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Protein interactions with myocilin
Kelly Wentz-Hunter1, Jun Ueda, Beatrice Y J T Yue
1Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago College of Medicine, Chicago, Illinois 60612, USA.
Purpose:
To identify factors that interact in vivo with myocilin, a glaucoma gene product.
Methods:
The yeast two-hybrid system with myocilin as the bait and a human skeletal muscle cDNA library as the prey was used to identify potential factors that interact with myocilin. Interactions were also examined in bovine trabecular meshwork (TM) cells through a mammalian two-hybrid system. Biochemical coimmunoprecipitation from both human TM cell lysate and in vitro translated proteins was also used to confirm results obtained from yeast analysis.
Results:
Twenty positive clones isolated through yeast two-hybrid screening were deemed potential myocilin partners. Sequence analysis determined that two of them encoded for myocilin from amino acids 64 to 268. Myocilin was also found to interact with a component of the myosin motor protein, myosin regulatory light chain (RLC). The myocilin-myocilin and myocilin-RLC interactions revealed by the yeast system were further confirmed and demonstrated in cultured TM cells, by means of a mammalian two-hybrid system, and through biochemical coimmunoprecipitation, subcellular fractionation, immunofluorescence, and immunogold double labeling.
Conclusions:
These results indicate that myocilin can form homomultimers in vivo, independent of the olfactomedin-like domain. Further analysis established that the leucine zipper motif of myocilin may be necessary for the myocilin-RLC interaction. The interaction of myocilin with RLC, a component of the myosin motor protein complex, implies a role for myocilin in the actomyosin system, linking in turn this novel protein to functional status of the TM.
Insights
Myocilin, a glaucoma gene product, interacts with itself and myosin regulatory light chain (RLC) in trabecular meshwork cells. This suggests myocilin
Area of Science:
- Ocular biology
- Molecular genetics
Background:
- Myocilin is a protein encoded by a gene associated with glaucoma.
- Its in vivo interactions and cellular functions remain incompletely understood.
Purpose of the Study:
- To identify proteins that interact with myocilin within cells.
- To investigate the functional implications of these interactions in the trabecular meshwork.
Main Methods:
- Yeast two-hybrid system to screen for myocilin-interacting proteins.
- Mammalian two-hybrid system and coimmunoprecipitation to confirm interactions in trabecular meshwork cells.
Main Results:
- Myocilin self-interaction (homomultimerization) was confirmed in vivo.
- Myocilin interacts with myosin regulatory light chain (RLC), a component of the myosin motor protein complex.
- These interactions were validated in human and bovine trabecular meshwork cells.
Conclusions:
- Myocilin forms homomultimers independently of its olfactomedin-like domain.
- The leucine zipper motif of myocilin is likely involved in its interaction with RLC.
- Myocilin's interaction with RLC suggests a role in the actomyosin system and trabecular meshwork function.