate macrophages via the CD209a receptor. Therefore, we aimed to investigate the effects of LECT2 on H. pylori-infected macrophages. Macrophages were treated with recombinant LECT2, and both their ability to kill H. pylori and produce nitric oxide were analyzed. Western blot was performed to determine nuclear translocation and protein phosphorylation of p65, a subunit of nuclear factor (NF)-kB. Transfection experiments were performed to analyze the signaling pathway of LECT2 in macrophages. We found that treatment with LECT2 enhanced H. pylori killing and nitric oxide production in macrophages. In addition, DNA-binding activity and nuclear translocation of p65 were up-regulated by LECT2 treatment. Furthermore, we found that NFkB activation by LECT2 was mediated by Raf-1 in macrophages, and Raf-1 phosphorylation was specifically altered in response to LECT2. Moreover, LECT2 induced Ser28 phosphorylation in the intracellular domain of CD209a. CD209a Ser28 phosphorylation was required for LECT2-induced Raf-1 and NF-kB activation in RAW264.7 macrophages. Our study showed that the effects of LECT2 on H. pylori killing and nitric oxide production were dependent on CD209a phosphorylation, Raf-1, and NF-kB activation. Together, these results demonstrate for the first time that exposure to LECT2 can modulate specific intracellular mechanisms downstream of CD209a to enhance H. pylori killing and nitric oxide production in macrophages.

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“…After 4 h, cells were washed twice with phosphate-buffered saline (PBS; 137 mM NaCl, 10 mM phosphate, 2.7 mM KCl, pH 7.4), and nuclei were prepared by lysis in hypotonic buffer (29). Protein-chromatin cross-linking was performed using an optimized two-step protocol with disuccinimidyl glutarate (Pierce) followed by formaldehyde (30).…”

Section: Identification Of Rela-binding Protein By Affinity Tandem Mamentioning

confidence: 99%

Modulation of Gene Expression Regulated by the Transcription Factor NF-κB/RelA

Zhao

Tian

et al. 2014

Journal of Biological Chemistry

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Background:Interacting proteins modulate the activity of NF-B/RelA transcription factor and expression of its targets. Results: By analyzing gene expression, protein binding, and DNA binding, we inferred and characterized 8349 such modulations. Conclusion: Different modulator groups affect separate pathways. Significance: We provide new insight into the activity of NF-B/RelA. Our inference model can be applied to other processes and pathways.

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Functional analysis of the nuclear proteome of human A549 alveolar epithelial cells by HPLC-high resolution 2-D gel electrophoresis

Cited by 35 publications

References 55 publications

Cross-Talk between ICAM-1 and GM-CSF Receptor Signaling Modulates Eosinophil Survival and Activation

Cross-Talk between ICAM-1 and GM-CSF Receptor Signaling Modulates Eosinophil Survival and Activation

LECT2 association with macrophage-mediated killing of Helicobacter pylori by activating NF-κB and nitric oxide production

Modulation of Gene Expression Regulated by the Transcription Factor NF-κB/RelA

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