Modification of erythropoietin structure by N-homocysteinylation affects its antiapoptotic and proliferative functions
Many patients under therapy with recombinant human erythropoietin (rhuEPO) show resistance to the treatment, an effect likely associated with the accumulation of tissue factors, especially in renal and cardiovascular diseases. Hyperhomocysteinemia due to high serum levels of homocysteine has been su...
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2018
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| Acceso en línea: | https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_1742464X_v285_n20_p3801_Schiappacasse http://hdl.handle.net/20.500.12110/paper_1742464X_v285_n20_p3801_Schiappacasse |
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paper:paper_1742464X_v285_n20_p3801_Schiappacasse2023-06-08T16:27:05Z Modification of erythropoietin structure by N-homocysteinylation affects its antiapoptotic and proliferative functions erythropoietin erythropoietin resistance hyperhomocysteinemia N-homocysteinylation protein structure congo red erythropoietin lysine n homocysteine thiolactonyl retinamido cobalamin oligomer polyacrylamide gel thioflavine alpha helix Article beta sheet capillary zone electrophoresis cardiorenal syndrome cell aggregation cell function cell proliferation circular dichroism conformational transition controlled study electrophoretic mobility eryptosis erythropoiesis human human cell n homocysteinylation photon correlation spectroscopy priority journal protein interaction protein modification protein secondary structure protein structure ultraviolet spectroscopy Many patients under therapy with recombinant human erythropoietin (rhuEPO) show resistance to the treatment, an effect likely associated with the accumulation of tissue factors, especially in renal and cardiovascular diseases. Hyperhomocysteinemia due to high serum levels of homocysteine has been suggested among the risk factors in those pathologies. Its main effect is the N-homocysteinylation of proteins due to the interaction between the highly reactive homocysteine thiolactone (HTL) and lysine residues. The aim of this study was to evaluate the effect of N-homocysteinylation on the erythropoietic and antiapoptotic abilities of EPO, which can be a consequence of structural changes in the modified protein. We found that both cellular functions were altered in the presence of HTL-EPO. A decreased net positive charge of HTL-EPO was detected by capillary zone electrophoresis, while analysis of polyacrylamide gel electropherograms suggested formation of aggregates. Far-UV spectra, obtained by Circular Dichroism Spectroscopy, indicated a switch of the protein's secondary structure from α-helix to β-sheet structures. Results of Congo red and Thioflavin T assays confirm the formation of repetitive β-sheet structures, which may account for aggregates. Accordingly, Dynamic Light Scattering analysis showed a markedly larger radius of the HTL-EPO structures, supporting the formation of soluble oligomers. These structural changes might interfere with the conformational adaptations necessary for efficient ligand-receptor interaction, thus affecting the proliferative and antiapoptotic functions of EPO. The present findings may contribute to explain the resistance exhibited by patients with cardio-renal syndrome to treatment with rhuEPO, as a consequence of structural modifications due to protein N-homocysteinylation. © 2018 Federation of European Biochemical Societies 2018 https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_1742464X_v285_n20_p3801_Schiappacasse http://hdl.handle.net/20.500.12110/paper_1742464X_v285_n20_p3801_Schiappacasse |
| institution |
Universidad de Buenos Aires |
| institution_str |
I-28 |
| repository_str |
R-134 |
| collection |
Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) |
| topic |
erythropoietin erythropoietin resistance hyperhomocysteinemia N-homocysteinylation protein structure congo red erythropoietin lysine n homocysteine thiolactonyl retinamido cobalamin oligomer polyacrylamide gel thioflavine alpha helix Article beta sheet capillary zone electrophoresis cardiorenal syndrome cell aggregation cell function cell proliferation circular dichroism conformational transition controlled study electrophoretic mobility eryptosis erythropoiesis human human cell n homocysteinylation photon correlation spectroscopy priority journal protein interaction protein modification protein secondary structure protein structure ultraviolet spectroscopy |
