Ankyrin repeat and suppressor of cytokine signaling box containing protein-10 is associated with ubiquitin-mediated degradation pathways in trabecular meshwork cells

Mol Vis. 2013 Jul 25;19:1639-55. Print 2013.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adolescent
  • Adult
  • Antibodies / metabolism
  • Autophagy
  • Biomarkers / metabolism
  • Child
  • Child, Preschool
  • Dermis / cytology
  • Fibroblasts / metabolism
  • Humans
  • Immunoprecipitation
  • Lysosomes / metabolism
  • Microscopy, Confocal
  • Middle Aged
  • Proteasome Endopeptidase Complex / metabolism
  • Protein Transport
  • Proteolysis*
  • Signal Transduction*
  • Suppressor of Cytokine Signaling Proteins / immunology
  • Suppressor of Cytokine Signaling Proteins / metabolism*
  • Trabecular Meshwork / cytology*
  • Trabecular Meshwork / metabolism*
  • Ubiquitin / metabolism*
  • Young Adult

Substances

  • ASB10 protein, human
  • Antibodies
  • Biomarkers
  • Suppressor of Cytokine Signaling Proteins
  • Ubiquitin
  • Proteasome Endopeptidase Complex