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Ubiquitin is a versatile scaffold protein for the generation of molecules with de novo binding and advantageous drug-like properties.

Job F, Settele F, Lorey S, Rundfeldt C, Baumann L, Beck-Sickinger AG, Haupts U, Lilie H, Bosse-Doenecke E - FEBS Open Bio (2015)

Bottom Line: In this work we provide evidence that ubiquitin is safe as tested experimentally in vivo.In contrast to previously published results, we show that, in our hands, ubiquitin does not act as a functional ligand of the chemokine receptor CXCR4.Furthermore, intravenous application to mice at high concentrations did not induce any detectable effect on cytokine levels or hematological parameters.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biochemistry and Biotechnology/Technical Biochemistry, Martin-Luther-University Halle-Wittenberg, Kurt-Mothes-Straße 3, D-06120 Halle (Saale), Germany.

ABSTRACT
In the search for effective therapeutic strategies, protein-based biologicals are under intense development. While monoclonal antibodies represent the majority of these drugs, other innovative approaches are exploring the use of scaffold proteins for the creation of binding molecules with tailor-made properties. Ubiquitin is especially suited for this strategy due to several key characteristics. Ubiquitin is a natural serum protein, 100% conserved across the mammalian class and possesses high thermal, structural and proteolytic stability. Because of its small size and lack of posttranslational modifications, it can be easily produced in Escherichia coli. In this work we provide evidence that ubiquitin is safe as tested experimentally in vivo. In contrast to previously published results, we show that, in our hands, ubiquitin does not act as a functional ligand of the chemokine receptor CXCR4. Cellular assays based on different signaling pathways of the receptor were conducted with the natural agonist SDF-1 as a benchmark. In none of the assays could a response to ubiquitin treatment be elicited. Furthermore, intravenous application to mice at high concentrations did not induce any detectable effect on cytokine levels or hematological parameters.

No MeSH data available.


Related in: MedlinePlus

Binding analysis and calcium signaling of ubiquitin on THP-1 and Jurkat cells. (A) Binding of 1 μM (left panel) or 10 μM (right panel) FITC-ubiquitin (R&D Systems) with (red) or without (green) competition with 30-fold molar excess of unlabeled ubiquitin (Sigma) to THP-1 cells was assessed by flow cytometry. Untreated cells are shown in grey. (B) Binding of 1 μM (left panel) or 10 μM (right panel) FITC-ubiquitin (R&D Systems) with (red) or without (green) competition with 30-fold molar excess of unlabeled ubiquitin (Sigma) to Jurkat cells was assessed by flow cytometry. Untreated cells are shown in grey. (C and D) THP-1 (C) or Jurkat (D) cells were labelled with the Calcium 5 fluorophore and incubated with different concentrations of SDF-1 or ubiquitin (Sigma). Increase in Calcium 5 signal intensity indicating calcium signaling was monitored in the FlexStation 3 instrument. Error bars represent SEM of four (THP-1) and three (Jurkat) independent experiments.
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f0020: Binding analysis and calcium signaling of ubiquitin on THP-1 and Jurkat cells. (A) Binding of 1 μM (left panel) or 10 μM (right panel) FITC-ubiquitin (R&D Systems) with (red) or without (green) competition with 30-fold molar excess of unlabeled ubiquitin (Sigma) to THP-1 cells was assessed by flow cytometry. Untreated cells are shown in grey. (B) Binding of 1 μM (left panel) or 10 μM (right panel) FITC-ubiquitin (R&D Systems) with (red) or without (green) competition with 30-fold molar excess of unlabeled ubiquitin (Sigma) to Jurkat cells was assessed by flow cytometry. Untreated cells are shown in grey. (C and D) THP-1 (C) or Jurkat (D) cells were labelled with the Calcium 5 fluorophore and incubated with different concentrations of SDF-1 or ubiquitin (Sigma). Increase in Calcium 5 signal intensity indicating calcium signaling was monitored in the FlexStation 3 instrument. Error bars represent SEM of four (THP-1) and three (Jurkat) independent experiments.

Mentions: Our in vivo experiments did not suggest any evidence for specific ubiquitin accumulation on the tested hematopoetic cells. However, previously it has been reported that extracellular ubiquitin is able to bind to and activate the CXCR4 receptor present on lymphocytic cell lines like THP-1 [48]. To corroborate our data from the in vivo studies and relate them to the existing literature we sought to investigate the binding of ubiquitin to CXCR4 in two relevant cell lines, THP-1 (Fig. 4A) and Jurkat (Fig. 4B). Cells were incubated with 1 or 10 μM FITC-labeled ubiquitin and binding was assessed through FACS measurements. To account for non-specific binding of FITC-ubiquitin to the cellular surface, unlabeled ubiquitin was added in a 30-fold molar excess to compete for specific binding. The addition of 10 μM labelled ubiquitin to both CXCR4 positive cell lines resulted in a small shift of the cell populations towards higher fluorescence intensities, whereas none or a very weak shift was observed after incubation with 1 μM FITC-ubiquitin in THP1 and Jurkat respectively. Binding at the higher FITC-ubiquitin concentration was most likely non-specific, as it could not be blocked with unlabeled ubiquitin and no decrease in the FACS signal was detected under these conditions. In addition, experiments with HEK293 overexpressing the CXCR4 were performed. Alexa Fluor 647 labelled SDF-1 showed increased binding to HEK293 cells after CXCR4 cDNA transfection (Fig. S3B), whereas no FITC-ubiquitin binding to the receptor could be detected (Fig. S3A). As a control, non-transfected HEK293 cells were used. These results are in contradiction to reports claiming that ubiquitin binds specifically to THP-1 or to CXCR4-transfected HEK293 cells at the examined conditions [48].


