Cargando…

The Molecular Determinants of NEDD8 Specific Recognition by Human SENP8

Although neuronal-precursor-cell-expressed developmentally downregulated protein-8 (NEDD8) and ubiquitin share the highest level of sequence identity and structural similarity among several known ubiquitin-like proteins, their conjugation to a protein leads to distinct biological consequences. In th...

Descripción completa

Detalles Bibliográficos
Autores principales: Shin, Yung-Cheng, Tang, Siao-Jing, Chen, Jou-Han, Liao, Pei-Han, Chang, Shih-Chung
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3215745/
https://www.ncbi.nlm.nih.gov/pubmed/22110750
http://dx.doi.org/10.1371/journal.pone.0027742
_version_ 1782216432699834368
author Shin, Yung-Cheng
Tang, Siao-Jing
Chen, Jou-Han
Liao, Pei-Han
Chang, Shih-Chung
author_facet Shin, Yung-Cheng
Tang, Siao-Jing
Chen, Jou-Han
Liao, Pei-Han
Chang, Shih-Chung
author_sort Shin, Yung-Cheng
collection PubMed
description Although neuronal-precursor-cell-expressed developmentally downregulated protein-8 (NEDD8) and ubiquitin share the highest level of sequence identity and structural similarity among several known ubiquitin-like proteins, their conjugation to a protein leads to distinct biological consequences. In the study, we first identified the NEDD8 protein of Chlamydomonas reinhardtii (CrNEDD8) and discovered that CrNEDD8 is fused at the C-terminus of a ubiquitin moiety (CrUb) in a head-to-tail arrangement. This CrUb-CrNEDD8 protein was termed CrRUB1 (related to ubiquitin 1) by analogy with a similar protein in Arabidopsis thaliana (AtRUB1). Since there is high sequence identity in comparison to the corresponding human proteins (97% for ubiquitin and 84% for NEDD8), a His-CrRUB1-glutathione S-transferase (GST) fusion construct was adopted as the alternative substrate to characterize the specificity of NEDD8-specific peptidase SENP8 for CrNEDD8. The data showed that SENP8 only cleaved the peptide bond beyond the di-glycine motif of CrNEDD8 and His-RUB1 was subsequently generated, confirming that SENP8 has exquisite specificity for CrNEDD8 but not CrUb. To further determine the basis of this specificity, site-directed mutagenesis at earlier reported putative molecular determinants of NEDD8 specific recognition by SENP8 was performed. We found that a single N51E mutation of CrNEDD8 completely inhibited its hydrolysis by SENP8. Conversely, a single E51N mutation of CrUb enabled this ubiquitin mutant to undergo hydrolysis by SENP8, revealing that a single residue difference at the position 51 contributes substantially to the substrate selectivity of SENP8. Moreover, the E51N/R72A double mutant of the CrUb subdomain can further increase the efficiency of cleavage by SENP8, indicating that the residue at position 72 is also important in substrate recognition. The E51N or R72A mutation of CrUb also inhibited the hydrolysis of CrUb by ubiquitin-specific peptidase USP2. However, USP2 cannot cleave the N51E/A72R double mutant of the CrNEDD8 subdomain, suggesting that USP2 requires additional recognition sites.
format Online
Article
Text
id pubmed-3215745
institution National Center for Biotechnology Information
language English
publishDate 2011
publisher Public Library of Science
record_format MEDLINE/PubMed
