September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
The Ubiquitin-Proteasome Pathway Generates Keratin 6A-Derived Antimicrobial Peptides to Mediate Antimicrobial Activities in Human Corneal Epithelial Cells
Author Affiliations & Notes
  • Jonathan K Chan
    Department of Ophthalmic Research, Cleveland Clinic Cole Eye Institute and Lerner Research Institute, Cleveland, Ohio, United States
  • Priscilla Too
    Department of Ophthalmic Research, Cleveland Clinic Cole Eye Institute and Lerner Research Institute, Cleveland, Ohio, United States
  • K P Connie Tam
    Department of Ophthalmic Research, Cleveland Clinic Cole Eye Institute and Lerner Research Institute, Cleveland, Ohio, United States
    Department of Ophthalmology, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, Ohio, United States
  • Footnotes
    Commercial Relationships   Jonathan Chan, None; Priscilla Too, None; K P Connie Tam, US 9187541 (P)
  • Footnotes
    Support  NIH Grant R01EY023000
Investigative Ophthalmology & Visual Science September 2016, Vol.57, No Pagination Specified. doi:
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      Jonathan K Chan, Priscilla Too, K P Connie Tam; The Ubiquitin-Proteasome Pathway Generates Keratin 6A-Derived Antimicrobial Peptides to Mediate Antimicrobial Activities in Human Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 201657(12):.

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

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Abstract

Purpose : Keratin-derived Antimicrobial Peptides (KAMPs) are constitutively derived from keratin 6A (K6A) in human corneal epithelial cells. We previously showed that purified bacterial antigens (i.e. Pseudomonas aeruginosa flagellin and LPS) induced disassembly of K6A filaments into the cytosolic pool and upregulation of KAMPs production. However, the proteolytic processing events of cytosolic K6A leading to KAMPs production are undefined. Here, we tested the hypothesis that the ubiquitin-proteasome pathway generates KAMPs through ubiquitination-coupled processing and degradation of cytosolic K6A.

Methods : Stratified telomerase-immortalized human corneal epithelial (hTCEpi) cells were pretreated with or without a specific 26S proteasome inhibitor (epoxomicin, 200nM) for 2 h, followed by 16 h incubation with purified Staphylococcus aureus lipoteichoic acid (LTA, 1 μg/ml). Cytosolic cell lysates were immunoblotted for KAMPs, as well as total and ubiquitinated K6A, or fractionated to isolate the KAMPs-enriched <10-kDa fractions. Antimicrobial activity of lysate fractions was determined against P. aeruginosa clinical isolate 6206 using viable counts after 3-h incubation. Mass spectrometry was used to identify endogenous ubiquitination sites on K6A.

Results : LTA stimulation concomitantly increased cytosolic K6A and KAMPs levels in stratified hTCEpi cells compared to untreated control. The <10-kDa lysate fraction of LTA-stimulated cells was potently bactericidal against P. aeruginosa. Epoxomicin treatment promoted accumulation of polyubiquitinated K6A in the LTA-stimulated cells but abolished the production of KAMPs. As such, the antimicrobial activity of the <10-kDa lysate fraction of epoxomicin-treated cells was significantly reduced (P = 0.006). Ubiquitination sites of K6A included the previously reported lysine-180, and two novel sites at lysine-194 and -204.

Conclusions : The data suggest that endogenous KAMPs are produced via ubiquitin-dependent proteasomal degradation of K6A. While proteasome processing is known to be involved in adaptive immunity, our data underscore the importance of ubiquitin-proteasome pathway in harnessing endogenous antimicrobial peptide production for epithelial innate defense. Further studies will determine the regulation of KAMPs production by specific ubiquitination sites and E3 ligases.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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