Abstract
PeRSPeCTIve Tubulin glutamylation: a skeleton key for neurodegenerative diseases Microtubules (MTs) are cytoskeletal elements formed by a non-cova-lent association of α-and β-tubulin heterodimers. They provide structure and shape to all eukaryotic cells and are implicated in a variety of fundamental cellular processes including cell motility, cell division, mechanotransduction as well as long-distance intracellular cargo transport. In neurons, they constitute the molecular frame that maintains the lengthy axonal projections. In view of the relative size of some ax-ons in the human body, which can reach up to 1 m, the active transport of e.g., vesicles over the MT arrays to the synaptic cleft, is of particular importance. Considering the numerous roles of MTs, it is not surprising that already 30 years ago, impairment of the MT-based system was proposed as a unifying hypothesis for the variable clinical presentations in Alzheimer's disease (Matsuyama and Jarvik, 1989). In this context, a key question is how the MT network accommodates all these different functions, often within the same cell? Current view is that every MT-dependent process is executed through the recruitment of a specific set of MT-associated proteins (MAPs) and molecular motors. Thus, it is of fundamental importance to understand how recruitment of these MAPs and motors is regulated. Since many of the MAPs and motors bind to the C-terminal tails of α-and β-tubulin, which are known to protrude from the MT surface, one important mechanism by which MTs may regulate the association of the effector proteins is through posttranslational modifications (PTMs). The modifications that occur on the C-terminal tails consists of either addition or removal of amino acids including polyglutamylation, polyglycylation and detyrosination. Very recently we have identified the members of the vasohibin family as cysteine proteases responsible for tubulin detyrosination (Aillaud et al., 2017), a modification, which consists of proteolytic removal of the very C-terminal tyrosine residue present on α-tubulin. The reverse reaction that consists of reattachment of a tyrosine residue is carried out by an enzyme called tubulin tyrosine ligase (TTL). Moreover, the C-terminal tails of both α-and β-tubulin are also subjected to polymodifications namely polyglutamylation and polyglycylation. These modifications are reversible and consist of the enzymatic addition of sidechains composed of either glutamate or glycine to the gamma carboxyl groups of primary sequence gluta-mates. The enzymes, involved in the addition of both glutamylation and glycylation side chains, share a homology domain with TTL and thus are called tubulin tyrosine ligase like (TTLL). The human genome contains thirteen TTLL related genes. Nine of them are involved in tubulin polyglutamylation (TTLL1, TTLL2, TTLL4, TTLL5, TTLL6, TTLL7, TTLL9, TTLL11 and TTLL13) and three in tubulin polyglyc-ylation (TTLL3, TTLL8 and TTLL10) (Rogowski et al., 2009), while one, TTLL12, remains without assigned function. On the other hand, tubulin deglutamylation has been shown to be catalyzed by a family of cytosolic carboxypeptidases (CCP), which is composed of six members (Rogowski et al., 2010). In contrast, the enzymes responsible for de-glycylation remain to be discovered. Overall, this complex enzymatic machinery allows for spatial and temporal fine-tuning of the physico-chemical properties of the MTs surface, ensuring functional diversification. In analogy to the "histone code", this regulatory system was originally coined as the "tubulin code" in a seminal review (Verhey and Gaertig, 2007). A proof of concept of the "tubulin code" was provided in the context of in vitro studies showing that PTMs confer unique biochemical properties, drive dynein and kinesin motor velocity, proces-sivity and the rates of MT depolymerisation (Sirajuddin et al., 2014). While polyglycylation appears to be specific to cilia and flagella, polyglutamylation and detyrosination are more ubiquitous. Biochemical characterization of MTs obtained from brain tissue revealed the presence of extensive PTMs on the protruding C-terminal tails of α-and β-tubulin with the most abundant modification being polyglutam-ylation. The first enzyme involved in glutamylation to be identified, TTLL1, was originally purified from mouse brain using classical biochemistry , and confirmed genetically by developing knockout Tetra-hymena cells, which lacked homologous gene and showed reduced level of glutamylation (Janke et al., 2005). A comprehensive follow up study demonstrated that in humans, apart from TTLL1, eight additional members of the TTLL family encode tubulin glutamylases. These enzymes are characterized by different specificities with some of them preferentially being involved in initiation while the others in the elongation of the glutamate chain. The identification of the reverse enzymes, the CCPs, came with the analysis of Purkinje cell degenera-tion (pcd) mouse model. These mice exhibit ataxia, which results from postnatal degeneration of almost all Purkinje cells in the cerebellum. Genetic analysis revealed that pcd mice carry a mutation in the CCP1 gene, which encodes a protein having tubulin deglutamylase activity. As such, pcd mice display abnormally high level of polyglutamylation in the cerebellar neurons. Stunningly, the Purkinje cell degeneration phenotype observed in the pcd mice was rescued by a knockdown of TTLL1 glutamylase, demonstrating that neuronal death is indeed mediated by tubulin hyperglutamylation. These observations provided the first molecular link between altered levels of tubulin glutamylation and neurodegeneration (Rogowski et al., 2010). Current view limits the regulation of tubulin glutamylation levels in the cells to direct competition between the forward and reverse enzymes and does not include additional regulators. Recently, we have identified cilia and spindle-associated protein (CSAP) as a master regulator of tubulin glutamylases (Bompard et al., 2018). We found that expression of CSAP enhances overall activity of all autonomously active glutamylating enzymes and in the case of TTLL5 and TTLL7 also potentiates their elongase activity. Moreover, biochemical analysis revealed that CSAP interacts with TTLL glutamylases and appears to regulate their protein abundance through stabilization (Figure 1A). In turn, due to its high affinity for MTs, CSAP redirects glutamylase activity from tubulin towards MTs. By exploring the human protein atlas (Uhlén et al., 2015), we found that CSAP has a striking distribution in human tissue and is preferentially expressed in brain (Figure 1B). Thus, we propose that neurons utilize the expression of CSAP, as a regulatory Figure 1 Cilia and spindle-associated protein (CSAP) function and tissue distribution in humans. (A) Schematic representation of the role of CSAP protein in glutamylation of microtubules. (B) Protein expression of CSAP in different organs of the human body. Data are obtained from Human Protein Atlas available from www.proteinatlas.org. TTLL: Tubulin tyrosine ligase like; CCP: cyto-solic carboxypeptidases. A B [Downloaded free from http://www.nrronline.org on Thursday, December 5, 2019, IP: 195.83.84.168]