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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

Ubiquitin impact on the CXCR4 signaling cascade. (A) THP-1 cells were incubated with SDF-1 or ubiquitin (Sigma) in the indicated concentrations. cAMP content was analyzed after cell lysis with the cAMP complete enzyme immunoassay kit, acetylated format for higher sensitivity. Error bars represent SEM of three independent experiments. (B) HEK293 cells expressing CXCR4 were transfected with a pGL4.29 reporter plasmid and stimulated after 48 h with forskolin without (dark grey bar) or in combination with SDF-1 (red bar) or ubiquitin (F45W Scil Proteins, blue bars) in indicated concentrations. Unstimulated cells (light grey bar) served as negative control. Data were normalized on luciferase activity for forskolin treatment set to 100%. Error bars represent SD from three experiments. (C and D) HEK293 (C) or COS-7 (D) cells were transiently transfected with CXCR4 and the chimeric G-protein G alphaΔ6qi4myr. Afterwards cells were labelled with 0.3 μCi 3H-myo-inositol for more than 16 h followed by incubation with SDF-1 or ubiquitin (Sigma) in the indicated concentrations. Accumulated IP3 was collected by anion exchange chromatography from cell extracts followed by a beta counter quantification. Error bars represent SEM from three (HEK) or four (COS-7) independent experiments. (E and F) THP-1 (E) or Jurkat (F) cells were seeded in the upper compartment of 96 HTS transwell plate chambers. The lower compartment was filled with PBS containing indicated concentrations of SDF-1 or ubiquitin (Sigma). After 3 h incubation cells in the lower chamber were collected and analyzed by flow cytometry. Chemotactic index was calculated as the ratio of the number of migrated cells at the indicated ligand concentration to the number of cells migrated to buffer. Error bars represent SEM from three independent experiments.
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f0025: Ubiquitin impact on the CXCR4 signaling cascade. (A) THP-1 cells were incubated with SDF-1 or ubiquitin (Sigma) in the indicated concentrations. cAMP content was analyzed after cell lysis with the cAMP complete enzyme immunoassay kit, acetylated format for higher sensitivity. Error bars represent SEM of three independent experiments. (B) HEK293 cells expressing CXCR4 were transfected with a pGL4.29 reporter plasmid and stimulated after 48 h with forskolin without (dark grey bar) or in combination with SDF-1 (red bar) or ubiquitin (F45W Scil Proteins, blue bars) in indicated concentrations. Unstimulated cells (light grey bar) served as negative control. Data were normalized on luciferase activity for forskolin treatment set to 100%. Error bars represent SD from three experiments. (C and D) HEK293 (C) or COS-7 (D) cells were transiently transfected with CXCR4 and the chimeric G-protein G alphaΔ6qi4myr. Afterwards cells were labelled with 0.3 μCi 3H-myo-inositol for more than 16 h followed by incubation with SDF-1 or ubiquitin (Sigma) in the indicated concentrations. Accumulated IP3 was collected by anion exchange chromatography from cell extracts followed by a beta counter quantification. Error bars represent SEM from three (HEK) or four (COS-7) independent experiments. (E and F) THP-1 (E) or Jurkat (F) cells were seeded in the upper compartment of 96 HTS transwell plate chambers. The lower compartment was filled with PBS containing indicated concentrations of SDF-1 or ubiquitin (Sigma). After 3 h incubation cells in the lower chamber were collected and analyzed by flow cytometry. Chemotactic index was calculated as the ratio of the number of migrated cells at the indicated ligand concentration to the number of cells migrated to buffer. Error bars represent SEM from three independent experiments.

Mentions: To further differentiate effects on the CXCR4 signaling cascade we made use of a cAMP ELISA kit (Enzo life sciences). Intracellular cAMP levels were quantified after incubation of the respective cells with the CXCR4 ligand SDF-1 or ubiquitin at two different concentrations, 1 μM and 100 nM. Since CXCR4 signals through G alpha i proteins, ligand binding and activation of the receptor should result in decreasing cAMP levels, which would correspond to an increase in signal intensity in this specific ELISA setup. Again, SDF-1 was able to decrease intracellular cAMP levels, whereas ubiquitin had no effect on intracellular cAMP levels (Fig. 5A). Because this is in contrast to already reported results, we verified several sources of experimental variability: (i) a THP-1 cell line from a different supplier (ATCC) revealed essentially the same result (Fig. S5A). (ii) To account for differences in incubation times we performed a time course experiment with increased incubation times of up to 60 min. For all tested conditions, there was no effect of ubiquitin on intracellular cAMP levels (Fig. S5B). (iii) Since we noticed slightly increased cAMP levels in some ubiquitin treated samples compared to controls, we looked at possible antagonistic effects of ubiquitin in the presence of SDF-1. Therefore, 1 μM SDF-1 and ubiquitin in various concentrations ranging from 1 to 10 μM were added simultaneously to THP-1 cells and incubated for 15 min followed by cAMP ELISA. In all tested concentrations ubiquitin had no antagonistic effect on SDF-1 treated THP-1 cells (Fig. S5C).


