TAU-PROTEOFORMS IN AQUEOUS HUMOUR OF HEALTHY AND GLAUCOMA SUBJECTS AND ANALYSIS OF THE MOLECULAR PROPERTIES OF UB-TAU SPECIES IN VITRO

Abstract

Purpose : Tau is a neuron-restricted microtubule-binding protein whose aggregation is pathognomonic of neurodegenerative diseases (tauopathies). Tau miss-sorting and accumulation has been proposed to be associated with glaucoma pathogenesis. Recently, tauopathies have been reported to display a distinguishable pattern of Tau ubiquitylation that affects protein sorting and aggregating fibril diversity. The conjugation of ubiquitin (Ub) to lysine (K) residues is a conserved post-synthetic modification each protein can be decorated within living cells, which regulates substrate sorting or clearance through the proteasome. Here we report identification of Ub-proteins and Tau proteoforms in the aqueous humor (AH) of healthy and glaucoma subjects and the characterization of the kinetics of digestion of enzymatically and semi-synthetically produced mono-Ub-Tau species by the 20S proteasome in vitro.

Methods : Western blotting and proteomics studies were applied to interrogate the repertoire of Ub-proteins and to identify Tau proteoforms in the AH of healthy (cataract) and glaucoma subjects (n=15). The Microtubule Binding Domain (MBD) of Tau has been conjugated to Ub (mono-) at different lysine residues, recapitulating proteoforms described in brain tissues post-mortem, through a semisynthetic strategy and fed to the 20S to figure out the dynamics of catalytic processing.

Results : Total Ub-proteins were frequent in the AH and display an average 2-fold increase in glaucoma vs healthy subjects (p<0.0001). Tau shows up as different proteoforms, including Ub-positive species matter of further investigation, in the AH of glaucoma subjects. Site-specific mono-ubiquitylated Tau-MBD species show greatly divergent kinetics of processing by the 20S.

Conclusions : Double the amounts of Ub-proteins are immune-detectable in the AH of glaucoma subjects compared to healthy controls. If confirmed on a larger sample size, the accumulation of specific Tau proteoforms, especially individual Ub-Tau species, may elucidate the mechanisms of metabolism within the retina and identify novel biomarkers for improved glaucoma management. The divergent pathogenicity of individual Ub-Tau proteoforms may also be associated with their dynamics of interaction with the 20S and differential propensity toward intrinsic resistance or optimized clearance to proteolysis

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.