Future Directions

This web page was produced as an assignment for Genetics 677, an undergraduate course at UW-Madison.

Future Directions:
    While working on this website, it became abundantly clear that the amount of research being conducted on glaucoma, in particular myocilin, is currently growing at a very steady pace. However, I found it especially interesting that even amidst all of this research, myocilin's normal function is still not well characterized. More specifically, the olfactomedin domain of this protein, which takes up a phenomenal 44% of the protein (see Protein-Domains page for visualization), is even less well understood . I found this quite striking as this domain is present not only in myocilin but many other proteins as well. Additionally, it is present in proteins of 44 species besides humans and is existent in every known myocilin homologue. Because of this, it is apparent that this olfactomedin domain is well-conserved and must be of great functional importance to many species and a subset of their proteins.
    With this in mind, I think further characterization of this domain could be of vast importance and well worth studying. To begin, truncation experiments involving the myocilin protein could elucidate the olfactomedin domain's role in myocilin localization. For this set of experiments, I would like to begin by truncating the protein at different amino acid positions, starting at the C-terminus (at which the olfactomedin domain is located) and working towards the N-terminus, tagging the truncated proteins with GFP, and monitoring [changes in] localization of the various proteins. Such experiments could show whether or not the olfactomedin domain, in particular specific portions of the domain, are needed for normal localization of myocilin. I would expect full length proteins and proteins in which the area with the highest number of mutations in the olfactomedin domain is NOT deleted should localize normally (see Main page for visualization of the location of mutations in myocilin associated with glaucoma). I imagine that once this "hot spot" of mutations is deleted, the proteins will localize abnormally; this abnormal localization would presumably include myocilin proteins in which the entire olfactomedin domain has been deleted.
    Second, further characterization of the proteins in myocilin's interacting protein network could demonstrate the role of various domains in these proteins' localization and activity. After sorting the proteins included in myocilin's interacting protein network (from STRING - see Protein Interactions page) using gene ontology terms, I found that only 5 of the 10 proteins localize to at least one of the same places as myocilin. These proteins include ANGPTL7, OLFM3, FBN1, FN1, CYP1B1. Out of these 5 proteins, only one of them, OLFM3, contains an olfactomedin domain similar to that of myocilin. Using this information, microarray experiments comparing the expression of MYOC and OLFM3 in wild type vs. glaucomatous patients could help narrow down the role and expression of the olfactomedin domain in glaucoma. For this set of experiments, I think it would be important to monitor expression of these two proteins in a few different species with known myocilin and olfactomedin-3 homologues such as Pan troglodytes and Mus musculus. For comparison, it would be particularly interesting to compare expression of MYOC and OLFM-3 in the chosen homologues to a line of Mus musculus missing the gene Pax6 (Mus musculus∆Pax6). This interest sparks from the fact that Pax6 controls eye development. In Mus musculus∆Pax6, I would expect a significant decrease in expression of these two genes as there would be no eyes for them to function in or to localize to; however, only replicates of the described microarray experiments could confirm this with certainty. As a negative control, a species that does not have eyes yet is known to have proteins containing an olfactomedin domain, such as Ciona intestinalis, could be used.
    Finally, observing the interaction between a drug used to treat glaucoma and the olfactomedin domain could help demonstrate the drug's mechanism of action and again, the importance of myocilin's olfactomedin domain. While the mechanistic pathway of some drugs used to treat glaucoma, including prostaglandin analogues, is somewhat understood, further characterization could be extremely helpful in generating new glaucoma drug therapies. To test for an interaction between the olfactomedin domain and a drug used to treat glaucoma, a chemical assay could be implemented. Using this, one could look for direct binding of the drug to a wild-type olfactomedin domain or possibly even direct binding of the drug to a mutated olfactomedin domain. If the drug binds to mutated olfactomedin domains, perhaps it aids these mutated proteins by correcting abnormal localization and directs them to areas of normal localization where they would no longer block the outflow of the aqueous humor. Via this hypothetical mechanism, the drug would not allow mutated proteins to accummulate in the aqueous humor outflow pathway, thereby preventing an increase in intraocular pressure.
    Clearly, myocilin research is easily validated as its associated disease, glaucoma, is currently the second leading cause of blindness worldwide (1). Any research that could help lower this stastic would be much welcomed and learning more about myocilin's olfactomedin domain may help uncover its role in other diseases that are associated with gene products containing a similar olfactomedin domain.

