Binase penetration into alveolar epithelial cells does not induce cell death

Cabrera-Fuentes, Héctor A.; Kalacheva, N. V.; Mukhametshina, Regina T.; Zelenikhin, P. V.; Kolpakov, A. I.; Barreto, Guillermo; Preissner, Klaus T.; Ilinskaya, О. N.

doi:10.1134/s199075081204004x

Cited by 4 publications

(5 citation statements)

References 21 publications

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“…Figure A shows a schematic representation of the experimental workflow. Primary ATII cells from adult mouse lung were isolated and cultured as previously described . Nonlabeled ATII cells or MLE-12 cells were mixed with lung tissue from SILAC-mice at a ratio of 1:1 (wet weight/wet weight).…”

Section: Resultsmentioning

confidence: 99%

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Quantitative Proteome Analysis of Alveolar Type-II Cells Reveals a Connection of Integrin Receptor Subunits Beta 2/6 and WNT Signaling

Mukhametshina

Ruhs²,

Singh³

et al. 2013

J. Proteome Res.

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Alveolar type-II cells (ATII cells) are lung progenitor cells responsible for regeneration of alveolar epithelium during homeostatic turnover and in response to injury. Characterization of ATII cells will have a profound impact on our understanding and treatment of lung disease. The identification of novel ATII cell-surface proteins can be used for sorting and enrichment of these cells for further characterization. Here we combined a high-resolution mass spectrometry-based membrane proteomic approach using lungs of the SILAC mice with an Affymetrix microarray-based transcriptome analysis of ATII cells. We identified 16 proteins that are enriched in the membrane fraction of ATII cells and whose genes are highly expressed in these cells. Interestingly, we confirmed our data for two of these genes, integrin beta 2 and 6 (Itgb2 and Itgb6), by qRT-PCR expression analysis and Western blot analysis of protein extracts. Moreover, flow cytometry and immunohistochemistry in adult lung revealed that ITGB2 and ITGB6 are present in subpopulations of surfactant-associated-protein-C-positive cells, suggesting the existence of different types of ATII cells. Furthermore, analysis of the Itgb2(-/-) mice showed that Itgb2 is required for proper WNT signaling regulation in the lung.

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Section: Resultsmentioning

confidence: 99%

“…Primary alveolar type II cells (ATII cells) were isolated from C57BL/6 mice, as previously described, with minor modifications. Crude cells suspensions from the lungs were prepared by intratracheal instillation of agarose containing dispase (BD Heidelberg, cat.…”

Section: Methodsmentioning

confidence: 99%

Quantitative Proteome Analysis of Alveolar Type-II Cells Reveals a Connection of Integrin Receptor Subunits Beta 2/6 and WNT Signaling

Mukhametshina

Ruhs²,

Singh³

et al. 2013

J. Proteome Res.

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“…Binase (monomeric form of 12.3 kDa, 109 amino acid residues, pI value of 9.5) was isolated as homogenous protein from cultural fluid, according to the procedure described by Makarov et al [ 22 ], and enzyme purity was confirmed by electrophoresis [ 7 , 8 , 12 , 13 ]. Catalytic activity of binase toward yeast RNA was 14 × 106 U/mg protein [ 23 ].…”

Section: Methodsmentioning

confidence: 99%

“…Xenogeneic, extracellular cytotoxic ribonucleases (RNases) of bacterial or eukaryotic origin, that trigger apoptosis/cytotoxicity in tumor and other cells, may have a great potential as anticancer drugs [ 7 – 11 ]. We earlier demonstrated the selective cytotoxic action of the RNase binase from Bacillus pumilus (formerly designated B. intermedius ) towards lung carcinoma and leukemic and ovarian cancer cells [ 8 , 12 – 14 ] and found that cell sensitivity towards xenogeneic RNases is related to expression of oncogenes ras, kit, and AML1 [ 15 – 17 ].…”

Section: Introductionmentioning

confidence: 99%

Antitumor Macrophage Response to Bacillus pumilus Ribonuclease (Binase)

Макеева

Rodriguez-Montesinos

Zelenikhin

et al. 2017

Mediators of Inflammation

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Extracellular bacterial ribonucleases such as binase from Bacillus pumilus possess cytotoxic activity against tumor cells with a potential for clinical application. Moreover, they may induce activation of tumor-derived macrophages either into the M1-phenotype with well-documented functions in the regulation of the antitumor immune response or into M2-macrophages that may stimulate tumor growth, metastasis, and angiogenesis. In this study, binase or endogenous RNase1 (but not RNA or short oligonucleotides) stimulated the expression of activated NF-κB p65 subunit in macrophages. Since no changes in MyD88 and TRIF adaptor protein expression were observed, toll-like receptors may not be involved in RNase-related NF-κB pathway activation. In addition, short exposure (0.5 hr) to binase induced the release of cytokines such as IL-6, МСР-1, or TNF-α (but not IL-4 and IL-10), indicative for the polarization into antitumor M1-macrophages. Thus, we revealed increased expression of activated NF-κB p65 subunit in macrophages upon stimulation by binase and RNase1, but not RNA or short oligonucleotides.

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“…After electrophoresis, the proteins were transferred to a nitrocellulose membrane by semi-dry electroblotting. Anti-binase antibodies were isolated from rabbit blood as described earlier [31] . Antibodies were visualised using anti-rabbit IgG-POD secondary antibodies (Sigma) and the LumiLight detection system (Roche Diagnostics).…”

Section: Methodsmentioning

confidence: 99%

New Insight into Secreted Ribonuclease Structure: Binase Is a Natural Dimer

et al. 2014

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The biological effects of ribonucleases (RNases), such as the control of the blood vessels growth, the toxicity towards tumour cells and antiviral activity, require a detailed explanation. One of the most intriguing properties of RNases which can contribute to their biological effects is the ability to form dimers, which facilitates efficient RNA hydrolysis and the evasion of ribonuclease inhibitor. Dimeric forms of microbial RNase binase secreted by Bacillus pumilus (former B. intermedius) have only been found in crystals to date. Our study is the first report directly confirming the existence of binase dimers in solution and under natural conditions of enzyme biosynthesis and secretion by bacilli. Using different variants of gel electrophoresis, immunoblotting, size-exclusion chromatography and mass-spectrometry, we revealed that binase is a stable natural dimer with high catalytic activity.

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Binase penetration into alveolar epithelial cells does not induce cell death

Cited by 4 publications

References 21 publications

Quantitative Proteome Analysis of Alveolar Type-II Cells Reveals a Connection of Integrin Receptor Subunits Beta 2/6 and WNT Signaling

Quantitative Proteome Analysis of Alveolar Type-II Cells Reveals a Connection of Integrin Receptor Subunits Beta 2/6 and WNT Signaling

Antitumor Macrophage Response to Bacillus pumilus Ribonuclease (Binase)

New Insight into Secreted Ribonuclease Structure: Binase Is a Natural Dimer

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