Mutant Myocilin Is Degraded by the Ubiquitin Proteasome System and Forms Aggresome

Abstract

Abstract: : Purpose: Mutant forms of myocilin expressed in human embryonic kidney (HEK) cells are relatively insoluble in Triton X-100 and are inefficiently secreted. Mutant myocilins may be retained in cells because they are misfolded. We wished to investigate the cellular fate of mutant myocilins with the aim of understanding pathogenic mechanisms of myocilin-associated glaucoma. Methods: Wild type or mutant forms of myocilin were transiently expressed in HEK cells and protein samples were analyzed by immunoprecipitation and immunoblotting. Myocilin was localized within cells by immunocytochemistry followed by confocal laser scanning microscopy. Results: Mutant myocilins readily accumulated in high molecular weight species that were retained in the stacking gel during SDS-PAGE, while almost all wild type protein migrated as monomers. About 15% of cells expressing mutant myocilin had juxtanuclear inclusion bodies. Inclusion bodies were found rarely (< 1%) in cells expressing only wild type myocilin. Co-expression of wild type and mutant myocilin substantially increased the accumulation of wild type protein in inclusion bodies. Inclusion bodies were observed in both HEK and human trabecular meshwork cells and exhibited the key characteristics of aggresomes; the inclusion bodies (1) localized close to centrosomes as determined by gamma-tubulin co-staining, (2) were coated by the intermediate filament protein, vimentin, and (3) were dependent for their formation upon intact microtubules. Unlike most aggresomes, the ER-resident chaperones protein disulfide isomerase and calnexin were recruited to myocilin aggresomes. Myocilin aggresomes stained for ubiquitin and mutant myocilin protein co-immunoprecipitated with ubiquitin. Chemical inhibition of 26S proteasome function led to a dramatic increase of both high molecular weight and monomeric species of mutant myocilin, indicating that mutation of myocilin destabilizes the protein and targets it for degradation by the ubiquitin proteasome system. Conclusions: These data may explain a toxic gain of function associated with disease-causing myocilin mutations in the pathogenesis of open angle glaucoma, which may share a common pathogenic mechanism with other late-onset dominant neurodegenerative diseases.

Keywords: protein structure/function • gene/expression • chaperones