June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
Ocular chemoproteomics for determining broad profiles of enzymatic activities
Author Affiliations & Notes
  • Steffi Daniel
    Ophthalmology, The University of Texas Southwestern Medical Center, Dallas, Texas, United States
  • John Hulleman
    Ophthalmology, The University of Texas Southwestern Medical Center, Dallas, Texas, United States
  • Footnotes
    Commercial Relationships   Steffi Daniel None; John Hulleman None
  • Footnotes
    Support  Roger and Dorothy Hirl Research Fund, NEI P30 grant (EY030413)
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 4606 – F0398. doi:
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      Steffi Daniel, John Hulleman; Ocular chemoproteomics for determining broad profiles of enzymatic activities. Invest. Ophthalmol. Vis. Sci. 2022;63(7):4606 – F0398.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Changes in expression of several enzymes have been reported in age-related and degenerative ocular diseases. However, the current ‘omics approaches (transcriptomics, proteomics) only determine abundance changes, which does not provide true enzyme functionality (e.g., in the case of propeptide processing or the presence of inhibitors). Chemoproteomics (e.g., activity-based protein profiling [ABPP]) is a versatile tool to broadly probe enzyme class activity and is a technique that has not been fully utilized in vision research. Employing ABPP, we performed an unbiased characterization of active serine hydrolases in mouse eyes and primary ocular cultures.

Methods : Neural retina (NR), RPE, and liver from WT C57BL/6 mice were homogenized and probed with a TAMARA-tagged fluorophosphonate (FP) probe that specifically and covalently labels the active-site of functional serine hydrolases. Samples were analyzed by fluorescent imaging after SDS-PAGE. NR, RPE, liver, and serum were also procured from fibulin-3 knockout (F3 KO) mice (a model that we speculated may have a different serine hydrolase activity landscape) and processed similarly. Lastly, samples (apical, basal, and lysates) from primary porcine RPE cells established on transwells were harvested by ABPP after 1 and 6 mo in culture.

Results : ABPP of serine hydrolases with the TAMARA FP probe revealed a number of differential activities between ocular tissues as well as liver in WT animals. Lower amounts of active serine hydrolases were detected in F3 KO animals in all of the tissues compared to WT littermates. Moreover, TAMARA FP probe was able to detect changes in serine hydrolase activity in polarized RPE cultures, highlighting that serine hydrolase activity is concentrated in the apical fraction, with little activity detected basally or in cell lysates.

Conclusions : Our data demonstrate the ability of ABPP to detect tissue-level differences in activity of serine hydrolases. We also identified polarity-specific differences in serine hydrolase activity of RPE cells and changes therein with culture age. We speculate that ABPPs for additional enzyme classes such as MMPs and cysteine proteases will yield important ocular pathobiology information and may serve as potential biomarkers for impending ocular diseases, enabling researchers to identify and monitor the active location of these enzymes, providing an additional dimension to conventional ‘omics approaches.

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

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