Abstract
Purpose :
Current therapeutics for dry eye disease mainly treat the symptoms rather than the underlying cause of the disease as its root cause is largely unknown. The purpose of this study is to determine the molecular events in the early stage of hyperosmotic stress and identify key molecules and/or processes that cause the loss of ocular homeostasis.
Methods :
A structural proteomic approach was employed to determine protein structural changes in human corneal and conjunctival epithelial cells upon hyperosmotic stress. Ocular cells were incubated in normal and hyperosmolar media (450 mOsm) for 5, 10, 20, 40, and 60 min, followed by labelling with an enrichable lysine-reactive probe. Proteins were extracted and digested into peptides. Labeled peptides were enriched using click chemistry and solid phase separation with a pH-sensitive handle. The samples were analyzed by liquid chromatography-mass spectrometry. The proteomic data were searched against the UniProt-SwissProt Human proteome database using the MSFragger software. The abundance of labeled amino acids was extracted using an in-house Python script. Statistical analysis was performed using an R package Limma. Finally, functional analysis was performed using the String database.
Results :
Within the first hour of hyperosmotic stress, no proteins in corneal epithelial cells showed significant abundance changes, but 2102 labeled sites of 1083 proteins showed significant changes, indicating structural changes. There were 136 proteins with differential expression within the first hour in human conjunctival epithelial cells. More significantly, 6088 labeled sites of 2030 proteins in conjunctival cells were indicated to change structures. Functional analysis showed the significantly changed proteins in both ocular cells were enriched in pathways such as cellular response to stimuli, cell cycle, and metabolism of proteins.
Conclusions :
Ocular cells respond to hyperosmotic stress rapidly by adapting their proteome homeostasis network. Our structural proteomic approach can capture such responses at a very early stage and likely before irreversible damage occurs. The information gained from this approach could help us understand ocular homeostasis in response to stress, further identify the key molecules/processes in maintaining or failing to maintain ocular homeostasis, and potentially identify new therapeutic targets and approaches.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.