Ubiquitin is a versatile scaffold protein for the generation of molecules with de novo binding and advantageous drug-like properties.

Job F, Settele F, Lorey S, Rundfeldt C, Baumann L, Beck-Sickinger AG, Haupts U, Lilie H, Bosse-Doenecke E - FEBS Open Bio (2015)

Binding analysis and calcium signaling of ubiquitin on THP-1 and Jurkat cells. (A) Binding of 1 μM (left panel) or 10 μM (right panel) FITC-ubiquitin (R&D Systems) with (red) or without (green) competition with 30-fold molar excess of unlabeled ubiquitin (Sigma) to THP-1 cells was assessed by flow cytometry. Untreated cells are shown in grey. (B) Binding of 1 μM (left panel) or 10 μM (right panel) FITC-ubiquitin (R&D Systems) with (red) or without (green) competition with 30-fold molar excess of unlabeled ubiquitin (Sigma) to Jurkat cells was assessed by flow cytometry. Untreated cells are shown in grey. (C and D) THP-1 (C) or Jurkat (D) cells were labelled with the Calcium 5 fluorophore and incubated with different concentrations of SDF-1 or ubiquitin (Sigma). Increase in Calcium 5 signal intensity indicating calcium signaling was monitored in the FlexStation 3 instrument. Error bars represent SEM of four (THP-1) and three (Jurkat) independent experiments.
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Related In: Results  -  Collection

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f0020: Binding analysis and calcium signaling of ubiquitin on THP-1 and Jurkat cells. (A) Binding of 1 μM (left panel) or 10 μM (right panel) FITC-ubiquitin (R&D Systems) with (red) or without (green) competition with 30-fold molar excess of unlabeled ubiquitin (Sigma) to THP-1 cells was assessed by flow cytometry. Untreated cells are shown in grey. (B) Binding of 1 μM (left panel) or 10 μM (right panel) FITC-ubiquitin (R&D Systems) with (red) or without (green) competition with 30-fold molar excess of unlabeled ubiquitin (Sigma) to Jurkat cells was assessed by flow cytometry. Untreated cells are shown in grey. (C and D) THP-1 (C) or Jurkat (D) cells were labelled with the Calcium 5 fluorophore and incubated with different concentrations of SDF-1 or ubiquitin (Sigma). Increase in Calcium 5 signal intensity indicating calcium signaling was monitored in the FlexStation 3 instrument. Error bars represent SEM of four (THP-1) and three (Jurkat) independent experiments.
Mentions: Our in vivo experiments did not suggest any evidence for specific ubiquitin accumulation on the tested hematopoetic cells. However, previously it has been reported that extracellular ubiquitin is able to bind to and activate the CXCR4 receptor present on lymphocytic cell lines like THP-1 [48]. To corroborate our data from the in vivo studies and relate them to the existing literature we sought to investigate the binding of ubiquitin to CXCR4 in two relevant cell lines, THP-1 (Fig. 4A) and Jurkat (Fig. 4B). Cells were incubated with 1 or 10 μM FITC-labeled ubiquitin and binding was assessed through FACS measurements. To account for non-specific binding of FITC-ubiquitin to the cellular surface, unlabeled ubiquitin was added in a 30-fold molar excess to compete for specific binding. The addition of 10 μM labelled ubiquitin to both CXCR4 positive cell lines resulted in a small shift of the cell populations towards higher fluorescence intensities, whereas none or a very weak shift was observed after incubation with 1 μM FITC-ubiquitin in THP1 and Jurkat respectively. Binding at the higher FITC-ubiquitin concentration was most likely non-specific, as it could not be blocked with unlabeled ubiquitin and no decrease in the FACS signal was detected under these conditions. In addition, experiments with HEK293 overexpressing the CXCR4 were performed. Alexa Fluor 647 labelled SDF-1 showed increased binding to HEK293 cells after CXCR4 cDNA transfection (Fig. S3B), whereas no FITC-ubiquitin binding to the receptor could be detected (Fig. S3A). As a control, non-transfected HEK293 cells were used. These results are in contradiction to reports claiming that ubiquitin binds specifically to THP-1 or to CXCR4-transfected HEK293 cells at the examined conditions [48].

Bottom Line: In this work we provide evidence that ubiquitin is safe as tested experimentally in vivo.In contrast to previously published results, we show that, in our hands, ubiquitin does not act as a functional ligand of the chemokine receptor CXCR4.Furthermore, intravenous application to mice at high concentrations did not induce any detectable effect on cytokine levels or hematological parameters.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biochemistry and Biotechnology/Technical Biochemistry, Martin-Luther-University Halle-Wittenberg, Kurt-Mothes-Straße 3, D-06120 Halle (Saale), Germany.

ABSTRACT
In the search for effective therapeutic strategies, protein-based biologicals are under intense development. While monoclonal antibodies represent the majority of these drugs, other innovative approaches are exploring the use of scaffold proteins for the creation of binding molecules with tailor-made properties. Ubiquitin is especially suited for this strategy due to several key characteristics. Ubiquitin is a natural serum protein, 100% conserved across the mammalian class and possesses high thermal, structural and proteolytic stability. Because of its small size and lack of posttranslational modifications, it can be easily produced in Escherichia coli. In this work we provide evidence that ubiquitin is safe as tested experimentally in vivo. In contrast to previously published results, we show that, in our hands, ubiquitin does not act as a functional ligand of the chemokine receptor CXCR4. Cellular assays based on different signaling pathways of the receptor were conducted with the natural agonist SDF-1 as a benchmark. In none of the assays could a response to ubiquitin treatment be elicited. Furthermore, intravenous application to mice at high concentrations did not induce any detectable effect on cytokine levels or hematological parameters.

No MeSH data available.


Related in: MedlinePlus