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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

Impact of high concentrations of ubiquitin on intracellular calcium levels in THP-1 and Jurkat cells. (A) THP-1 or (B) Jurkat cells were labelled with the Calcium 5 fluorophore and incubated with high concentrations of ubiquitin (5 μM, 10 μM, 20 μM (Sigma); 50 μM (R&D Systems)). As positive control cells were incubated with 50 nM SDF-1 and unstimulated cells (PBS) served as negative control. Calcium 5 fluorescence signal was monitored using the FlexStation 3 instrument. A representative experiment for each cell line from two independent measurements is shown. Error bars represent SD of triplicates.
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f0045: Impact of high concentrations of ubiquitin on intracellular calcium levels in THP-1 and Jurkat cells. (A) THP-1 or (B) Jurkat cells were labelled with the Calcium 5 fluorophore and incubated with high concentrations of ubiquitin (5 μM, 10 μM, 20 μM (Sigma); 50 μM (R&D Systems)). As positive control cells were incubated with 50 nM SDF-1 and unstimulated cells (PBS) served as negative control. Calcium 5 fluorescence signal was monitored using the FlexStation 3 instrument. A representative experiment for each cell line from two independent measurements is shown. Error bars represent SD of triplicates.


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)

Impact of high concentrations of ubiquitin on intracellular calcium levels in THP-1 and Jurkat cells. (A) THP-1 or (B) Jurkat cells were labelled with the Calcium 5 fluorophore and incubated with high concentrations of ubiquitin (5 μM, 10 μM, 20 μM (Sigma); 50 μM (R&D Systems)). As positive control cells were incubated with 50 nM SDF-1 and unstimulated cells (PBS) served as negative control. Calcium 5 fluorescence signal was monitored using the FlexStation 3 instrument. A representative experiment for each cell line from two independent measurements is shown. Error bars represent SD of triplicates.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4522466&req=5

f0045: Impact of high concentrations of ubiquitin on intracellular calcium levels in THP-1 and Jurkat cells. (A) THP-1 or (B) Jurkat cells were labelled with the Calcium 5 fluorophore and incubated with high concentrations of ubiquitin (5 μM, 10 μM, 20 μM (Sigma); 50 μM (R&D Systems)). As positive control cells were incubated with 50 nM SDF-1 and unstimulated cells (PBS) served as negative control. Calcium 5 fluorescence signal was monitored using the FlexStation 3 instrument. A representative experiment for each cell line from two independent measurements is shown. Error bars represent SD of triplicates.
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