advertisement
Abstract #54514 Published in IGR 15-3
Ankyrin repeat and suppressor of cytokine signaling box containing protein-10 is associated with ubiquitin-mediated degradation pathways in trabecular meshwork cells
Keller KE; Yang YF; Sun YY; Sykes R; Acott TS; Wirtz MKMolecular Vision 2013; 19: 1639-1655
PURPOSE: Ankyrin repeat and suppressor of cytokine signaling (SOCS) box containing protein-10 (ASB10) was recently identified as a gene that causes primary open-angle glaucoma. Here, we investigated endogenous ASB10 protein expression in human trabecular meshwork (HTM) cells to provide the first clues to the biologic function of this protein. METHODS: Primary HTM cells were cultured and immunostained with anti-ASB10 and various biomarkers of the ubiquitin-mediated proteasomal and autophagy-lysosomal degradation pathways. Cells were imaged with confocal and high-resolution structured illumination microscopy. Colocalization was quantified using Imaris Bitplane software, which generated a Pearson's correlation coefficient value. Coimmunoprecipitation of ASB10-transfected cells was performed. RESULTS: Immunofluorescence and confocal analysis showed that ASB10 was localized in intracellular structures in HTM cells. Two populations were observed: small, spherical vesicles and larger, less abundant structures. In the ASB10-silenced cells, the number of large structures was significantly decreased. ASB10 partially colocalized with biomarkers of the ubiquitin-mediated proteasomal pathway including ubiquitin and the α4 subunit of the 20S proteasome. However, ASB10 itself was not ubiquitinated. ASB10 also colocalized with numerous biomarkers of specific autophagic structures: aggresomes (histone deacetylase 6 [HDAC6] and heat shock protein 70 [HSP70]), autophagosomes (light chain 3 [LC3] and p62), amphisomes (Rab7), and lysosomes (lysosomal-associated membrane protein 1 [LAMP1]). Pearson coefficients indicated strong colocalization of large ASB10-stained structures with the α4 subunit of the 20S proteasome, K48 and K63-linked ubiquitin antibodies, p62, HSP70, and HDAC6 (Pearson's range, 0.59-0.82). Coimmunoprecipitation assays showed a positive interaction of ASB10 with HSP70 and with the α4 subunit of the 20S proteasome. Super-resolution structured illumination confocal microscopy suggested that the smaller ASB10-stained vesicles aggregated into the larger structures, which resembled aggresome-like induced structures. Treatment of HTM cells with an autophagy activator (MG132) or inhibitors (wortmannin, bafilomycin A1) significantly increased and decreased the number of small ASB10-stained vesicles, respectively. No discernible differences in the colocalization of large ASB10-stained structures with ubiquitin or HDAC6 were observed between dermal fibroblasts derived from a normal individual and a patient with primary open-angle glaucoma carrying a synonymous ASB10 mutation. CONCLUSIONS: Our evidence suggests that ASB10 may play a role in ubiquitin-mediated degradation pathways in TM cells.
Casey Eye Institute, Oregon Health & Science University, 3181 SW Sam Jackson Park Rd, Portland, OR 97239, USA. gregorka@ohsu.edu
Classification:
2.5.1 Trabecular meshwork (Part of: 2 Anatomical structures in glaucoma > 2.5 Meshwork)
3.5 Molecular biology incl. SiRNA (Part of: 3 Laboratory methods)
3.4.2 Gene studies (Part of: 3 Laboratory methods > 3.4 Molecular genetics)
