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The cytosolic carboxypeptidases CCP2 and CCP3 catalyze posttranslational removal of acidic amino acids.

Tort O, Tanco S, Rocha C, Bièche I, Seixas C, Bosc C, Andrieux A, Moutin MJ, Avilés FX, Lorenzo J, Janke C - Mol. Biol. Cell (2014)

Bottom Line: Here we complete the functional characterization of this protein family by demonstrating that CCP2 and CCP3 are deglutamylases, with CCP3 being able to hydrolyze aspartic acids with similar efficiency.In addition, we show that CCP2 and CCP3 are highly regulated proteins confined to ciliated tissues.The characterization of two novel enzymes for carboxy-terminal protein modification provides novel insights into the broadness of this barely studied process.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biotecnologia i de Biomedicina, Department of Biochemistry and Molecular Biology, Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona), Spain Institut Curie, 91405 Orsay, France.

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Structural modeling of the carboxypeptidase domains of CCP2 and CCP3. (A) Modeled structure of human CCP3 (as a convention, all residues are numbered according to the corresponding active-site residues in bovine carboxypeptidase A1 after propeptide cleavage). The catalytic E270, the main substrate-specificity determining R255, and putative secondary binding-site residues are indicated. (B) Scheme of the substrate-binding subsites in the active site of carboxypeptidases (Schechter and Berger, 1967). (C) Vacuum electrostatics surface representation of the active site of hCCP3. Basic residues are indicated in blue and acidic residues in red. Positions 198 and 201 (corresponding to H462 and K465 in hCCP3) shape the S1 binding site in the hCCP3 model. Positions 127 and 145 (R414 and R424 in hCCP3) define the S2 subsite. Position 200 (R464 in hCCP3) is oriented toward the outer part of the active site, possibly defining an additional, positively charged S3 subsite. Note that although S1′ is defined by different residues (Supplemental Figure S1B), we here only depicted positions 250 and 255 because they are the major determinants of substrate specificity for CCPs. (D) Overlapped model structures of hCCP2 (cyan) and hCCP3 (green) demonstrate the conserved positions in the active-site residues (key residues E270 and R255 are shown). (E) Immunoblots of extracts of HEK293T cells expressing YFP-tagged murine and human CCP proteins. Expression of YFP-CCPs was analyzed with anti-GFP, and deglutamylating activity was visualized with anti–∆2-tubulin labeling on endogenous α‑tubulin. α‑Tubulin levels were controlled with 12G10 antibody. mCCP3_opt is a codon-optimized synthetic gene construct; hCCP3 is the human 73-kDa isoform of CCP3 (Q8NEM8-2 Uniprot). Note the presence of a specific degradation product (*) of mCCP1. Images of structures in A, C, and D were generated with PyMOL 1.3. (–), control without transfection.
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Figure 1: Structural modeling of the carboxypeptidase domains of CCP2 and CCP3. (A) Modeled structure of human CCP3 (as a convention, all residues are numbered according to the corresponding active-site residues in bovine carboxypeptidase A1 after propeptide cleavage). The catalytic E270, the main substrate-specificity determining R255, and putative secondary binding-site residues are indicated. (B) Scheme of the substrate-binding subsites in the active site of carboxypeptidases (Schechter and Berger, 1967). (C) Vacuum electrostatics surface representation of the active site of hCCP3. Basic residues are indicated in blue and acidic residues in red. Positions 198 and 201 (corresponding to H462 and K465 in hCCP3) shape the S1 binding site in the hCCP3 model. Positions 127 and 145 (R414 and R424 in hCCP3) define the S2 subsite. Position 200 (R464 in hCCP3) is oriented toward the outer part of the active site, possibly defining an additional, positively charged S3 subsite. Note that although S1′ is defined by different residues (Supplemental Figure S1B), we here only depicted positions 250 and 255 because they are the major determinants of substrate specificity for CCPs. (D) Overlapped model structures of hCCP2 (cyan) and hCCP3 (green) demonstrate the conserved positions in the active-site residues (key residues E270 and R255 are shown). (E) Immunoblots of extracts of HEK293T cells expressing YFP-tagged murine and human CCP proteins. Expression of YFP-CCPs was analyzed with anti-GFP, and deglutamylating activity was visualized with anti–∆2-tubulin labeling on endogenous α‑tubulin. α‑Tubulin levels were controlled with 12G10 antibody. mCCP3_opt is a codon-optimized synthetic gene construct; hCCP3 is the human 73-kDa isoform of CCP3 (Q8NEM8-2 Uniprot). Note the presence of a specific degradation product (*) of mCCP1. Images of structures in A, C, and D were generated with PyMOL 1.3. (–), control without transfection.

Mentions: After the discovery that four (CCP1, CCP4, CCP5, CCP6) of the six murine CCPs are deglutamylases (Rogowski et al., 2010), attention turned to the potential functions of CCP2 and CCP3. Considering that enzymes for deglycylation and detyrosination of tubulin have not been identified so far and that the reverse enzymes for these two reactions are all members of the same TTLL family, one expectation was that CCP2 and CCP3 could be involved in either or both of these PTMs. Indeed, an initial report (Sahab et al., 2011) attributed a detyrosinating activity to CCP2; however, unambiguous evidence for this activity was not provided, and overexpression of CCP2 did not lead to a strong increase in detyrosinated tubulin. To gain more insight into the substrate preferences of CCP2 and CCP3, we modeled the catalytic domains of the two enzymes based on the CCP crystal structures of Pseudomonas aeruginosa (PDB 4a37; Otero et al., 2012), Burkholderia mallei (PDB 3k2k), and Shewanella denitrificans (PDB 3l2n; Figure 1A and Supplemental Figure S1A).