spelling pubmed-32157452011-11-21 The Molecular Determinants of NEDD8 Specific Recognition by Human SENP8 Shin, Yung-Cheng Tang, Siao-Jing Chen, Jou-Han Liao, Pei-Han Chang, Shih-Chung PLoS One Research Article Although neuronal-precursor-cell-expressed developmentally downregulated protein-8 (NEDD8) and ubiquitin share the highest level of sequence identity and structural similarity among several known ubiquitin-like proteins, their conjugation to a protein leads to distinct biological consequences. In the study, we first identified the NEDD8 protein of Chlamydomonas reinhardtii (CrNEDD8) and discovered that CrNEDD8 is fused at the C-terminus of a ubiquitin moiety (CrUb) in a head-to-tail arrangement. This CrUb-CrNEDD8 protein was termed CrRUB1 (related to ubiquitin 1) by analogy with a similar protein in Arabidopsis thaliana (AtRUB1). Since there is high sequence identity in comparison to the corresponding human proteins (97% for ubiquitin and 84% for NEDD8), a His-CrRUB1-glutathione S-transferase (GST) fusion construct was adopted as the alternative substrate to characterize the specificity of NEDD8-specific peptidase SENP8 for CrNEDD8. The data showed that SENP8 only cleaved the peptide bond beyond the di-glycine motif of CrNEDD8 and His-RUB1 was subsequently generated, confirming that SENP8 has exquisite specificity for CrNEDD8 but not CrUb. To further determine the basis of this specificity, site-directed mutagenesis at earlier reported putative molecular determinants of NEDD8 specific recognition by SENP8 was performed. We found that a single N51E mutation of CrNEDD8 completely inhibited its hydrolysis by SENP8. Conversely, a single E51N mutation of CrUb enabled this ubiquitin mutant to undergo hydrolysis by SENP8, revealing that a single residue difference at the position 51 contributes substantially to the substrate selectivity of SENP8. Moreover, the E51N/R72A double mutant of the CrUb subdomain can further increase the efficiency of cleavage by SENP8, indicating that the residue at position 72 is also important in substrate recognition. The E51N or R72A mutation of CrUb also inhibited the hydrolysis of CrUb by ubiquitin-specific peptidase USP2. However, USP2 cannot cleave the N51E/A72R double mutant of the CrNEDD8 subdomain, suggesting that USP2 requires additional recognition sites. Public Library of Science 2011-11-14 /pmc/articles/PMC3215745/ /pubmed/22110750 http://dx.doi.org/10.1371/journal.pone.0027742 Text en Shin et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Shin, Yung-Cheng
Tang, Siao-Jing
Chen, Jou-Han
Liao, Pei-Han
Chang, Shih-Chung
The Molecular Determinants of NEDD8 Specific Recognition by Human SENP8
title The Molecular Determinants of NEDD8 Specific Recognition by Human SENP8
title_full The Molecular Determinants of NEDD8 Specific Recognition by Human SENP8
title_fullStr The Molecular Determinants of NEDD8 Specific Recognition by Human SENP8
title_full_unstemmed The Molecular Determinants of NEDD8 Specific Recognition by Human SENP8
title_short The Molecular Determinants of NEDD8 Specific Recognition by Human SENP8
title_sort molecular determinants of nedd8 specific recognition by human senp8
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3215745/
https://www.ncbi.nlm.nih.gov/pubmed/22110750
http://dx.doi.org/10.1371/journal.pone.0027742
work_keys_str_mv AT shinyungcheng themoleculardeterminantsofnedd8specificrecognitionbyhumansenp8
AT tangsiaojing themoleculardeterminantsofnedd8specificrecognitionbyhumansenp8
AT chenjouhan themoleculardeterminantsofnedd8specificrecognitionbyhumansenp8
AT liaopeihan themoleculardeterminantsofnedd8specificrecognitionbyhumansenp8
AT changshihchung themoleculardeterminantsofnedd8specificrecognitionbyhumansenp8
AT shinyungcheng moleculardeterminantsofnedd8specificrecognitionbyhumansenp8
AT tangsiaojing moleculardeterminantsofnedd8specificrecognitionbyhumansenp8
AT chenjouhan moleculardeterminantsofnedd8specificrecognitionbyhumansenp8
AT liaopeihan moleculardeterminantsofnedd8specificrecognitionbyhumansenp8
AT changshihchung moleculardeterminantsofnedd8specificrecognitionbyhumansenp8