Anti-beta II Tubulin (aa 107-137) polyclonal antibody

A combination of structural and protein-chemistry approaches along with phylogenetic analyses provide insights into the specific activities of mouse tubulin tyrosine ligase-like enzymes as initiases or elongases of glutamylation. Glutamylation, introduced by tubulin tyrosine ligase-like (TTLL) enzymes, is the most abundant modification of brain tubulin. Essential effector proteins read the tubulin glutamylation pattern, and its misregulation causes neurodegeneration. TTLL glutamylases post-translationally add glutamates to internal glutamates in tubulin carboxy-terminal tails (branch initiation, through an isopeptide bond), and additional glutamates can extend these (elongation). TTLLs are thought to specialize in initiation or elongation, but the mechanistic basis for regioselectivity is unknown. We present cocrystal structures of murine TTLL6 bound to tetrahedral intermediate analogs that delineate key active-site residues that make this enzyme an elongase. We show that TTLL4 is exclusively an initiase and, through combined structural and phylogenetic analyses, engineer TTLL6 into a branch-initiating enzyme. TTLL glycylases add glycines post-translationally to internal glutamates, and we find that the same active-site residues discriminate between initiase and elongase glycylases. These active-site specializations of TTLL glutamylases and glycylases ultimately yield the chemical complexity of cellular microtubules.

Microtubules (MTs) are known to be post-translationally modified at the neuromuscular junction (NM)), hence increasing their stability. To date however, the function(s) of the dynamic MT network and its relative stability in the formation and maintenance of NMJs remain poorly described. Stabilization of the MT is dependent in part on its acetylation status, and HDAC6 is capable of reversing this post-translational modification. Here, we report that HDAC6 preferentially accumulates at NMJs and that it contributes to the organization and the stability of NMJs. Indeed, pharmacological inhibition of HDAC6 protects against MT disorganization and reduces the size of acetylcholine receptor (AChR) clusters. Moreover, the endogenous HDAC6 inhibitor paxillin interacts with HDAC6 in skeletal muscle cells, colocalizes with AChR aggregates, and regulates the formation of AChR. Our findings indicate that the focal insertion of AChRs into the postsynaptic membrane is regulated by stable MTs and highlight how an MT/HDAC6/paxillin axis participates in the regulation of AChR insertion and removal to control the structure of NMJs.