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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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Optimized forms of mCCP2 and mCCP3 reveal deglutamylating activity. (A) Scheme of generic CCP; full-length and truncated forms of mCCP2 and mCCP3, and the 73‑kDa hCCP3 isoform. The green boxes indicate in the conserved N-terminal domain (Nt) specific to CCPs, with FESGNL, WYYY, and YPYTY conserved motifs (Rodriguez de la Vega et al, 2007). The blue box shows the conserved carboxypeptidase domain (CP) with the catalytic residue E270 (E-cat). Gray lines are nonconserved sequences that were partially truncated in the optimization. The shortest truncated versions that are still enzymatically active are mCCP2_Z1703 and mCCP3_Z1670 (red boxes). They are shown in comparison to the full-length versions and to hCCP3 (orange box). (B) Immunoblot of cell extracts from HEK293T cells expressing YFP-tagged, full-length mCCP2 and mCCP3, their optimized truncated forms (A), and enzymatically dead versions as controls. ∆2-Tubulin was used as readout for deglutamylating activity. Detyrosination and deglutamylase activities were followed by generation of deTyr- and Δ2-tubulin. Active enzymes generate ∆2-tubulin. (C) Immunocytochemistry of HEK293T cells transfected with active and inactive truncated YFP-tagged mCCP2 and mCCP3, as well as with GFP-mCCP1. After fixation of the cells with a protocol preserving microtubule structures (Bell and Safiejko-Mroczka, 1995), ∆2-tubulin was detected. Images were collected on an inverted confocal microscope (Leica SP5; Leica, Wetzlar, Germany) using a 63× objective at 25ºC and analyzed with LAF AS Lite 1.8.1 (Leica). Scale bar, 20 μm.
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Figure 2: Optimized forms of mCCP2 and mCCP3 reveal deglutamylating activity. (A) Scheme of generic CCP; full-length and truncated forms of mCCP2 and mCCP3, and the 73‑kDa hCCP3 isoform. The green boxes indicate in the conserved N-terminal domain (Nt) specific to CCPs, with FESGNL, WYYY, and YPYTY conserved motifs (Rodriguez de la Vega et al, 2007). The blue box shows the conserved carboxypeptidase domain (CP) with the catalytic residue E270 (E-cat). Gray lines are nonconserved sequences that were partially truncated in the optimization. The shortest truncated versions that are still enzymatically active are mCCP2_Z1703 and mCCP3_Z1670 (red boxes). They are shown in comparison to the full-length versions and to hCCP3 (orange box). (B) Immunoblot of cell extracts from HEK293T cells expressing YFP-tagged, full-length mCCP2 and mCCP3, their optimized truncated forms (A), and enzymatically dead versions as controls. ∆2-Tubulin was used as readout for deglutamylating activity. Detyrosination and deglutamylase activities were followed by generation of deTyr- and Δ2-tubulin. Active enzymes generate ∆2-tubulin. (C) Immunocytochemistry of HEK293T cells transfected with active and inactive truncated YFP-tagged mCCP2 and mCCP3, as well as with GFP-mCCP1. After fixation of the cells with a protocol preserving microtubule structures (Bell and Safiejko-Mroczka, 1995), ∆2-tubulin was detected. Images were collected on an inverted confocal microscope (Leica SP5; Leica, Wetzlar, Germany) using a 63× objective at 25ºC and analyzed with LAF AS Lite 1.8.1 (Leica). Scale bar, 20 μm.

Mentions: We thus generated a series of truncated forms of murine CCP2 and CCP3 in order to determine the minimal active size of these enzymes (Supplemental Table S1). The YFP-tagged truncated forms were expressed in HEK293T cells, and their activity was assessed by immunoblot for Δ2-tubulin (Supplemental Figure S2, A and B). Indeed, the truncated versions of both murine CCP2 and CCP3 showed a clear ∆2-tubulin–generating activity, demonstrating that both can act as deglutamylating enzymes. The shortest and most active versions are CCP2_Z1703 and CCP3_Z1670 (Figure 2A), which were obtained by truncating nonconserved N- and C-terminal sequences to obtain 65-kDa proteins that were similar in size and domain structure to the highly active deglutamylase CCP6 (Figure 2B and Supplemental Figure S2). Immunocytochemical analysis of HEK293T cells expressing CCP2 and CCP3 further showed a specific ∆2-tubulin labeling associated with the MTs in YFP-positive cells (Figure 2C). To demonstrate that the observed ∆2-tubulin generation after CCP2 and CCP3 expression is directly catalyzed by their active carboxypeptidase (CP) domains, we generated enzymatically dead versions by mutating the essential catalytic residues Glu-593 in mouse CCP2 and Glu-540 in mouse CCP3 (Glu-270 in bovine CPA and Glu-1094 in mouse CCP1; Wu et al., 2012). Enzymatically dead mutants of full-length and truncated versions of CCP2 and CCP3 were expressed at similar levels as the active forms but did not generate Δ2-tubulin (Figure 2B). Strikingly, a very faint ∆2-tubulin band present in the control cells is now absent in the cells overexpressing the enzymatically dead enzymes (Figure 2B), suggesting that these enzymes can act as dominant negative competitors for endogenous enzymes. These experiments demonstrate that the enzymatic activity of CCP2 and CCP3 is involved in the observed deglutamylation reactions on tubulin, leading to ∆2-tubulin.


