INCREASED UBIQUITYLATION OF β-TrCP SUBUNIT OF SCF E3-LIGASE IN PERIPHERAL BLOOD MONONUCLEAR CELLS OF GLAUCOMA SUBJECTS: A PROTEOMIC STUDY

Abstract

Purpose : Glaucoma is a chronic optic neuropathy characterized by progressive degeneration of optic nerve and death of retinal ganglion cells.
Although the pathogenesis of glaucoma is uncharacterized yet, molecular signatures of disease progression have been tracked in blood elements.
Peripheral blood mononuclear cells (PBMC), which are mainly represented by lymphocytes, monocytes and natural killer, have been studied to search for biomarkers and molecular clues of disease pathogenesis.
Accordingly, this pilot study has been dedicated to profiling the repertoire of ubiquitylated proteins in PBMC of glaucoma and healthy subjects by proteomic approaches.
Ubiquitin (Ub) conjugation is a major post-synthetic modification that is known to regulate the turnover and biological properties (e.g., trafficking, sorting, activity etc) of proteins and global metabolism.

Methods : PBMC of glaucoma (n=8) and control (cataract, n=8) subjects have been isolated by density-gradient sedimentation. All procedures have been performed in accordance with Helsinki declaration and the Good Clinical Practices. Enrolled subjects have been matched for age and gender.
A proteome profiler Ub array has been used to compare the ubiquitylation pattern of 49 different proteins between groups.
A “diGLY” proteomic workflow is being run to profiling the first global ubiquitylome of PBMC in glaucoma vs control subjects.

Results : The (Ub) array test has allowed to figure out a nearly 10-fold increase (by mean of signal intensity, *p<0.0001, Student τ test) of Ub-β-TrCP subunit of SCF E3-ligase in PBMCs of glaucoma subjects vs control subjects. Western blotting analysis of samples has allowed to uncover comparable basal level of non-Ub β-TrCP.
Application of diGLY proteomic workflow (in progress) is allowing to uncover global alterations of Ub-signalling in PBMC of glaucoma vs control subjects.

Conclusions : The Ub-proteome array has allowed to uncover a differential ubiquitylation pattern of β-TrCP subunit of SCF, which is an E3-ligase with a pivotal role in inflammation, between glaucoma and control subjects. Self-ubiquitylation of β-TrCP is expected to have regulatory functions, but the biological role remains to be characterized.
Profiling of the PBMC ubiquitylome is an unprecedented strategy to figure out molecular determinants of glaucoma pathogenesis.

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