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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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Analysis of mCCP2 and mCCP3 expression and of the Agbl2/Agbl3 double-KO mice. (A) Relative expression levels of mCCP2 and mCCP3 in different organs of wild-type (WT) mice as determined by qRT-PCR. Average values relative to the Tbp gene expression are represented, and error bars represent SD of five independent experiments. (B) Cumulative view of the relative expression levels of all six CCP genes in different murine tissues (values from A and Supplemental Figure S4A). (C) qRT-PCR analysis of expression levels of all six CCP genes during ciliogenesis in cultured IMCD3 cells. Cells were serum starved and analyzed at 0, 3, and 5 d after starvation. Mean values of expression levels relative to Tbp expression and relative to 0 d from three to five independent experiments are shown; error bars show SD. Two-way analysis of variance was used to determine significance levels (**p < 0.01; ***p < 0.001). (D) Comparative immunoblot analysis of protein extracts from different organs of WT and Agbl2/Agbl3 double-KO mice with polyE. Total tubulin levels were detected with anti–α-tubulin antibody (12G10). For sperm that showed the highest polyE levels in the tubulin region, the experiment was repeated with half of the initial protein load (lanes labeled with *). (E) Quantitative analysis of the polyE signal relative to the 12G10 signal Agbl2/Agbl3 double-KO mice relative to WT. Four of each WT and double KO were analyzed (Supplemental Figure S5), and relative values were plotted (WT was set to 1); error bars show SD. Student's t test was used to determine significance levels (*p < 0.05; **p < 0.01).
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Figure 4: Analysis of mCCP2 and mCCP3 expression and of the Agbl2/Agbl3 double-KO mice. (A) Relative expression levels of mCCP2 and mCCP3 in different organs of wild-type (WT) mice as determined by qRT-PCR. Average values relative to the Tbp gene expression are represented, and error bars represent SD of five independent experiments. (B) Cumulative view of the relative expression levels of all six CCP genes in different murine tissues (values from A and Supplemental Figure S4A). (C) qRT-PCR analysis of expression levels of all six CCP genes during ciliogenesis in cultured IMCD3 cells. Cells were serum starved and analyzed at 0, 3, and 5 d after starvation. Mean values of expression levels relative to Tbp expression and relative to 0 d from three to five independent experiments are shown; error bars show SD. Two-way analysis of variance was used to determine significance levels (**p < 0.01; ***p < 0.001). (D) Comparative immunoblot analysis of protein extracts from different organs of WT and Agbl2/Agbl3 double-KO mice with polyE. Total tubulin levels were detected with anti–α-tubulin antibody (12G10). For sperm that showed the highest polyE levels in the tubulin region, the experiment was repeated with half of the initial protein load (lanes labeled with *). (E) Quantitative analysis of the polyE signal relative to the 12G10 signal Agbl2/Agbl3 double-KO mice relative to WT. Four of each WT and double KO were analyzed (Supplemental Figure S5), and relative values were plotted (WT was set to 1); error bars show SD. Student's t test was used to determine significance levels (*p < 0.05; **p < 0.01).

Mentions: Glycylation is a posttranslational modification similar to polyglutamylation, performed by members of the TTLL family (Rogowski et al., 2009). Nothing is known about the enzymes responsible for shortening or removing posttranslationally generated glycine chains from tubulin. The unique deglycylase identified so far is an M20 peptidase family member in Giardia duodenalis (Lalle et al., 2011). Because this enzyme deglycylates the 14-3-3 proteins, and because M20 peptidases are not found in the mammalian genome, other candidates were considered as potential deglyclases in mammals, among them CCP2 and CCP3. Glycylation is typically enriched on the axonemes of motile cilia and flagella (Redeker et al., 1994). Thus the presence of CCP2 and CCP3 genes in ciliated organisms (Rodríguez de la Vega Otazo et al., 2013) and their increased expression levels in mouse tissues with motile cilia such as testis and trachea (Figure 4A) provided a potential link to glycylation. Although the structural model already suggested that both enzymes are rather specific to acidic amino acids (Figure 1), we wanted to obtain additional experimental proof to confirm this. Our attempts to directly test deglycylating activity in cells with glycylated MTs, which could be generated by expression of glycylating enzymes, failed due to the toxicity of this treatment for the cells. Thus we developed an alternative test for deglycylating activity, in which we coexpressed a chimeric telokin containing four Gly residues on the very C-terminus. This construct is specifically detected with the polyclonal antibody polyG, and removal of only one Gly residue completely abolished detection (Supplemental Figure S3B). The chimeric telokin-Gly was coexpressed together with CCP2_Z1703 or CCP3_Z1670, and a similar experiment was performed with a polyE antibody and a telokin with three Glu residues as positive control (Supplemental Figure S3C). Whereas both CCP2_Z1703 and CCP3_Z1670 were perfectly able to remove C-terminal glutamates, the unchanged polyG signals strongly suggest that C-terminally located Gly residues cannot be hydrolyzed by these enzymes. Thus CCP2 and CCP3 do not carry an intrinsic deglycylase activity.