The cytosolic carboxypeptidases CCP2 and CCP3 catalyze posttranslational removal of acidic amino acids.

Tort O, Tanco S, Rocha C, Bièche I, Seixas C, Bosc C, Andrieux A, Moutin MJ, Avilés FX, Lorenzo J, Janke C - Mol. Biol. Cell (2014)

Structural modeling of the carboxypeptidase domains of CCP2 and CCP3. (A) Modeled structure of human CCP3 (as a convention, all residues are numbered according to the corresponding active-site residues in bovine carboxypeptidase A1 after propeptide cleavage). The catalytic E270, the main substrate-specificity determining R255, and putative secondary binding-site residues are indicated. (B) Scheme of the substrate-binding subsites in the active site of carboxypeptidases (Schechter and Berger, 1967). (C) Vacuum electrostatics surface representation of the active site of hCCP3. Basic residues are indicated in blue and acidic residues in red. Positions 198 and 201 (corresponding to H462 and K465 in hCCP3) shape the S1 binding site in the hCCP3 model. Positions 127 and 145 (R414 and R424 in hCCP3) define the S2 subsite. Position 200 (R464 in hCCP3) is oriented toward the outer part of the active site, possibly defining an additional, positively charged S3 subsite. Note that although S1′ is defined by different residues (Supplemental Figure S1B), we here only depicted positions 250 and 255 because they are the major determinants of substrate specificity for CCPs. (D) Overlapped model structures of hCCP2 (cyan) and hCCP3 (green) demonstrate the conserved positions in the active-site residues (key residues E270 and R255 are shown). (E) Immunoblots of extracts of HEK293T cells expressing YFP-tagged murine and human CCP proteins. Expression of YFP-CCPs was analyzed with anti-GFP, and deglutamylating activity was visualized with anti–∆2-tubulin labeling on endogenous α‑tubulin. α‑Tubulin levels were controlled with 12G10 antibody. mCCP3_opt is a codon-optimized synthetic gene construct; hCCP3 is the human 73-kDa isoform of CCP3 (Q8NEM8-2 Uniprot). Note the presence of a specific degradation product (*) of mCCP1. Images of structures in A, C, and D were generated with PyMOL 1.3. (–), control without transfection.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 1: Structural modeling of the carboxypeptidase domains of CCP2 and CCP3. (A) Modeled structure of human CCP3 (as a convention, all residues are numbered according to the corresponding active-site residues in bovine carboxypeptidase A1 after propeptide cleavage). The catalytic E270, the main substrate-specificity determining R255, and putative secondary binding-site residues are indicated. (B) Scheme of the substrate-binding subsites in the active site of carboxypeptidases (Schechter and Berger, 1967). (C) Vacuum electrostatics surface representation of the active site of hCCP3. Basic residues are indicated in blue and acidic residues in red. Positions 198 and 201 (corresponding to H462 and K465 in hCCP3) shape the S1 binding site in the hCCP3 model. Positions 127 and 145 (R414 and R424 in hCCP3) define the S2 subsite. Position 200 (R464 in hCCP3) is oriented toward the outer part of the active site, possibly defining an additional, positively charged S3 subsite. Note that although S1′ is defined by different residues (Supplemental Figure S1B), we here only depicted positions 250 and 255 because they are the major determinants of substrate specificity for CCPs. (D) Overlapped model structures of hCCP2 (cyan) and hCCP3 (green) demonstrate the conserved positions in the active-site residues (key residues E270 and R255 are shown). (E) Immunoblots of extracts of HEK293T cells expressing YFP-tagged murine and human CCP proteins. Expression of YFP-CCPs was analyzed with anti-GFP, and deglutamylating activity was visualized with anti–∆2-tubulin labeling on endogenous α‑tubulin. α‑Tubulin levels were controlled with 12G10 antibody. mCCP3_opt is a codon-optimized synthetic gene construct; hCCP3 is the human 73-kDa isoform of CCP3 (Q8NEM8-2 Uniprot). Note the presence of a specific degradation product (*) of mCCP1. Images of structures in A, C, and D were generated with PyMOL 1.3. (–), control without transfection.
Mentions: After the discovery that four (CCP1, CCP4, CCP5, CCP6) of the six murine CCPs are deglutamylases (Rogowski et al., 2010), attention turned to the potential functions of CCP2 and CCP3. Considering that enzymes for deglycylation and detyrosination of tubulin have not been identified so far and that the reverse enzymes for these two reactions are all members of the same TTLL family, one expectation was that CCP2 and CCP3 could be involved in either or both of these PTMs. Indeed, an initial report (Sahab et al., 2011) attributed a detyrosinating activity to CCP2; however, unambiguous evidence for this activity was not provided, and overexpression of CCP2 did not lead to a strong increase in detyrosinated tubulin. To gain more insight into the substrate preferences of CCP2 and CCP3, we modeled the catalytic domains of the two enzymes based on the CCP crystal structures of Pseudomonas aeruginosa (PDB 4a37; Otero et al., 2012), Burkholderia mallei (PDB 3k2k), and Shewanella denitrificans (PDB 3l2n; Figure 1A and Supplemental Figure S1A).

Bottom Line: Here we complete the functional characterization of this protein family by demonstrating that CCP2 and CCP3 are deglutamylases, with CCP3 being able to hydrolyze aspartic acids with similar efficiency.In addition, we show that CCP2 and CCP3 are highly regulated proteins confined to ciliated tissues.The characterization of two novel enzymes for carboxy-terminal protein modification provides novel insights into the broadness of this barely studied process.

View Article: PubMed Central - PubMed

Affiliation: Institut de Biotecnologia i de Biomedicina, Department of Biochemistry and Molecular Biology, Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona), Spain Institut Curie, 91405 Orsay, France.

Show MeSH