| spellingShingle |
erythropoietin erythropoietin resistance hyperhomocysteinemia N-homocysteinylation protein structure congo red erythropoietin lysine n homocysteine thiolactonyl retinamido cobalamin oligomer polyacrylamide gel thioflavine alpha helix Article beta sheet capillary zone electrophoresis cardiorenal syndrome cell aggregation cell function cell proliferation circular dichroism conformational transition controlled study electrophoretic mobility eryptosis erythropoiesis human human cell n homocysteinylation photon correlation spectroscopy priority journal protein interaction protein modification protein secondary structure protein structure ultraviolet spectroscopy Modification of erythropoietin structure by N-homocysteinylation affects its antiapoptotic and proliferative functions |
| topic_facet |
erythropoietin erythropoietin resistance hyperhomocysteinemia N-homocysteinylation protein structure congo red erythropoietin lysine n homocysteine thiolactonyl retinamido cobalamin oligomer polyacrylamide gel thioflavine alpha helix Article beta sheet capillary zone electrophoresis cardiorenal syndrome cell aggregation cell function cell proliferation circular dichroism conformational transition controlled study electrophoretic mobility eryptosis erythropoiesis human human cell n homocysteinylation photon correlation spectroscopy priority journal protein interaction protein modification protein secondary structure protein structure ultraviolet spectroscopy |
| description |
Many patients under therapy with recombinant human erythropoietin (rhuEPO) show resistance to the treatment, an effect likely associated with the accumulation of tissue factors, especially in renal and cardiovascular diseases. Hyperhomocysteinemia due to high serum levels of homocysteine has been suggested among the risk factors in those pathologies. Its main effect is the N-homocysteinylation of proteins due to the interaction between the highly reactive homocysteine thiolactone (HTL) and lysine residues. The aim of this study was to evaluate the effect of N-homocysteinylation on the erythropoietic and antiapoptotic abilities of EPO, which can be a consequence of structural changes in the modified protein. We found that both cellular functions were altered in the presence of HTL-EPO. A decreased net positive charge of HTL-EPO was detected by capillary zone electrophoresis, while analysis of polyacrylamide gel electropherograms suggested formation of aggregates. Far-UV spectra, obtained by Circular Dichroism Spectroscopy, indicated a switch of the protein's secondary structure from α-helix to β-sheet structures. Results of Congo red and Thioflavin T assays confirm the formation of repetitive β-sheet structures, which may account for aggregates. Accordingly, Dynamic Light Scattering analysis showed a markedly larger radius of the HTL-EPO structures, supporting the formation of soluble oligomers. These structural changes might interfere with the conformational adaptations necessary for efficient ligand-receptor interaction, thus affecting the proliferative and antiapoptotic functions of EPO. The present findings may contribute to explain the resistance exhibited by patients with cardio-renal syndrome to treatment with rhuEPO, as a consequence of structural modifications due to protein N-homocysteinylation. © 2018 Federation of European Biochemical Societies |
| title |
Modification of erythropoietin structure by N-homocysteinylation affects its antiapoptotic and proliferative functions |
| title_short |
Modification of erythropoietin structure by N-homocysteinylation affects its antiapoptotic and proliferative functions |
| title_full |
Modification of erythropoietin structure by N-homocysteinylation affects its antiapoptotic and proliferative functions |
| title_fullStr |
Modification of erythropoietin structure by N-homocysteinylation affects its antiapoptotic and proliferative functions |
| title_full_unstemmed |
Modification of erythropoietin structure by N-homocysteinylation affects its antiapoptotic and proliferative functions |
| title_sort |
modification of erythropoietin structure by n-homocysteinylation affects its antiapoptotic and proliferative functions |
| publishDate |
2018 |
| url |
https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_1742464X_v285_n20_p3801_Schiappacasse http://hdl.handle.net/20.500.12110/paper_1742464X_v285_n20_p3801_Schiappacasse |
| _version_ |
1768544704689864704 |