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