The TTLL10 Polyglycylase Requires Monoglycylation for Microtubule Binding and is Inhibited by Polyglycylation

Core Concepts

TTLL8 is a monoglycylase that adds glycines to multiple sites on both α- and β-tubulin, while TTLL10 is a polyglycylase that can only elongate pre-existing monoglycine branches. TTLL10 binding to microtubules is stimulated by monoglycylation but inhibited by polyglycylation in a chain length-dependent manner, suggesting an autonomous mechanism for polyglycine chain length control.

Abstract

This study investigates the substrate specificity and regulation of the tubulin glycylation enzymes TTLL8 and TTLL10.

Key findings:

• TTLL8 is a monoglycylase that adds single glycines to multiple sites on both α- and β-tubulin tails.
• TTLL10 is a polyglycylase that can only elongate pre-existing monoglycine branches on tubulin, and does not initiate new glycine chains.
• TTLL10 binding to microtubules requires monoglycylation and is stimulated by this modification.
• In contrast, TTLL10 binding is progressively inhibited as polyglycine chains are elongated, suggesting an autonomous mechanism for controlling polyglycine chain length.
• Tubulin glutamylation by TTLL6 enhances the recruitment of TTLL10 to microtubules, providing a biochemical basis for the sequential deposition of these tubulin modifications during cilia development.
• The ability to generate differentially glycylated microtubules in vitro provides a tool to study the effects of the tubulin code on microtubule function.

Stats

Monoglycylation levels on α-tubulin ranged from α ∼ 0.1 to 1.1, and on β-tubulin from β ∼ 0.8 to 2.8.
Polyglycylation levels on α-tubulin ranged from α ∼ 3.0 to 3.3, and on β-tubulin from β ∼ 4.3 to 7.7.
Glutamylation levels on α-tubulin were estimated to be α ∼ 18, and on β-tubulin β ∼ 4.

Quotes

"TTLL8 is strictly a glycyl initiase that catalyzes the addition of monoglycines at multiple positions on both α and β-tubulin tails, while TTLL10 is exclusively a tubulin glycyl elongase that catalyzes the addition of glycines only to pre-existing glycine branches on either α or β-tubulin."
"TTLL10 binding to microtubules is progressively inhibited as polyglycine chains are elongated, proportional with polyglycine chain length, suggesting an autonomous mechanism of polyglycine chain length control."
"TTLL10 recruitment to microtubules is stimulated by TTLL6 polyglutamylation, demonstrating the interplay between these two modifications and providing a biochemical basis for the sequential deposition of these tubulin modifications during cilia development."

Key Insights Distilled From

The TTLL10 polyglycylase is stimulated by tubulin glutamylation and inhibited by polyglycylation

by Cummings,S.,... at www.biorxiv.org 03-31-2024

https://www.biorxiv.org/content/10.1101/2024.03.31.587457v1

Deeper Inquiries

How might the interplay between tubulin glycylation and glutamylation be leveraged to regulate microtubule-based processes in cells

The interplay between tubulin glycylation and glutamylation can be leveraged to regulate microtubule-based processes in cells by providing a mechanism for sequential deposition of these modifications on the axoneme. Glutamylation, which precedes glycylation during cilia biogenesis, enhances the recruitment of TTLL10 to microtubules. This sequential deposition ensures that TTLL10 is effectively recruited to microtubules that have been glutamylated first, allowing for the controlled addition of glycines by TTLL10. By modulating the levels of glutamylation and glycylation, cells can regulate the activity and localization of TTLL10, influencing the formation, stability, and function of cilia. This interplay between tubulin modifications provides a mechanism for fine-tuning the tubulin code and regulating microtubule dynamics in various cellular processes.

What other tubulin modifications or binding partners could potentially influence the activity and localization of TTLL10 and other glycylation enzymes

In addition to tubulin glycylation and glutamylation, other tubulin modifications or binding partners could potentially influence the activity and localization of TTLL10 and other glycylation enzymes. For example, tubulin acetylation, phosphorylation, or detyrosination could impact the substrate specificity or catalytic activity of TTLL10. Binding partners such as microtubule-associated proteins (MAPs) or motor proteins could also interact with glycylation enzymes, modulating their activity or recruitment to microtubules. Furthermore, posttranslational modifications on other proteins involved in the glycylation pathway, such as chaperones or regulatory proteins, could affect the function of TTLL10 and the overall glycylation process. Understanding the crosstalk between different tubulin modifications and their regulators is essential for deciphering the complex network of interactions that govern microtubule dynamics and cellular processes.

Could the autonomous control of polyglycine chain length by TTLL10 inhibition be exploited to engineer microtubules with specific glycylation patterns for in vitro studies or therapeutic applications

The autonomous control of polyglycine chain length by TTLL10 inhibition could be exploited to engineer microtubules with specific glycylation patterns for in vitro studies or therapeutic applications. By manipulating the levels of TTLL10 and other glycylation enzymes, researchers could generate microtubules with tailored glycylation patterns, allowing for the study of how different glycylation states affect microtubule function and interactions with cellular effectors. These engineered microtubules could serve as valuable tools for investigating the role of glycylation in cellular processes, such as cilia maintenance, cell division, or intracellular transport. Moreover, understanding the mechanisms that control polyglycine chain length could lead to the development of novel therapeutic strategies targeting tubulin modifications for the treatment of diseases associated with dysregulated microtubule dynamics.