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Lysine-Less Variants of Spinal Muscular Atrophy SMN and SMNΔ7 Proteins Are Degraded by the Proteasome Pathway
Spinal muscular atrophy is due to mutations affecting the SMN1 gene coding for the full-length protein (survival motor neuron; SMN) and the SMN2 gene that preferentially generates an exon 7-deleted protein (SMNΔ7) by alternative splicing. To study SMN and SMNΔ7 degradation in the cell, we have used...
Autores principales: | , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5751269/ https://www.ncbi.nlm.nih.gov/pubmed/29292768 http://dx.doi.org/10.3390/ijms18122667 |
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author | Sánchez-Lanzas, Raúl Castaño, José G. |
author_facet | Sánchez-Lanzas, Raúl Castaño, José G. |
author_sort | Sánchez-Lanzas, Raúl |
collection | PubMed |
description | Spinal muscular atrophy is due to mutations affecting the SMN1 gene coding for the full-length protein (survival motor neuron; SMN) and the SMN2 gene that preferentially generates an exon 7-deleted protein (SMNΔ7) by alternative splicing. To study SMN and SMNΔ7 degradation in the cell, we have used tagged versions at the N- (Flag) or C-terminus (V5) of both proteins. Transfection of those constructs into HeLa cells and treatment with cycloheximide showed that those protein constructs were degraded. Proteasomal degradation usually requires prior lysine ubiquitylation. Surprisingly, lysine-less variants of both proteins tagged either at N- (Flag) or C-terminus (V5) were also degraded. The degradation of the endogenous SMN protein, and the protein constructs mentioned above, was mediated by the proteasome, as it was blocked by lactacystin, a specific and irreversible proteasomal inhibitor. The results obtained allowed us to conclude that SMN and SMNΔ7 proteasomal degradation did not absolutely require internal ubiquitylation nor N-terminal ubiquitylation (prevented by N-terminal tagging). While the above conclusions are firmly supported by the experimental data presented, we discuss and justify the need of deep proteomic techniques for the study of SMN complex components (orphan and bound) turn-over to understand the physiological relevant mechanisms of degradation of SMN and SMNΔ7 in the cell. |
format | Online Article Text |
id | pubmed-5751269 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-57512692018-01-08 Lysine-Less Variants of Spinal Muscular Atrophy SMN and SMNΔ7 Proteins Are Degraded by the Proteasome Pathway Sánchez-Lanzas, Raúl Castaño, José G. Int J Mol Sci Article Spinal muscular atrophy is due to mutations affecting the SMN1 gene coding for the full-length protein (survival motor neuron; SMN) and the SMN2 gene that preferentially generates an exon 7-deleted protein (SMNΔ7) by alternative splicing. To study SMN and SMNΔ7 degradation in the cell, we have used tagged versions at the N- (Flag) or C-terminus (V5) of both proteins. Transfection of those constructs into HeLa cells and treatment with cycloheximide showed that those protein constructs were degraded. Proteasomal degradation usually requires prior lysine ubiquitylation. Surprisingly, lysine-less variants of both proteins tagged either at N- (Flag) or C-terminus (V5) were also degraded. The degradation of the endogenous SMN protein, and the protein constructs mentioned above, was mediated by the proteasome, as it was blocked by lactacystin, a specific and irreversible proteasomal inhibitor. The results obtained allowed us to conclude that SMN and SMNΔ7 proteasomal degradation did not absolutely require internal ubiquitylation nor N-terminal ubiquitylation (prevented by N-terminal tagging). While the above conclusions are firmly supported by the experimental data presented, we discuss and justify the need of deep proteomic techniques for the study of SMN complex components (orphan and bound) turn-over to understand the physiological relevant mechanisms of degradation of SMN and SMNΔ7 in the cell. MDPI 2017-12-08 /pmc/articles/PMC5751269/ /pubmed/29292768 http://dx.doi.org/10.3390/ijms18122667 Text en © 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Sánchez-Lanzas, Raúl Castaño, José G. Lysine-Less Variants of Spinal Muscular Atrophy SMN and SMNΔ7 Proteins Are Degraded by the Proteasome Pathway |
title | Lysine-Less Variants of Spinal Muscular Atrophy SMN and SMNΔ7 Proteins Are Degraded by the Proteasome Pathway |
title_full | Lysine-Less Variants of Spinal Muscular Atrophy SMN and SMNΔ7 Proteins Are Degraded by the Proteasome Pathway |
title_fullStr | Lysine-Less Variants of Spinal Muscular Atrophy SMN and SMNΔ7 Proteins Are Degraded by the Proteasome Pathway |
title_full_unstemmed | Lysine-Less Variants of Spinal Muscular Atrophy SMN and SMNΔ7 Proteins Are Degraded by the Proteasome Pathway |
title_short | Lysine-Less Variants of Spinal Muscular Atrophy SMN and SMNΔ7 Proteins Are Degraded by the Proteasome Pathway |
title_sort | lysine-less variants of spinal muscular atrophy smn and smnδ7 proteins are degraded by the proteasome pathway |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5751269/ https://www.ncbi.nlm.nih.gov/pubmed/29292768 http://dx.doi.org/10.3390/ijms18122667 |
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