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)

Ubiquitin impact on the CXCR4 signaling cascade. (A) THP-1 cells were incubated with SDF-1 or ubiquitin (Sigma) in the indicated concentrations. cAMP content was analyzed after cell lysis with the cAMP complete enzyme immunoassay kit, acetylated format for higher sensitivity. Error bars represent SEM of three independent experiments. (B) HEK293 cells expressing CXCR4 were transfected with a pGL4.29 reporter plasmid and stimulated after 48 h with forskolin without (dark grey bar) or in combination with SDF-1 (red bar) or ubiquitin (F45W Scil Proteins, blue bars) in indicated concentrations. Unstimulated cells (light grey bar) served as negative control. Data were normalized on luciferase activity for forskolin treatment set to 100%. Error bars represent SD from three experiments. (C and D) HEK293 (C) or COS-7 (D) cells were transiently transfected with CXCR4 and the chimeric G-protein G alphaΔ6qi4myr. Afterwards cells were labelled with 0.3 μCi 3H-myo-inositol for more than 16 h followed by incubation with SDF-1 or ubiquitin (Sigma) in the indicated concentrations. Accumulated IP3 was collected by anion exchange chromatography from cell extracts followed by a beta counter quantification. Error bars represent SEM from three (HEK) or four (COS-7) independent experiments. (E and F) THP-1 (E) or Jurkat (F) cells were seeded in the upper compartment of 96 HTS transwell plate chambers. The lower compartment was filled with PBS containing indicated concentrations of SDF-1 or ubiquitin (Sigma). After 3 h incubation cells in the lower chamber were collected and analyzed by flow cytometry. Chemotactic index was calculated as the ratio of the number of migrated cells at the indicated ligand concentration to the number of cells migrated to buffer. Error bars represent SEM from three independent experiments.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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Show All Figures
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f0025: Ubiquitin impact on the CXCR4 signaling cascade. (A) THP-1 cells were incubated with SDF-1 or ubiquitin (Sigma) in the indicated concentrations. cAMP content was analyzed after cell lysis with the cAMP complete enzyme immunoassay kit, acetylated format for higher sensitivity. Error bars represent SEM of three independent experiments. (B) HEK293 cells expressing CXCR4 were transfected with a pGL4.29 reporter plasmid and stimulated after 48 h with forskolin without (dark grey bar) or in combination with SDF-1 (red bar) or ubiquitin (F45W Scil Proteins, blue bars) in indicated concentrations. Unstimulated cells (light grey bar) served as negative control. Data were normalized on luciferase activity for forskolin treatment set to 100%. Error bars represent SD from three experiments. (C and D) HEK293 (C) or COS-7 (D) cells were transiently transfected with CXCR4 and the chimeric G-protein G alphaΔ6qi4myr. Afterwards cells were labelled with 0.3 μCi 3H-myo-inositol for more than 16 h followed by incubation with SDF-1 or ubiquitin (Sigma) in the indicated concentrations. Accumulated IP3 was collected by anion exchange chromatography from cell extracts followed by a beta counter quantification. Error bars represent SEM from three (HEK) or four (COS-7) independent experiments. (E and F) THP-1 (E) or Jurkat (F) cells were seeded in the upper compartment of 96 HTS transwell plate chambers. The lower compartment was filled with PBS containing indicated concentrations of SDF-1 or ubiquitin (Sigma). After 3 h incubation cells in the lower chamber were collected and analyzed by flow cytometry. Chemotactic index was calculated as the ratio of the number of migrated cells at the indicated ligand concentration to the number of cells migrated to buffer. Error bars represent SEM from three independent experiments.
Mentions: To further differentiate effects on the CXCR4 signaling cascade we made use of a cAMP ELISA kit (Enzo life sciences). Intracellular cAMP levels were quantified after incubation of the respective cells with the CXCR4 ligand SDF-1 or ubiquitin at two different concentrations, 1 μM and 100 nM. Since CXCR4 signals through G alpha i proteins, ligand binding and activation of the receptor should result in decreasing cAMP levels, which would correspond to an increase in signal intensity in this specific ELISA setup. Again, SDF-1 was able to decrease intracellular cAMP levels, whereas ubiquitin had no effect on intracellular cAMP levels (Fig. 5A). Because this is in contrast to already reported results, we verified several sources of experimental variability: (i) a THP-1 cell line from a different supplier (ATCC) revealed essentially the same result (Fig. S5A). (ii) To account for differences in incubation times we performed a time course experiment with increased incubation times of up to 60 min. For all tested conditions, there was no effect of ubiquitin on intracellular cAMP levels (Fig. S5B). (iii) Since we noticed slightly increased cAMP levels in some ubiquitin treated samples compared to controls, we looked at possible antagonistic effects of ubiquitin in the presence of SDF-1. Therefore, 1 μM SDF-1 and ubiquitin in various concentrations ranging from 1 to 10 μM were added simultaneously to THP-1 cells and incubated for 15 min followed by cAMP ELISA. In all tested concentrations ubiquitin had no antagonistic effect on SDF-1 treated THP-1 cells (Fig. S5C).

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