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References:
1. Fuse, Nobuo. "Genetic Bases for Glaucoma." The Tohoku Journal of Experimental Medicine 221 (2010): 1-10. PubMed. Web. 22 Jan. 2011
http://www.jstage.jst.go.jp/article/tjem/221/1/1/_pdf
2. Rao, Kollu N., Srujana Nagireddy, and Subhabrata Chakrabarti. "Complex Genetic Mechanisms in Glaucoma: An Overview." Indian Journal of Opthamology 59.7 (2011): 31-42. PubMed. Web. 22 Jan. 2011.
http://www.ijo.in/article.asp?issn=0301-4738;year=2011;volume=59;issue=7;spage=31;epage=42;aulast=Rao
3. American Optometric Association. Answers To Your Questions About Glaucoma. St. Louis: American Optometric Association. Print.
4. http://www.forbes.com/2009/06/26/life-saving-devices-technology-personal-monitors_slide_7.html
5. http://www.youtube.com/watch?v=OJMEfGFbFMI
6. "MYOC - Myocilin, Trabecular Meshwork Inducible Glucocorticoid Response - Genetics Home Reference." Genetics Home Reference - Your Guide to Understanding Genetic Conditions. 6 Feb. 2011. Web. 08 Feb. 2011. http://ghr.nlm.nih.gov/gene/MYOC
7. Saura, Maria, Montse Cabana, Carmen Ayuso, and Diana Valverde. "European Journal of Human Genetics - Mutations including the Promoter Region of Myocilin//TIGR Gene." Nature Publishing Group : Science Journals, Jobs, and Information. 13 Oct. 2004. Web. 08 Feb. 2011. http://www.nature.com/ejhg/journal/v13/n3/full/5201299a.html
8. Fingert, John H., Elise Heon, Jefferey M. Liebmann, Tetsuya Yamamoto, Jaime E. Craig, Julian Rait, Kazuhide Kawase, Sek-Tien Hoh, Yvonne M. Buys, Joanne Dickinson, Robin R. Hockey, Donna Williams-Lyn, Graham Trope, Yoshiaki Kitazawa, Robert Ritch, David A. Mackey, Wallace L.M. Alward, Val C. Sheffield, and Edwin M. Stoned. "Analysis of Myocilin Mutations in 1703 Glaucoma Patients From Five Different Populations — Hum Mol Genet." Oxford Journals | Life Sciences & Medicine | Human Molecular Genetics. 14 Mar. 1999. Web. 08 Feb. 2011. http://hmg.oxfordjournals.org/content/8/5/899.full
9. McMahon, C., Semina, E.V., Link, B.A. "Using zebrafish to study the complex genetics of glaucoma." Comparative Biochemistry and Physiology Part C: Toxicology and Parmacology. vol 138-3. July 2004. Web. 24 Feb 2011. http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W89-4CDJG6X-1&_user=443835&_coverDate=07%2F31%2F2004&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_acct=C000020958&_version=1&_urlVersion=0&_userid=443835&md5=525db1a96bf7be7620dabbcd34c426f9&search#toc2
10. Kubota, R., Noda, S., Wang, Y., Minoshima, S., Asakawa, S., Kudoh, J., Mashima, Y., Oguchi, Y., Shimizu, N. "A novel myosin-like protein (myocilin) expressed in the connecting cilium of the photoreceptor: molecular cloning, tissue expression, and chromosomal mapping." Genomics. vol 41-3.1 May 1997, Pages 360-369 Department of Molecular Biology, Keio University School of Medicine, Tokyo, Japan. http://www.sciencedirect.com.ezproxy.library.wisc.edu/science?_ob=ArticleURL&_udi=B6WG1-45M8YTG-C5&_user=443835&_coverDate=05%2F01%2F1997&_rdoc=1&_fmt=high&_orig=gateway&_origin=gateway&_sort=d&_docanchor=&view=c&_acct=C000020958&_version=1&_urlVersion=0&_userid=443835&md5=1562ae0f98031b9d89c1c8377a859b1e&search