Abstract
Purpose::
Increased accumulation and aggregation of oxidatively modified proteins, partly resulting from a dysfunction of the ubiquitin-proteasome pathway, have been described as a hallmark of various age-related degenerative disorders. The objective of this study was to investigate proteasome function and protein oxidation in pseudoexfoliation (PEX) syndrome, which is characterized by a progressive accumulation of abnormally aggregated extracellular fibrils and increased oxidative stress.
Methods::
Twelve eyes with PEX syndrome without and with glaucoma and 12 age-matched control eyes without PEX, either obtained at autopsy (post mortem time < 8 hours) or at surgical enucleation, were used. Proteasome activity was measured in ciliary body extracts by using fluorogenic peptides as substrates for the three different protease activities of the 26S proteasome; a proteasome activity inhibitor (epoxomicin) was used as control. Total proteasome content was determined by Western Blot analysis using antibodies against specific proteasomal subunits. The expression of ubiquitin-conjugating enzymes, which are members of the ubiquitin-proteasome system, was analyzed in anterior segment tissues by Real-time PCR and immunohistochemistry. Immunohistochemical analysis of oxidative protein modifications in PEX tissues was performed using antibodies against dinitrophenol (DNP).
Results::
Both the chymotrypsin-like and trypsin-like proteasome activities were significantly reduced (up to 55%; p<0.001) in ciliary body extracts from PEX eyes compared with control eyes, whereas the caspase-like activity was unchanged. This reduction in proteasome activity was independent of the presence of glaucoma in PEX eyes and was not associated with a decrease in proteasome expression. In contrast to the ubiquitin-conjugating enzymes UBE2D1, UBE2D2 and UBE2D3, the expression of the ubiquitin carrier enzymes UBE2A and UBE2B was significantly reduced both on the mRNA (about 60%; p<0.001) and protein level in ciliary body and iris specimens of PEX eyes compared with control eyes. An accumulation of oxidatively modified proteins was observed within PEX material deposits in anterior segment tissues of PEX eyes.
Conclusions::
These findings suggest that compromise of the proteasome system may be involved in the pathophysiology of PEX syndrome and may contribute to the accumulation of oxidatively modified proteins within the abnormal extracellular matrix deposits.
Keywords: oxidation/oxidative or free radical damage • extracellular matrix • pathology: human