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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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Substrate specificity of mCCP2 and mCCP3. (A) Schematic representation of the experimental setup. PolyE antibody recognizes telokin constructs with three or more consecutive C-terminal glutamate residues, whereas anti-Δ2-tubulin antibody detects specifically the C-terminal –GE epitope. C-terminal degradation is detected by generation of the Δ2-tubulin epitope, and absence or strong decrease of the polyE signal on telokin (Rogowski et al, 2010). (B) Immunoblot analysis of HEK293T extracts after coexpression of different YFP-CCPs and YFP-telokin variants. Activity is monitored as shown in A. The activity of truncated mCCP2 and mCCP3 (Figure 2A) is tested with telokin variants with different numbers of consecutive glutamate residues to test processivity and with an aspartate residue to test specificity. The activities are compared with mCCP1, an established deglutamylase (Rogowski et al, 2010), and a dead version of mCCP3 as negative control. Note that only mCCP3 removes aspartate efficiently. (C) Bioinformatic analysis of the number of proteins with uninterrupted C-terminal acid sequence stretches. Each column represents the total number of proteins per category (mixed Asp and Glu stretches), and the darker insets represent uninterrupted Glu stretches.
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Figure 3: Substrate specificity of mCCP2 and mCCP3. (A) Schematic representation of the experimental setup. PolyE antibody recognizes telokin constructs with three or more consecutive C-terminal glutamate residues, whereas anti-Δ2-tubulin antibody detects specifically the C-terminal –GE epitope. C-terminal degradation is detected by generation of the Δ2-tubulin epitope, and absence or strong decrease of the polyE signal on telokin (Rogowski et al, 2010). (B) Immunoblot analysis of HEK293T extracts after coexpression of different YFP-CCPs and YFP-telokin variants. Activity is monitored as shown in A. The activity of truncated mCCP2 and mCCP3 (Figure 2A) is tested with telokin variants with different numbers of consecutive glutamate residues to test processivity and with an aspartate residue to test specificity. The activities are compared with mCCP1, an established deglutamylase (Rogowski et al, 2010), and a dead version of mCCP3 as negative control. Note that only mCCP3 removes aspartate efficiently. (C) Bioinformatic analysis of the number of proteins with uninterrupted C-terminal acid sequence stretches. Each column represents the total number of proteins per category (mixed Asp and Glu stretches), and the darker insets represent uninterrupted Glu stretches.

Mentions: To further characterize whether CCP2 and CCP3 can remove subsequent glutamate residues from longer stretches of glutamates, such as those generated by enzymatic polyglutamylation (Audebert et al., 1993; van Dijk et al., 2007) or genetically encoded as in the case of MLCK, we used C-terminally engineered chimeras of telokin, a short version of MLCK, which is one of the few known substrates of CCP1 (Rogowski et al., 2010). Truncated active versions of CCP2 and CCP3 were coexpressed in HEK293T cells together with different C-terminal variants of YFP-telokin. The deglutamylation (removal for long glutamate chains) was monitored using the polyE antibody in immunoblot analysis (Shang, 2002), and the final deglutamylation product (ending with only one glutamate) was detected with the anti–Δ2-tubulin antibody (Figure 3A). Both CCP2_Z1703 and CCP3_Z1670 were able to trim long (7-Glu) and shorter polyglutamate chains from the C-terminus of chimeric telokin, as shown by decreased polyE signals and increased Δ2-tubulin immunoreactivity (Figure 3B). CCP1, known to shorten long glutamate chains, was used as positive control.


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)

Substrate specificity of mCCP2 and mCCP3. (A) Schematic representation of the experimental setup. PolyE antibody recognizes telokin constructs with three or more consecutive C-terminal glutamate residues, whereas anti-Δ2-tubulin antibody detects specifically the C-terminal –GE epitope. C-terminal degradation is detected by generation of the Δ2-tubulin epitope, and absence or strong decrease of the polyE signal on telokin (Rogowski et al, 2010). (B) Immunoblot analysis of HEK293T extracts after coexpression of different YFP-CCPs and YFP-telokin variants. Activity is monitored as shown in A. The activity of truncated mCCP2 and mCCP3 (Figure 2A) is tested with telokin variants with different numbers of consecutive glutamate residues to test processivity and with an aspartate residue to test specificity. The activities are compared with mCCP1, an established deglutamylase (Rogowski et al, 2010), and a dead version of mCCP3 as negative control. Note that only mCCP3 removes aspartate efficiently. (C) Bioinformatic analysis of the number of proteins with uninterrupted C-terminal acid sequence stretches. Each column represents the total number of proteins per category (mixed Asp and Glu stretches), and the darker insets represent uninterrupted Glu stretches.
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Related In: Results  -  Collection

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Figure 3: Substrate specificity of mCCP2 and mCCP3. (A) Schematic representation of the experimental setup. PolyE antibody recognizes telokin constructs with three or more consecutive C-terminal glutamate residues, whereas anti-Δ2-tubulin antibody detects specifically the C-terminal –GE epitope. C-terminal degradation is detected by generation of the Δ2-tubulin epitope, and absence or strong decrease of the polyE signal on telokin (Rogowski et al, 2010). (B) Immunoblot analysis of HEK293T extracts after coexpression of different YFP-CCPs and YFP-telokin variants. Activity is monitored as shown in A. The activity of truncated mCCP2 and mCCP3 (Figure 2A) is tested with telokin variants with different numbers of consecutive glutamate residues to test processivity and with an aspartate residue to test specificity. The activities are compared with mCCP1, an established deglutamylase (Rogowski et al, 2010), and a dead version of mCCP3 as negative control. Note that only mCCP3 removes aspartate efficiently. (C) Bioinformatic analysis of the number of proteins with uninterrupted C-terminal acid sequence stretches. Each column represents the total number of proteins per category (mixed Asp and Glu stretches), and the darker insets represent uninterrupted Glu stretches.
Mentions: To further characterize whether CCP2 and CCP3 can remove subsequent glutamate residues from longer stretches of glutamates, such as those generated by enzymatic polyglutamylation (Audebert et al., 1993; van Dijk et al., 2007) or genetically encoded as in the case of MLCK, we used C-terminally engineered chimeras of telokin, a short version of MLCK, which is one of the few known substrates of CCP1 (Rogowski et al., 2010). Truncated active versions of CCP2 and CCP3 were coexpressed in HEK293T cells together with different C-terminal variants of YFP-telokin. The deglutamylation (removal for long glutamate chains) was monitored using the polyE antibody in immunoblot analysis (Shang, 2002), and the final deglutamylation product (ending with only one glutamate) was detected with the anti–Δ2-tubulin antibody (Figure 3A). Both CCP2_Z1703 and CCP3_Z1670 were able to trim long (7-Glu) and shorter polyglutamate chains from the C-terminus of chimeric telokin, as shown by decreased polyE signals and increased Δ2-tubulin immunoreactivity (Figure 3B). CCP1, known to shorten long glutamate chains, was used as positive control.

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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Related in: MedlinePlus