Professor, Vice Chair for Research
Ph.D., Baylor College of Medicine, Houston, Texas
Association for Research in Vision and Ophthalmology (ARVO)
American Society for Cell Biology (ASCB)
International Society for Eye Research (ISER)
Federation of American Societies for Experimental Biology (FASEB)
RESEARCH SUMMARY Research in my laboratory focuses on understanding the role of a protein in normal retinal function and how mutations can influence that function leading to the disease phenotype.
Role of extracellular matrix in retinal homeostasis and diseases.
The overall objective of this research is to investigate how the extracellular matrix (ECM) supports retinal health and determine its contribution to the acceleration of the degenerative process in inherited retinal diseases.
To study retinal ECM, we focused on tyrosine sulfated proteins. Tyrosine sulfation is a post-translational modification that occurs only on secreted and membrane proteins. It is catalyzed by two trans-Golgi tyrosine sulfotransferases (TPST-1 and TPST-2). TPSTs transfer a sulfate group from the sulfate donor phosphoadenosine 5'-phosphosulfate to the hydroxyl group of tyrosine to form a tyrosine-O-sulfate ester. Sulfated proteins have been identified among adhesion molecules, coagulation factors, matrix proteins, serpins, enzymes and proteins of unknown functions. Sulfation has been shown to be important for ligand-receptor interactions, rate of a protein’s secretion, or a protein’s binding affinity.
We have recently shown that the retina contains many tyrosine-sulfated proteins and expresses transcripts for both TPST genes. We have also evaluated retinal function in Tpst1-/-, Tpst2-/- and double knockout (DKO) mice. The DKO animals exhibited drastic reduction in electroretinographic (ERG) responses associated with abnormal photoreceptor ultrastructure. This shows for the first time the role ECM plays in ERG responses, specifically and in retinal function in general. Since the DKO mice die early, we generated a conditional knockout mouse to determine the long term effects of lack of sulfation. We are also systematically identifying sulfated proteins in the retina using classic immunoaffinity purification techniques and proteomics analysis. Most of the proteins identified so far are involved in human retinal diseases such as retinitis pigmentosa and age related macular degeneration.
Role of P53 in retinal development and maintenance.
Because of its role in cell cycle regulation, cancer and apoptosis, one would think that P53 is involved in maintaining the post-mitotic state of the adult retina. Furthermore, due to their role in phototransduction, retinal cells are under constant oxidative stress that can result in DNA damage. If not repaired, this damage can result in cell death and vision loss.
We found out that transcript and protein levels for P53 in the normal eye were highest at E17 and 18, respectively. However, both P53 transcript and protein precipitously dropped thereafter, and no protein was detected on immunoblots after P3. Immunohistochemistry (IHC) analysis of the developing eye showed that P53 is abundantly expressed at E18 in all layers of the retinal neuroblast.
To determine the role of P53, if any, in the adult retina, we utilize the super P53 mouse that over-expresses P53 driven by its endogenous promoter. We also generated transgenic mice that over-express P53 in retinal photoreceptors.
Overexpression of P53 early in development in the super P53 mouse led to increased developmental retinal apoptosis that ultimately resulted in retinal structure that lacks several layers of photoreceptors. However, there were no structural or functional effects in the postmitotic retina suggesting that the transgene was regulated in a manner similar to the endogenous gene.
In another line of transgenic mice, expression of P53 was driven by the human interphotoreceptor retinoid binding protein (IRBP) promoter which is limited to the photoreceptors. P53 over-expression led to increased developmental apoptosis as well. Since P53 expression continued into adulthood, photoreceptor degeneration continued past the developmental stages and was transgene levels-dependent. Combined, these results strongly support a role for P53 in apoptosis of retinal cells during development. However, the most interesting finding is that certain genes that are only expressed in differentiated retinal cells are regulated by P53. This result demonstrates a role for P53 in the postmitotic retina other than in the regulation of cell cycle.
Role of glycosylation in the function of rhodopsin.
Rhodopsin (RHO), the rod photoreceptor’s most abundant protein, is glycosylated at Asn-2 and 15 on its extracellular N-terminus. RHO resides in the membrane of the individual discs as well as in the plasma membrane. In its latter location, RHO interacts with the extracellular matrix.
