A structural and functional analysis of Nna1 inPurkinje cell degeneration(pcd) mice

Abstract

The axotomy-inducible enzyme Nna1 defines a subfamily of M14 metallocarboxypeptidases, and its mutation underlies the Purkinje cell degeneration (pcd) mouse. However, the relationship among its catalytic activity, substrate specificities, and the critical processes of neurodegeneration/axon regeneration is incompletely understood. Here we used a transgenic rescue strategy targeting expression of modified forms of Nna1 to Purkinje cells in pcd mice to determine structure-activity relationships for neuronal survival and in parallel characterized the enzymatic properties of purified recombinant Nna1. The Nna1 subfamily uniquely shares conserved substrate-determining residues with aspartoacylase that, when mutated, cause Canavan disease. Homologous mutations (D1007E and R1078E) inactivate Nna1 in vivo, as does mutation of its catalytic glutamate (E1094A), which implies that metabolism of acidic substrates is essential for neuronal survival. Consistent with reports that Nna1 is a tubulin glutamylase, recombinant Nna1-but not the catalytic mutants-removes glutamate from tubulin. Recombinant Nna1 metabolizes synthetic substrates with 2 or more C-terminal glutamate (but not aspartate) residues (V(max) for 3 glutamates is ∼7-fold higher than 2 glutamates although K(M) is similar). Catalysis is not ATP/GTP dependent, and mutating the ATP/GTP binding site of Nna1 has no effect in vivo. Nna1 is a monomeric enzyme essential for neuronal survival through hydrolysis of polyglutamate-containing substrates.

C-terminomics Screen for Natural Substrates of Cytosolic Carboxypeptidase 1 Reveals Processing of Acidic Protein C termini

“…Two of these peptides indicate CCP1-mediated removal of a single amino acid from the protein's C terminus: release of glutamate from the C terminus in case of the eukaryotic translation initiation factor 4H (eIF4H), and of aspartate in the case of stathmin. Previous reports demonstrated the action of CCP1 on C-terminal glutamates (15,36,37), and it was suggested that CCP1 does not recognize aspartate (15,37). Our observed trimming of the C terminus of stathmin indicates that CCP1 is indeed able to release C-terminal aspartates.…”

“…the C-terminal Asp and two or three subsequent Glu residues were removed, Table I). Given that M14 MCPs release only one amino acid at a time, it is expected that CCP1 sequentially releases these amino acids (37). The TRAF-type zinc finger domain-containing protein (TRAD1) was also identified as a putative CCP1 substrate.…”

“…N-terminal Strep-and HA-tagged TRAD1 was overexpressed in HEK293F cells together with CCP1 or an inactive form of CCP1 (CCP1-E270Q). The latter contains a mutation of a key catalytic residue of M14 metallocarboxypeptidases (E270Q using the numbering system of bovine carboxypeptidase A, E1102Q in human CCP1) that polarizes the metal-bound water molecule responsible for attack of the scissile bond (37,40). Because of the low sensitivity of the polyE antibody, we enriched TRAD1 by means of its N-terminal Strep-tag.…”

Cytosolic Carboxypeptidase 5 Removes α- and γ-Linked Glutamates from Tubulin

“…1), further fractionation on ion exchange columns that resulted in increased purity of the CCP5 protein led to the complete loss of activity toward either tubulin or peptide (data not shown). Similarly, we could not further purify CCP1 on ion exchange columns without complete loss of activity (19), and gel filtration of CCP1 does not result in homogenous protein (34). The CCPs are not inherently unstable and do not lose much activity after incubation for several h at 4°C, the length of time required to run the ion exchange column.…”

“…Recently, purified CCP1 was reported to release Glu from small peptides containing 2-3 Glu residues and a blocked N terminus (34). The structure of a bacterial CCP, as determined by x-ray crystallography, shows classical metallocarboxypeptidase-like motifs, including the presence of an active site and substrate-binding pockets generally similar to those of enzymatically active metallocarboxypeptidases, such as mammalian carboxypeptidases A and B (35).…”