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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

Analysis of cAMP accumulation. (A) THP-1 cells (purchased from ATCC) were incubated with SDF-1 (red bars) or ubiquitin (Sigma, blue bars) in indicated concentrations. cAMP content was analyzed after cell lysis. Error bars represent SD of a triplicate measurement. (B) THP-1 cells were incubated with 1 μM ubiquitin (Sigma) for indicated time points. cAMP content was analyzed after cell lysis. Error bars represent SD. (C) THP-1 cells were incubated with SDF-1 alone (red) or in combination with ubiquitin (Sigma) in indicated concentrations (purple). cAMP content was analyzed after cell lysis. Error bars represent SD. All samples (A–C) were analyzed after cell lysis with the cAMP complete enzyme immunoassay kit, acetylated format. (D) HEK293 cells expressing CXCR4 were transfected with 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 F4A mutant (R&D Systems, dark blue bars) or V70A mutant (R&D Systems, light blue bars) in indicated concentrations. Cells incubated with 100 nM SDF-1 and 10 μM of the CXCR4 inhibitor AMD3100 (light grey bars) served as control. Luciferase activity was set 100 % for the forskolin treated sample (n = 1). (E) 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 100 nM SDF-1 (red bar) or ubiquitin (F45W ubiquitin Scil Proteins, light green bars; F45W di-ubiquitin Scil Proteins, green bars; ubiquitin R&D Systems, orange bars; ubiquitin Sigma, blue bars) in indicated concentrations. Cells incubated with 100 nM SDF-1 and 10 μM of the CXCR4 inhibitor AMD3100 (light grey bars) served as control. Data were normalized on luciferase activity for forskolin treatment set to 100%. Error bars represent SD from triplicates.
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f0050: Analysis of cAMP accumulation. (A) THP-1 cells (purchased from ATCC) were incubated with SDF-1 (red bars) or ubiquitin (Sigma, blue bars) in indicated concentrations. cAMP content was analyzed after cell lysis. Error bars represent SD of a triplicate measurement. (B) THP-1 cells were incubated with 1 μM ubiquitin (Sigma) for indicated time points. cAMP content was analyzed after cell lysis. Error bars represent SD. (C) THP-1 cells were incubated with SDF-1 alone (red) or in combination with ubiquitin (Sigma) in indicated concentrations (purple). cAMP content was analyzed after cell lysis. Error bars represent SD. All samples (A–C) were analyzed after cell lysis with the cAMP complete enzyme immunoassay kit, acetylated format. (D) HEK293 cells expressing CXCR4 were transfected with 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 F4A mutant (R&D Systems, dark blue bars) or V70A mutant (R&D Systems, light blue bars) in indicated concentrations. Cells incubated with 100 nM SDF-1 and 10 μM of the CXCR4 inhibitor AMD3100 (light grey bars) served as control. Luciferase activity was set 100 % for the forskolin treated sample (n = 1). (E) 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 100 nM SDF-1 (red bar) or ubiquitin (F45W ubiquitin Scil Proteins, light green bars; F45W di-ubiquitin Scil Proteins, green bars; ubiquitin R&D Systems, orange bars; ubiquitin Sigma, blue bars) in indicated concentrations. Cells incubated with 100 nM SDF-1 and 10 μM of the CXCR4 inhibitor AMD3100 (light grey bars) served as control. Data were normalized on luciferase activity for forskolin treatment set to 100%. Error bars represent SD from 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)

Analysis of cAMP accumulation. (A) THP-1 cells (purchased from ATCC) were incubated with SDF-1 (red bars) or ubiquitin (Sigma, blue bars) in indicated concentrations. cAMP content was analyzed after cell lysis. Error bars represent SD of a triplicate measurement. (B) THP-1 cells were incubated with 1 μM ubiquitin (Sigma) for indicated time points. cAMP content was analyzed after cell lysis. Error bars represent SD. (C) THP-1 cells were incubated with SDF-1 alone (red) or in combination with ubiquitin (Sigma) in indicated concentrations (purple). cAMP content was analyzed after cell lysis. Error bars represent SD. All samples (A–C) were analyzed after cell lysis with the cAMP complete enzyme immunoassay kit, acetylated format. (D) HEK293 cells expressing CXCR4 were transfected with 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 F4A mutant (R&D Systems, dark blue bars) or V70A mutant (R&D Systems, light blue bars) in indicated concentrations. Cells incubated with 100 nM SDF-1 and 10 μM of the CXCR4 inhibitor AMD3100 (light grey bars) served as control. Luciferase activity was set 100 % for the forskolin treated sample (n = 1). (E) 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 100 nM SDF-1 (red bar) or ubiquitin (F45W ubiquitin Scil Proteins, light green bars; F45W di-ubiquitin Scil Proteins, green bars; ubiquitin R&D Systems, orange bars; ubiquitin Sigma, blue bars) in indicated concentrations. Cells incubated with 100 nM SDF-1 and 10 μM of the CXCR4 inhibitor AMD3100 (light grey bars) served as control. Data were normalized on luciferase activity for forskolin treatment set to 100%. Error bars represent SD from triplicates.
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f0050: Analysis of cAMP accumulation. (A) THP-1 cells (purchased from ATCC) were incubated with SDF-1 (red bars) or ubiquitin (Sigma, blue bars) in indicated concentrations. cAMP content was analyzed after cell lysis. Error bars represent SD of a triplicate measurement. (B) THP-1 cells were incubated with 1 μM ubiquitin (Sigma) for indicated time points. cAMP content was analyzed after cell lysis. Error bars represent SD. (C) THP-1 cells were incubated with SDF-1 alone (red) or in combination with ubiquitin (Sigma) in indicated concentrations (purple). cAMP content was analyzed after cell lysis. Error bars represent SD. All samples (A–C) were analyzed after cell lysis with the cAMP complete enzyme immunoassay kit, acetylated format. (D) HEK293 cells expressing CXCR4 were transfected with 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 F4A mutant (R&D Systems, dark blue bars) or V70A mutant (R&D Systems, light blue bars) in indicated concentrations. Cells incubated with 100 nM SDF-1 and 10 μM of the CXCR4 inhibitor AMD3100 (light grey bars) served as control. Luciferase activity was set 100 % for the forskolin treated sample (n = 1). (E) 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 100 nM SDF-1 (red bar) or ubiquitin (F45W ubiquitin Scil Proteins, light green bars; F45W di-ubiquitin Scil Proteins, green bars; ubiquitin R&D Systems, orange bars; ubiquitin Sigma, blue bars) in indicated concentrations. Cells incubated with 100 nM SDF-1 and 10 μM of the CXCR4 inhibitor AMD3100 (light grey bars) served as control. Data were normalized on luciferase activity for forskolin treatment set to 100%. Error bars represent SD from 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