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
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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.
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.