ALTERED PROCESSING OF MYOCILIN THROUGH THE UBIQUITIN-PROTEASOME SYSTEM INTRODUCES THE FIRST PROFILING OF THE GLOBAL UBIQUITYLOME OF TRABECULAR MESHWORK CELLS IN A GLAUCOMA MODEL

Abstract

Purpose : Dysregulation of Trabecular Meshwork Cells (TMCs) metabolism is involved in glaucoma pathogenesis, but the molecular mechanisms are largely unknown yet.
Ubiquitin (Ub) conjugation by E1-E2-E3 enzymes (the E3-ligase confers substrate-specificity) is a major post-synthetic modification that finely regulates the degradation (by proteasome) and biological properties of proteins along with global metabolism, indeed.
Processing of glycoprotein myocilin (MYOC/TIGR gene) through Ub-proteasome system was first found impaired in a primary human TMCs (hTMC) strain challenged with dexamethasone (dexa) [a model of steroid-induced glaucoma in vitro].
Hence, this study aims at:
1) Identifying the myoc:E3-ligase pair;
2) Profiling of the TMCs ubiquitylome and its perturbations;

Methods : A hTMC strain has been challenged with 500 ng/mL dexa or vehicle (4 days) and the proteasome inhibitor epoxomicin (epox) delivered to enrich Ub-proteins and to monitor their turnover.
Native-gel electrophoresis, Western blotting (Wb) and RT-PCR have been applied to follow protein content/expression. Bioinformatic (UbiBrowser software) and proteomic studies have been used to identify candidate myoc-E3-ligase pairs. Delivery of 27-mer siRNAs has been used to silence E3-ligase expression. A proteomic workflow (i.e., diGLY proteomics) is being completed (work in progress) to profiling the first TMCs ubiquitylome.

Results : Differently from untreated cells, dexa-treated TMCs show no accumulation of myoc either free or associated with intact proteasome particles after epox delivery (Wb and native-gel, *p<0.0001, one-way ANOVA-Tukey's test).
Bioinformatic and proteomic studies suggest that synoviolin (SYVN1) and CHIP E3-ligases may conjugate Ub to myoc.
CHIP, but not SYVN1, is downregulated by dexa in TMCs and CHIP silencing induces myoc accumulation even in the absence of dexa (RT-PCR and Wb, *p<0.0001, one-way ANOVA-Tukey's test).
Pilot diGLY proteomic studies highlight perturbations of global ubiquitylome in dexa-treated TMCs.

Conclusions : 1) Myoc accumulation in dexa-treated TMCs may originate also from impaired proteasome turnover;
2) CHIP likely is the E3-ligase of myoc and is downregulated by dexa;
3) Identification of E3-ligases-substrate pairs and ubiquitylome profiling is expected to introduce molecular insights on TMC metabolism and glaucoma pathogenesis.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.