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

Abstract

Cytosolic carboxypeptidases (CCPs) constitute a new subfamily of M14 metallocarboxypeptidases associated to axonal regeneration and neuronal degeneration, among others. CCPs are deglutamylating enzymes, able to catalyze the shortening of polyglutamate side-chains and the gene-encoded C termini of tubulin, telokin, and myosin light chain kinase. The functions of these enzymes are not entirely understood, in part because of the lack of information about C-terminal protein processing in the cell and its functional implications. By means of C-terminal COFRADIC, a positional proteomics approach, we searched for cellular substrates targets of CCP1, the most relevant member of this family. We here identified seven new putative CCP1 protein substrates, including ribosomal proteins, translation factors, and high mobility group proteins. Furthermore, we showed for the first time that CCP1 processes both glutamates as well as C-terminal aspartates. The implication of these C termini in molecular interactions furthermore suggests that CCP1-mediated shortening of acidic protein tails might regulate proteinprotein and protein-DNA interactions. Molecular & Cellular

An Approach to Incorporate Multi‐Enzyme Digestion into C‐TAILS for C‐Terminomics Studies

“…Recently, Tanco et al. identified seven new putative Cytosolic carboxy peptidases 1 protein substrates using C‐terminal COFRADIC method …”

Excessive tubulin polyglutamylation causes neurodegeneration and perturbs neuronal transport

“…We next asked whether accumulation of polyglutamylation, rather than the impairment of the second enzymatic activity of CCP1, the removal of gene-encoded C-terminal acidic amino acids from tubulins or other proteins Tanco et al, 2015), is causative for the degeneration of neurons lacking CCP1. To specifically reduce polyglutamylation exclusively in the Purkinje cells of pcd mice, we deleted the major brain polyglutamylase, TTLL1 (Janke et al, 2005), in these neurons by combining the pcd (Ccp1 À/À ), Ttll1 flox/flox , and L7-cre (Rico et al, 2004) alleles ( Fig 1B).…”

“…The recent discovery of a large number of tubulin-modifying enzymes now allows to directly address the role of tubulin PTMs (Janke, 2014). Cytosolic carboxypeptidases (CCPs) (Kalinina et al, 2007;Rodriguez de la Vega et al, 2007) are a class of tubulinmodifying enzymes that remove acidic amino acid residues from the carboxy-termini of peptide chains Berezniuk et al, 2012), thus controlling two major types of PTMs: First, they reverse polyglutamylation, a PTM catalyzed by polyglutamylases from the tubulin-tyrosine ligase-like (TTLL) family (Janke et al, 2005;van Dijk et al, 2007), and second, CCPs can remove C-terminal, gene-encoded glutamate residues, which converts a-tubulin into D2and D3-tubulins Aillaud et al, 2016) or affects other proteins with acidic C-termini (Tanco et al, 2015). Impaired deglutamylase activity was initially linked to neurodegeneration in a mouse model with early loss of the Purkinje cells in the cerebellum, the Purkinje cell degeneration (pcd) mouse (Mullen et al, 1976).…”

Multiple Architectures and Mechanisms of Latency in Metallopeptidase Zymogens

“…One protein was reported from the bacterium Lysinibacillus sphaericus, which is involved in the metabolism of the bacterial cell wall (M14C). More recently, the intracellular cytosolic carboxypeptidases (M14D) were discovered in mammals and classified into a distinct group 70,71 . Porcine pancreatic pro-carboxypeptidase B1 was the first MP, for which the zymogen structure was solved in 1991 72 , and to date sixteen more pro-funnelin structures have been published, all from A/B-type zymogens (Table 1).…”