Cytosolic carboxypeptidase 1: Tubulin processing and neurodegeneration

Abstract

Purkinje cell degeneration (pcd) is a classical mouse mutant which has been used as a model of neurodegenerative disorder for decades. pcd mouse has a disruption in the gene encoding cytosolic carboxypeptidase 1 (CCP1), an enzyme which was initially discovered to be linked to axonal regeneration. Expression of wild-type CCP1 cDNA, but not CCP1 with mutated catalytic amino acids, rescues from the pcd phenotype indicating that CCP1 is an active enzyme which is important for Purkinje cell survival. Although pcd mice have been studied for more than 40 years, the precise function of CCP1 and the mechanisms of neurodegeneration remain to be understood.;We proposed two potential functions for CCP1: degradation of intracellular peptides and processing of tubulin. In the present study we tested our hypothesis. First, we investigated the levels of intracellular peptides in the brains of adult wild-type and pcd mice. We detected accumulation of hundreds of cytosolic peptides in adult pcd mouse brains, which raised the possibility that CCP1 functions in peptide degradation. However, contrary to adult mouse brain, young pcd brain and adult heart and spleen did not show accumulation of intracellular peptides, suggesting that peptide degradation is not a primary CCP1 function.;The role of CCP1 in tubulin processing was also studied. Overexpression or knock-down of CCP1 in cultured cells does not affect the levels of most intracellular peptides but alters the levels of delta2-tubulin. Analysis of purified CCP1 activity toward purified brain tubulin showed that CCP1 removes glutamates from polyglutamylated chains of alpha- and beta-tubulin, and cleaves glutamates from the C-terminus of alpha-tubulin. Consistent with this, both alpha- and beta-tubulin are hyper-glutamylated in the pcd mouse brains. Tubulin hyper-glutamylation and subsequent Purkinje cell degeneration in pcd mice are rescued by the knockout of the tubulin tyrosine ligase-like-1 gene.;Collectively, our in vitro and in vivo data indicate that the primary function of CCP1 is processing of alpha- and beta-tubulin, but not protesome-generated protein fragments. The role of CCP1 in tubulin posttranslational modifications, and therefore in intracellular tubulin transport, is consistent with the numerous cellular defects observed in pcd mice.