Isolevuglandin-modified proteins, including elevated levels of inactive calpain-1, accumulate in glaucomatous trabecular meshwork.
We report that protein adducts of iso[4]levuglandin E2 (iso[4]LGE2), a highly reactive product of free radical-induced lipid oxidation, accumulate in human glaucomatous trabecular meshwork (TM) but not in controls. Reactive oxygen species play a pathogenic role in primary open angle glaucoma by fostering changes that reduce permeability of the TM tissue and consequently impede aqueous humor outflow resulting in elevated intraocular pressure. IsoLGs covalently modify proteins and are especially effective in causing protein-protein cross-linking. We found elevated levels of calpain-1 in glaucomatous TM. However, calpain activity in glaucomatous TM is only about 50% of that in controls. This paradox is explicable by the fact that modification by isoLGs renders calpain-1 inactive. Thus, treatment of calpain-1 with iso[4]LGE2 in vitro results in covalent modification, inactivation, the formation of high molecular weight aggregates (as determined by Western and dynamic light scattering analyses), and resistance to proteasomal digestion. Iso[4]LGE2-modified calpain-1 undergoes ubiquitination, and its loading impairs the cellular proteasome activity, consistent with competitive inhibition and formation of suicidal high molecular weight aggregates. These data suggest that interference with proteasomal activity, owing to protein modification by isoLGs, could contribute to glaucoma pathophysiology by decreasing the ability of the TM to modulate outflow resistance.
Govindarajan
Bharathi
B
Department of Chemistry, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Laird
James
J
Salomon
Robert G
RG
Bhattacharya
Sanjoy K
SK
D002154:Calpain / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D005231:Fatty Acids, Unsaturated / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D005902:Glaucoma, Open-Angle / Q000201:enzymology*; D006367:HeLa Cells; D006801:Humans; D008027:Light; D009900:Optic Nerve / Q000201:enzymology; D010447:Peptide Hydrolases / Q000378:metabolism; D011458:Prostaglandins E / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D014176:Protein Biosynthesis; D020836:Protein Structure, Quaternary; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012542:Scattering, Radiation; D014020:Tissue Extracts; D014129:Trabecular Meshwork / Q000201:enzymology* / Q000473:pathology*; D054875:Ubiquitination
D005231:Fatty Acids, Unsaturated; D011458:Prostaglandins E; D012333:RNA, Messenger; D014020:Tissue Extracts; C526910:iso(4)levuglandin E2; C047684:levuglandin E2; D010447:Peptide Hydrolases; D002154:Calpain; C484806:CAPN1 protein, human; D046988:Proteasome Endopeptidase Complex