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)

Optimized forms of mCCP2 and mCCP3 reveal deglutamylating activity. (A) Scheme of generic CCP; full-length and truncated forms of mCCP2 and mCCP3, and the 73‑kDa hCCP3 isoform. The green boxes indicate in the conserved N-terminal domain (Nt) specific to CCPs, with FESGNL, WYYY, and YPYTY conserved motifs (Rodriguez de la Vega et al, 2007). The blue box shows the conserved carboxypeptidase domain (CP) with the catalytic residue E270 (E-cat). Gray lines are nonconserved sequences that were partially truncated in the optimization. The shortest truncated versions that are still enzymatically active are mCCP2_Z1703 and mCCP3_Z1670 (red boxes). They are shown in comparison to the full-length versions and to hCCP3 (orange box). (B) Immunoblot of cell extracts from HEK293T cells expressing YFP-tagged, full-length mCCP2 and mCCP3, their optimized truncated forms (A), and enzymatically dead versions as controls. ∆2-Tubulin was used as readout for deglutamylating activity. Detyrosination and deglutamylase activities were followed by generation of deTyr- and Δ2-tubulin. Active enzymes generate ∆2-tubulin. (C) Immunocytochemistry of HEK293T cells transfected with active and inactive truncated YFP-tagged mCCP2 and mCCP3, as well as with GFP-mCCP1. After fixation of the cells with a protocol preserving microtubule structures (Bell and Safiejko-Mroczka, 1995), ∆2-tubulin was detected. Images were collected on an inverted confocal microscope (Leica SP5; Leica, Wetzlar, Germany) using a 63× objective at 25ºC and analyzed with LAF AS Lite 1.8.1 (Leica). Scale bar, 20 μm.
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Related In: Results  -  Collection

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Figure 2: Optimized forms of mCCP2 and mCCP3 reveal deglutamylating activity. (A) Scheme of generic CCP; full-length and truncated forms of mCCP2 and mCCP3, and the 73‑kDa hCCP3 isoform. The green boxes indicate in the conserved N-terminal domain (Nt) specific to CCPs, with FESGNL, WYYY, and YPYTY conserved motifs (Rodriguez de la Vega et al, 2007). The blue box shows the conserved carboxypeptidase domain (CP) with the catalytic residue E270 (E-cat). Gray lines are nonconserved sequences that were partially truncated in the optimization. The shortest truncated versions that are still enzymatically active are mCCP2_Z1703 and mCCP3_Z1670 (red boxes). They are shown in comparison to the full-length versions and to hCCP3 (orange box). (B) Immunoblot of cell extracts from HEK293T cells expressing YFP-tagged, full-length mCCP2 and mCCP3, their optimized truncated forms (A), and enzymatically dead versions as controls. ∆2-Tubulin was used as readout for deglutamylating activity. Detyrosination and deglutamylase activities were followed by generation of deTyr- and Δ2-tubulin. Active enzymes generate ∆2-tubulin. (C) Immunocytochemistry of HEK293T cells transfected with active and inactive truncated YFP-tagged mCCP2 and mCCP3, as well as with GFP-mCCP1. After fixation of the cells with a protocol preserving microtubule structures (Bell and Safiejko-Mroczka, 1995), ∆2-tubulin was detected. Images were collected on an inverted confocal microscope (Leica SP5; Leica, Wetzlar, Germany) using a 63× objective at 25ºC and analyzed with LAF AS Lite 1.8.1 (Leica). Scale bar, 20 μm.
Mentions: We thus generated a series of truncated forms of murine CCP2 and CCP3 in order to determine the minimal active size of these enzymes (Supplemental Table S1). The YFP-tagged truncated forms were expressed in HEK293T cells, and their activity was assessed by immunoblot for Δ2-tubulin (Supplemental Figure S2, A and B). Indeed, the truncated versions of both murine CCP2 and CCP3 showed a clear ∆2-tubulin–generating activity, demonstrating that both can act as deglutamylating enzymes. The shortest and most active versions are CCP2_Z1703 and CCP3_Z1670 (Figure 2A), which were obtained by truncating nonconserved N- and C-terminal sequences to obtain 65-kDa proteins that were similar in size and domain structure to the highly active deglutamylase CCP6 (Figure 2B and Supplemental Figure S2). Immunocytochemical analysis of HEK293T cells expressing CCP2 and CCP3 further showed a specific ∆2-tubulin labeling associated with the MTs in YFP-positive cells (Figure 2C). To demonstrate that the observed ∆2-tubulin generation after CCP2 and CCP3 expression is directly catalyzed by their active carboxypeptidase (CP) domains, we generated enzymatically dead versions by mutating the essential catalytic residues Glu-593 in mouse CCP2 and Glu-540 in mouse CCP3 (Glu-270 in bovine CPA and Glu-1094 in mouse CCP1; Wu et al., 2012). Enzymatically dead mutants of full-length and truncated versions of CCP2 and CCP3 were expressed at similar levels as the active forms but did not generate Δ2-tubulin (Figure 2B). Strikingly, a very faint ∆2-tubulin band present in the control cells is now absent in the cells overexpressing the enzymatically dead enzymes (Figure 2B), suggesting that these enzymes can act as dominant negative competitors for endogenous enzymes. These experiments demonstrate that the enzymatic activity of CCP2 and CCP3 is involved in the observed deglutamylation reactions on tubulin, leading to ∆2-tubulin.

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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