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Assembly and proteolytic processing of mycobacterial ClpP1 and ClpP2
BACKGROUND: Caseinolytic proteases (ClpPs) are barrel-shaped self-compartmentalized peptidases involved in eliminating damaged or short-lived regulatory proteins. The Mycobacterium tuberculosis (MTB) genome contains two genes coding for putative ClpPs, ClpP1 and ClpP2 respectively, that are likely t...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2011
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3258218/ https://www.ncbi.nlm.nih.gov/pubmed/22132756 http://dx.doi.org/10.1186/1471-2091-12-61 |
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author | Benaroudj, Nadia Raynal, Bertrand Miot, Marika Ortiz-Lombardia, Miguel |
author_facet | Benaroudj, Nadia Raynal, Bertrand Miot, Marika Ortiz-Lombardia, Miguel |
author_sort | Benaroudj, Nadia |
collection | PubMed |
description | BACKGROUND: Caseinolytic proteases (ClpPs) are barrel-shaped self-compartmentalized peptidases involved in eliminating damaged or short-lived regulatory proteins. The Mycobacterium tuberculosis (MTB) genome contains two genes coding for putative ClpPs, ClpP1 and ClpP2 respectively, that are likely to play a role in the virulence of the bacterium. RESULTS: We report the first biochemical characterization of ClpP1 and ClpP2 peptidases from MTB. Both proteins were produced and purified in Escherichia coli. Use of fluorogenic model peptides of diverse specificities failed to show peptidase activity with recombinant mycobacterial ClpP1 or ClpP2. However, we found that ClpP1 had a proteolytic activity responsible for its own cleavage after the Arg8 residue and cleavage of ClpP2 after the Ala12 residue. In addition, we showed that the absence of any peptidase activity toward model peptides was not due to an obstruction of the entry pore by the N-terminal flexible extremity of the proteins, nor to an absolute requirement for the ClpX or ClpC ATPase complex. Finally, we also found that removing the putative propeptides of ClpP1 and ClpP2 did not result in cleavage of model peptides. We have also shown that recombinant ClpP1 and ClpP2 do not assemble in the conventional functional tetradecameric form but in lower order oligomeric species ranging from monomers to heptamers. The concomitant presence of both ClpP1 and ClpP2 did not result in tetradecameric assembly. Deleting the amino-terminal extremity of ClpP1 and ClpP2 (the putative propeptide or entry gate) promoted the assembly in higher order oligomeric species, suggesting that the flexible N-terminal extremity of mycobacterial ClpPs participated in the destabilization of interaction between heptamers. CONCLUSION: Despite the conservation of a Ser protease catalytic triad in their primary sequences, mycobacterial ClpP1 and ClpP2 do not have conventional peptidase activity toward peptide models and display an unusual mechanism of self-assembly. Therefore, the mechanism underlying their peptidase and proteolytic activities might differ from that of other ClpP proteolytic complexes. |
format | Online Article Text |
id | pubmed-3258218 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2011 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-32582182012-01-14 Assembly and proteolytic processing of mycobacterial ClpP1 and ClpP2 Benaroudj, Nadia Raynal, Bertrand Miot, Marika Ortiz-Lombardia, Miguel BMC Biochem Research Article BACKGROUND: Caseinolytic proteases (ClpPs) are barrel-shaped self-compartmentalized peptidases involved in eliminating damaged or short-lived regulatory proteins. The Mycobacterium tuberculosis (MTB) genome contains two genes coding for putative ClpPs, ClpP1 and ClpP2 respectively, that are likely to play a role in the virulence of the bacterium. RESULTS: We report the first biochemical characterization of ClpP1 and ClpP2 peptidases from MTB. Both proteins were produced and purified in Escherichia coli. Use of fluorogenic model peptides of diverse specificities failed to show peptidase activity with recombinant mycobacterial ClpP1 or ClpP2. However, we found that ClpP1 had a proteolytic activity responsible for its own cleavage after the Arg8 residue and cleavage of ClpP2 after the Ala12 residue. In addition, we showed that the absence of any peptidase activity toward model peptides was not due to an obstruction of the entry pore by the N-terminal flexible extremity of the proteins, nor to an absolute requirement for the ClpX or ClpC ATPase complex. Finally, we also found that removing the putative propeptides of ClpP1 and ClpP2 did not result in cleavage of model peptides. We have also shown that recombinant ClpP1 and ClpP2 do not assemble in the conventional functional tetradecameric form but in lower order oligomeric species ranging from monomers to heptamers. The concomitant presence of both ClpP1 and ClpP2 did not result in tetradecameric assembly. Deleting the amino-terminal extremity of ClpP1 and ClpP2 (the putative propeptide or entry gate) promoted the assembly in higher order oligomeric species, suggesting that the flexible N-terminal extremity of mycobacterial ClpPs participated in the destabilization of interaction between heptamers. CONCLUSION: Despite the conservation of a Ser protease catalytic triad in their primary sequences, mycobacterial ClpP1 and ClpP2 do not have conventional peptidase activity toward peptide models and display an unusual mechanism of self-assembly. Therefore, the mechanism underlying their peptidase and proteolytic activities might differ from that of other ClpP proteolytic complexes. BioMed Central 2011-12-01 /pmc/articles/PMC3258218/ /pubmed/22132756 http://dx.doi.org/10.1186/1471-2091-12-61 Text en Copyright ©2011 Benaroudj et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Research Article Benaroudj, Nadia Raynal, Bertrand Miot, Marika Ortiz-Lombardia, Miguel Assembly and proteolytic processing of mycobacterial ClpP1 and ClpP2 |
title | Assembly and proteolytic processing of mycobacterial ClpP1 and ClpP2 |
title_full | Assembly and proteolytic processing of mycobacterial ClpP1 and ClpP2 |
title_fullStr | Assembly and proteolytic processing of mycobacterial ClpP1 and ClpP2 |
title_full_unstemmed | Assembly and proteolytic processing of mycobacterial ClpP1 and ClpP2 |
title_short | Assembly and proteolytic processing of mycobacterial ClpP1 and ClpP2 |
title_sort | assembly and proteolytic processing of mycobacterial clpp1 and clpp2 |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3258218/ https://www.ncbi.nlm.nih.gov/pubmed/22132756 http://dx.doi.org/10.1186/1471-2091-12-61 |
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