Evolutionary Divergence of Enzymatic Mechanisms for Posttranslational Polyglycylation

Abstract

Polyglycylation is a posttranslational modification that generates glycine side chains on proteins. Here we identify a family of evolutionarily conserved glycine ligases that modify tubulin using different enzymatic mechanisms. In mammals, two distinct enzyme types catalyze the initiation and elongation steps of polyglycylation, whereas Drosophila glycylases are bifunctional. We further show that the human elongating glycylase has lost enzymatic activity due to two amino acid changes, suggesting that the functions of protein glycylation could be sufficiently fulfilled by monoglycylation. Depletion of a glycylase in Drosophila using RNA interference results in adult flies with strongly decreased total glycylation levels and male sterility associated with defects in sperm individualization and axonemal maintenance. A more severe RNAi depletion is lethal at early developmental stages, indicating that protein glycylation is essential. Together with the observation that multiple proteins are glycylated, our functional data point towards a general role of glycylation in protein functions.

Crystal structure of tubulin tyrosine ligase-like 3 reveals essential architectural elements unique to tubulin monoglycylases

“…In mice, loss of TTLL3-mediated glycylation in colon epithelial cells results in increased cell proliferation and the potentiation of cancerous cell growth (21). Consistent with this phenotype, TTLL3-inactivating mutations have been identified in colon cancer patients (12,23). Glycylation is also important during development.…”

“…cysts, randomized multicilia orientation, and defects in ciliary fluid flow (13,24), whereas knockout of TTLL3B is lethal in flies (12).…”

“…The glycyl chain length on either α-or β-tubulin tails in axonemes is highly variable (16)(17)(18), ranging from 1 to 40 posttranslationally added glycines (19). Of the 13 TTLL enzymes in mammals (10, 11), three are glycylases: TTLL3, -8, and -10 (12,13,20). Cellular overexpression studies suggest that these enzymes are specialized for either initiating (TTLL3 and TTLL8) or elongating the glycyl chain (TTLL10) (12,20).…”

“…Of the 13 TTLL enzymes in mammals (10, 11), three are glycylases: TTLL3, -8, and -10 (12,13,20). Cellular overexpression studies suggest that these enzymes are specialized for either initiating (TTLL3 and TTLL8) or elongating the glycyl chain (TTLL10) (12,20). Such specialization would not be surprising, as initiation requires the enzyme to accommodate a glutamate and an incoming glycine in its active site and catalyze formation of an isopeptide bond, whereas elongation requires it to accommodate two glycines and catalyze a standard peptide bond.…”

Polyglutamylation des microtubules et neurodégénérescence

“…Les glutamylases ont été identifiées il y a quelques années [2,3]. Elles font partie d'une famille de protéines nommées tubuline tyrosine ligase-like (TTLL) qui regroupent les diverses enzymes en charge de l'addition de résidus sur le carboxy-terminus de la tubuline : la tubuline tyrosine ligase (TTL, Figure 1B [4]), les glutamylases ( Figure 1A) et les glycyclases [5,6]. Au cours de l'évolution, cette famille d'enzymes est apparue avec les cils (au moment de l'émergence des eucaryotes), suggérant un rôle majeur de ces modifications dans l'assemblage de ces organites.…”