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Kelly Wentz–Hunter, Jun Ueda, Beatrice Y. J. T. Yue; Protein Interactions with Myocilin. Invest. Ophthalmol. Vis. Sci. 2002;43(1):176-182.
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purpose. To identify factors that interact in vivo with myocilin, a glaucoma
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.
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