PROJECT SUMMARY Through mass spectrometry, glutamylation is becoming recognized as an important post-translational modifi- cation (PTM) in polarized neuronal cells such as the retinal photoreceptor and hippocampal pyramidal cells. Despite this greater awareness of glutamylation, there exist gaps in our understanding of (1) how glutamyla- tion specifically occurs in the photoreceptor, (2) which specific glutamylase(s) are necessary for hippocampal development, and (3) how a neurodegenerative state affects glutamylation levels. In addressing those gaps, the long-term goal is to understand how glutamylation annotates the cytoskeleton for specialized roles. The central hypothesis is that the homeostasis of glutamylation, the most abundant post-translational modification in the nervous system, is essential to neuronal and retinal health and that aberrant glutamylation levels may serve as an adjunctive biomarker. The central hypothesis will be challenged by three specific aims: (1) gain a structural understanding of how a glutamylase, Tubulin Tyrosine Ligase-Like 5 (TTLL5) recognizes a novel non-tubulin target (RPGR-ORF15, the most commonly mutated gene in retinitis pigmentosa (RP)) in the reti- nal photoreceptor and assess how point mutations informed by the structural complex impacts in oculo RPGR-ORF15 glutamylation and opsin trafficking. (2) Identify which of the nine glutamylases are necessary for hippocampal pyramidal cytoskeleton development, function, and maintenance and to determine which glu- tamylase(s) could be perturbed in a neurodegenerative state like Alzheimer's disease (AD), and (3) examine how glutamylation levels are affected in neurodegenerative samples. This juxtaposition of RP and AD high- lights the fundamental role glutamylation plays in maintaining cytoskeletal structure and functions across var- ied polarized cell types such as photoreceptors and hippocampal neurons. The proposed research is signifi- cant because defining the structural requirements of glutamylation in the photoreceptor will have relevance to basic photoreceptor biology and help RP patients who carry mutations in TTLL5's Cofactor Interaction Do- main (CID) or RPGR-ORF15's Basic Domain (BD) identified by Next Generation Sequencing (NGS). Be- cause rational therapeutics is structure dependent, a structure of the CID-BD is needed especially since we currently possess no homology models for CID. More broadly, a structural understanding of how a glutamyl- ase recognizes a non-tubulin retinal target will lay the groundwork to understand how TTLL paracatalytic re- gions functions as recognition adaptors for substrate targets. This will emerge as a unifying theme for the TTLL superfamily as the family is further explored and implicated as an etiology of disease by NGS. Likewise, defining the genetic requirements of glutamylation in the hippocampal cells will broaden our understanding of which glutamylases play a role in the development and maintenance of the hippocamp...