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)

Analysis of mCCP2 and mCCP3 expression and of the Agbl2/Agbl3 double-KO mice. (A) Relative expression levels of mCCP2 and mCCP3 in different organs of wild-type (WT) mice as determined by qRT-PCR. Average values relative to the Tbp gene expression are represented, and error bars represent SD of five independent experiments. (B) Cumulative view of the relative expression levels of all six CCP genes in different murine tissues (values from A and Supplemental Figure S4A). (C) qRT-PCR analysis of expression levels of all six CCP genes during ciliogenesis in cultured IMCD3 cells. Cells were serum starved and analyzed at 0, 3, and 5 d after starvation. Mean values of expression levels relative to Tbp expression and relative to 0 d from three to five independent experiments are shown; error bars show SD. Two-way analysis of variance was used to determine significance levels (**p < 0.01; ***p < 0.001). (D) Comparative immunoblot analysis of protein extracts from different organs of WT and Agbl2/Agbl3 double-KO mice with polyE. Total tubulin levels were detected with anti–α-tubulin antibody (12G10). For sperm that showed the highest polyE levels in the tubulin region, the experiment was repeated with half of the initial protein load (lanes labeled with *). (E) Quantitative analysis of the polyE signal relative to the 12G10 signal Agbl2/Agbl3 double-KO mice relative to WT. Four of each WT and double KO were analyzed (Supplemental Figure S5), and relative values were plotted (WT was set to 1); error bars show SD. Student's t test was used to determine significance levels (*p < 0.05; **p < 0.01).
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Figure 4: Analysis of mCCP2 and mCCP3 expression and of the Agbl2/Agbl3 double-KO mice. (A) Relative expression levels of mCCP2 and mCCP3 in different organs of wild-type (WT) mice as determined by qRT-PCR. Average values relative to the Tbp gene expression are represented, and error bars represent SD of five independent experiments. (B) Cumulative view of the relative expression levels of all six CCP genes in different murine tissues (values from A and Supplemental Figure S4A). (C) qRT-PCR analysis of expression levels of all six CCP genes during ciliogenesis in cultured IMCD3 cells. Cells were serum starved and analyzed at 0, 3, and 5 d after starvation. Mean values of expression levels relative to Tbp expression and relative to 0 d from three to five independent experiments are shown; error bars show SD. Two-way analysis of variance was used to determine significance levels (**p < 0.01; ***p < 0.001). (D) Comparative immunoblot analysis of protein extracts from different organs of WT and Agbl2/Agbl3 double-KO mice with polyE. Total tubulin levels were detected with anti–α-tubulin antibody (12G10). For sperm that showed the highest polyE levels in the tubulin region, the experiment was repeated with half of the initial protein load (lanes labeled with *). (E) Quantitative analysis of the polyE signal relative to the 12G10 signal Agbl2/Agbl3 double-KO mice relative to WT. Four of each WT and double KO were analyzed (Supplemental Figure S5), and relative values were plotted (WT was set to 1); error bars show SD. Student's t test was used to determine significance levels (*p < 0.05; **p < 0.01).
Mentions: Glycylation is a posttranslational modification similar to polyglutamylation, performed by members of the TTLL family (Rogowski et al., 2009). Nothing is known about the enzymes responsible for shortening or removing posttranslationally generated glycine chains from tubulin. The unique deglycylase identified so far is an M20 peptidase family member in Giardia duodenalis (Lalle et al., 2011). Because this enzyme deglycylates the 14-3-3 proteins, and because M20 peptidases are not found in the mammalian genome, other candidates were considered as potential deglyclases in mammals, among them CCP2 and CCP3. Glycylation is typically enriched on the axonemes of motile cilia and flagella (Redeker et al., 1994). Thus the presence of CCP2 and CCP3 genes in ciliated organisms (Rodríguez de la Vega Otazo et al., 2013) and their increased expression levels in mouse tissues with motile cilia such as testis and trachea (Figure 4A) provided a potential link to glycylation. Although the structural model already suggested that both enzymes are rather specific to acidic amino acids (Figure 1), we wanted to obtain additional experimental proof to confirm this. Our attempts to directly test deglycylating activity in cells with glycylated MTs, which could be generated by expression of glycylating enzymes, failed due to the toxicity of this treatment for the cells. Thus we developed an alternative test for deglycylating activity, in which we coexpressed a chimeric telokin containing four Gly residues on the very C-terminus. This construct is specifically detected with the polyclonal antibody polyG, and removal of only one Gly residue completely abolished detection (Supplemental Figure S3B). The chimeric telokin-Gly was coexpressed together with CCP2_Z1703 or CCP3_Z1670, and a similar experiment was performed with a polyE antibody and a telokin with three Glu residues as positive control (Supplemental Figure S3C). Whereas both CCP2_Z1703 and CCP3_Z1670 were perfectly able to remove C-terminal glutamates, the unchanged polyG signals strongly suggest that C-terminally located Gly residues cannot be hydrolyzed by these enzymes. Thus CCP2 and CCP3 do not carry an intrinsic deglycylase activity.

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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