A Family of Protein-Deglutamylating Enzymes Associated with Neurodegeneration

Abstract

Polyglutamylation is a posttranslational modification that generates glutamate side chains on tubulins and other proteins. Although this modification has been shown to be reversible, little is known about the enzymes catalyzing deglutamylation. Here we describe the enzymatic mechanism of protein deglutamylation by members of the cytosolic carboxypeptidase (CCP) family. Three enzymes (CCP1, CCP4, and CCP6) catalyze the shortening of polyglutamate chains and a fourth (CCP5) specifically removes the branching point glutamates. In addition, CCP1, CCP4, and CCP6 also remove gene-encoded glutamates from the carboxyl termini of proteins. Accordingly, we show that these enzymes convert detyrosinated tubulin into Δ2-tubulin and also modify other substrates, including myosin light chain kinase 1. We further analyze Purkinje cell degeneration (pcd) mice that lack functional CCP1 and show that microtubule hyperglutamylation is directly linked to neurodegeneration. Taken together, our results reveal that controlling the length of the polyglutamate side chains on tubulin is critical for neuronal survival.

Comparison of the Enzymatic and Functional Properties of Three Cytosolic Carboxypeptidase Family Members

“…Recombinant Nna1, CCP4, and CCP6 Metabolize the Polyglutamate Side Chain of Tubulin-Previous enzymatic studies of Nna1 family members were performed using tubulin as substrate and lysates of cells transfected with CCP4 and CCP6 as a source of enzyme with detection using an antibody to the polyglutamate chain of tubulin (10). As it is not practical to use this approach to determine details of enzyme kinetics or substrate preferences, we generated and purified N-terminally histidinetagged versions of Nna1 (7) and CCP4 and CCP6 (Fig.…”

“…We suggested that this might be a consequence of overlapping expression or redundancy with other Nna1 subfamily members, such as CCP4 and CCP6 (3), that are reported to catalyze the identical glutamase reaction in vitro (10). The primary objectives of this study were to determine whether the three CCPs have identical enzymatic and functional properties and whether these properties are consistent with the enzymes contributing to the selective neurodegeneration in pcd mice.…”

“…In parallel, Nna1 and its related family members, CCP4 -6, were demonstrated to be glutamases (9,10), enzymes that catabolize runs of glutamic acid either at the C terminus of proteins or in covalently attached side chains that are added to some proteins (notably tubulin) through a post-translational process termed polyglutamylation (reviewed in Ref. 11).…”

“…Polyglutamylation is a dynamic process in which glutamate residues are added to a branch point glutamate in the primary amino acid chain of substrate proteins by tubulin-tyrosine ligase-like (TTLL) enzymes and removed by the Nna1 subfamily of glutamases (7,9,10). These studies suggested that Nna1 functions in polyglutamylation by trimming polyglutamate chains and that disruption of this equilibrium in pcd mice leads to increased glutamate chain length on critical proteins such as tubulin and subsequent neurodegeneration (7,10). However, other features of the pcd mouse suggest that the situation in vivo may be more complex.…”

A missense mutation in AGTPBP1 was identified in sheep with a lower motor neuron disease

“…This protein has a role in protein turnover by cleaving proteasome-generated peptides into amino acids (Berezniuk et al, 2010), enzymatically removing geneencoded glutamic acids or tyrosine from the C-terminus of proteins (Rogowski et al, 2010, Kalinina et al, 2007 and also has a role in neuronal bioenergetics at least in mouse brains (Chakrabarti et al, 2010). Similar to other cytosolic carboxypeptidases, the mouse Nna1 protein is broadly expressed in the brain, pituitary, eye, testis and other tissues (Kalinina et al, 2007).…”