A Proteome-Wide Glutamylation Profiling Platform Reveals a Novel Gene Expression Regulatory Axis

Abstract

Protein glutamylation is a reversible post-translational modification in which one or more glutamate residues are appended to the side-chain carboxylate of a glutamate within a target protein. The modification is installed by tubulin tyrosine ligase-like (TTLL) writers, removed by ATP/GTP binding protein-like (AGBL/CCP) erasers, and has been studied for four decades almost exclusively on tubulin. Mutations in glutamylation enzymes cause inherited retinal dystrophies, and several family members emerge as selective dependencies in human cancers, yet the substrates, sites, and biology beyond microtubules have remained largely inaccessible. Glutamylation forms branched chains, which make it hard to detect through standard proteomic approaches. Also, the available antibody cannot resolve modification sites in a branch type-agnostic manner at the scale of the proteome. This thesis develops new tools for proteome-wide glutamylation site discovery and uses them to define a previously hidden layer of gene-expression regulation linked to disease. Chapter 1 situates glutamylation within the broader landscape of protein aminoacidylation. We start the thesis surveying the chemistry, enzymology, and biology of amino acid additions to protein N-termini, lysines, C-termini, and acidic side chains. Importantly, we articulate the analytical bottleneck that has kept glutamylation an exception within the post-translational modification toolkit. Chapter 2 introduces DeGlu, an end-to-end platform that combines metal-oxide affinity enrichment of acidic peptides, on-bead enzymatic deglutamylation by purified AGBL/CCPs in H218O. We also introduce Heavy-O Mapper, a computational framework that discriminates 18O incorporation at branch points from chain elongation through fragment-level voting in data-independent acquisition mass spectrometry. The platform, DeGlu potentiated by Heavy-O Mapper, yields the first whole-proteome inventory of endogenous glutamylation sites. Chapter 3 applies DeGlu to two disease contexts. In retinal pigment epithelial cells depleted of AGBL5, an enzyme mutated in autosomal recessive retinitis pigmentosa, I identify 123 hyperglutamylated sites enriched on chromatin-binding factors, including core and linker histones, splicing regulators, and POLR2A. AGBL5 loss reshapes chromatin compaction, dysregulates splicing, distorts immediate-early gene kinetics upon mitogenic stimulation, and bidirectionally rewires the transcriptome, downregulating retinal identity programs while upregulating proliferative networks. These results reposition AGBL5-associated retinal disease from a strictly ciliary disorder toward disrupted gene-expression machinery. Pan-cancer dependency analysis identifies glutamylation enzymes as context-selective vulnerabilities, and we show that colorectal adenocarcinoma cells are sensitive to the hypo-glutamylation state induced by TTLL11 perturbation. Chapter 4 synthesizes these findings into a conceptual model in which glutamylation tunes acidic-patch interactions on disordered protein surfaces, outlines the technical limitations and biological open questions that remain, and proposes a broader frame. The field's long-standing focus on microtubules underlies the tubulin code, but the chemistry catalogued here suggests a more general acidity code, through which distinct acidic side chains across the proteome encode regulatory potential for gene expression.