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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

Bioactivity of ubiquitin species. Ubiquitin variants show in vitro ubiquitination of Glow-Fold™ substrate by CHIP E3 ligase on a western blot. Reactions contained all components as described in the manufacturer’s protocol and ubiquitination was initiated by the addition of indicated ubiquitin variants. After 1 h at 37 °C reactions were terminated with SDS-PAGE sample buffer containing DTT and separated on a 10% TGX stain-free gel (Bio-Rad) shown in part as loading control in the lower panel. Ubiquitination of Glow-Fold™ substrate was monitored via western blot analysis using alpha-Glow-Fold™ primary antibody and HRP-labeled secondary antibody (R&D Systems, HAF017) shown in the upper panel. The non-ubiquitinated substrate is marked with arrows. Protein with different ubiquitination grades is visible as smear or bands at higher molecular weights.
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f0060: Bioactivity of ubiquitin species. Ubiquitin variants show in vitro ubiquitination of Glow-Fold™ substrate by CHIP E3 ligase on a western blot. Reactions contained all components as described in the manufacturer’s protocol and ubiquitination was initiated by the addition of indicated ubiquitin variants. After 1 h at 37 °C reactions were terminated with SDS-PAGE sample buffer containing DTT and separated on a 10% TGX stain-free gel (Bio-Rad) shown in part as loading control in the lower panel. Ubiquitination of Glow-Fold™ substrate was monitored via western blot analysis using alpha-Glow-Fold™ primary antibody and HRP-labeled secondary antibody (R&D Systems, HAF017) shown in the upper panel. The non-ubiquitinated substrate is marked with arrows. Protein with different ubiquitination grades is visible as smear or bands at higher molecular weights.


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)

Bioactivity of ubiquitin species. Ubiquitin variants show in vitro ubiquitination of Glow-Fold™ substrate by CHIP E3 ligase on a western blot. Reactions contained all components as described in the manufacturer’s protocol and ubiquitination was initiated by the addition of indicated ubiquitin variants. After 1 h at 37 °C reactions were terminated with SDS-PAGE sample buffer containing DTT and separated on a 10% TGX stain-free gel (Bio-Rad) shown in part as loading control in the lower panel. Ubiquitination of Glow-Fold™ substrate was monitored via western blot analysis using alpha-Glow-Fold™ primary antibody and HRP-labeled secondary antibody (R&D Systems, HAF017) shown in the upper panel. The non-ubiquitinated substrate is marked with arrows. Protein with different ubiquitination grades is visible as smear or bands at higher molecular weights.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

f0060: Bioactivity of ubiquitin species. Ubiquitin variants show in vitro ubiquitination of Glow-Fold™ substrate by CHIP E3 ligase on a western blot. Reactions contained all components as described in the manufacturer’s protocol and ubiquitination was initiated by the addition of indicated ubiquitin variants. After 1 h at 37 °C reactions were terminated with SDS-PAGE sample buffer containing DTT and separated on a 10% TGX stain-free gel (Bio-Rad) shown in part as loading control in the lower panel. Ubiquitination of Glow-Fold™ substrate was monitored via western blot analysis using alpha-Glow-Fold™ primary antibody and HRP-labeled secondary antibody (R&D Systems, HAF017) shown in the upper panel. The non-ubiquitinated substrate is marked with arrows. Protein with different ubiquitination grades is visible as smear or bands at higher molecular weights.
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