In vitro studies on the functional significance of RHO glycosylation have provided conflicting data. To understand the role of RHO’s glycosylated N-terminus in vivo, two transgenic mouse models were generated and characterized.
The No-glycosylation (NOG) model expresses a mutant form of RHO that is unable to be glycosylated at either of the two asparagines. We showed that lack of glycosylation triggers a dominant form of retinal degeneration. Electron microscopic (EM) examination of the transgenic retinas at postnatal day (P) 30 revealed the presence of vacuolar structures that partially distorted the rod photoreceptor outer segment (OS). Degeneration progressed with age, and by P120 the animals lost ~90% of their retinal function. IHC analysis determined no mislocalization of RHO and, therefore, suggests that the phenotype is due to the NOG expression. When bred into the rho-/- background, we found that the protein appears unstable and is regulated by ubiquitin-mediated proteasomal degradation. We are currently investigating where NOG is ubiquitinated and degraded.
The second transgenic mouse model was designed to investigate the role of the entire N-terminus in the function of RHO. Transgenic mice were generated that expresses a RHO-Beta-3 adrenergic (β3AR) chimeric protein, designated NBC. The NBC protein contains the body of the β3AR as well as the N- and C-termini of RHO. Establishment and subsequent characterization of the NBC model in the rho-/- genetic background revealed that the glycosylated N-terminus of RHO is sufficient for OS morphogenesis.
Kanan,Y., Hoffhines, A., Rauhauser, A., Murray, A., and Al-Ubaidi, M.R. (2009) Protein tyrosine-O-sulfation in the retina. Exp. Eye Res. 89; 559-67 Sherry, D.M., Murray, A.R., Kanan, Y., Arbogast, K.L., Hamilton, R.A., Fliesler, S.J., Burns, M.E., Moore, K.L. and Al-Ubaidi, M.R. (2010) Lack of protein-tyrosine sulfation disrupts photoreceptor outer segment morphogenesis, retinal function and retinal anatomy. European Journal of Neuroscience 32: 1461-72.
Vuong, L., Brobst D.E., Saadi, A., Ivanovic, I. and Al-Ubaidi, M.R. (2012) Pattern of expression of p53, its family members, and regulators during early ocular development and in the post-mitotic retina. Invest. Ophthal. Vis. Sci. 53:4821-31. Sherry, D.M., Kanan, Y., Hamilton, R., Hoffhines, A., Arbogast, K.L., Fliesler, S.J., Naash, M.I., Moore, K.L. and Al-Ubaidi, M.R. (2012) Differential developmental deficits in retinal function in the absence of either protein tyrosine sulfotransferase-1 or -2. PLoS One 7:e39702. Vuong, L., Brobst, D.E., Ivanovic, I., Sherry, D.M., Al-Ubaidi1, M.R. (2013) p53 selectively regulates developmental apoptosis of rod photoreceptors. PLoS One 8:e67381. Kanan, Y., Brobst, D., Han, Z., Naash, M.I. and Al-Ubaidi M.R. (2014) Fibulin 2, a tyrosine O-sulfated protein is up-regulated following retinal detachment. J. Biol. Chem. 289:13419-13433. Kanan, Y., Siefert, J.C., Kinter, M. and Al-Ubaidi, M.R. (2014) Complement Factor H, Vitronectin and Opticin are Tyrosine-Sulfated Proteins of The Retinal Pigment Epithelium. PLoS One e105409. Mitra, R.N., Merwin, M.J., Han, Z., Conley, S.M., Al-Ubaidi, M.R. and Naash, M.I. (2014) Yttrium oxide nanoparticles prevent photoreceptor death in a light-damage model of retinal degeneration. Free Radical Biology and Medicine 75C:140-148. Kelley, R.A., Al-Ubaidi, M.R. and Naash, M.I (2014) Retbindin is an Extracellular Riboflavin Binding Protein Found at the Photoreceptor/Retinal Pigment Epithelium Interface. J. Biol. Chem. (Epub ahead of print). Murray, A.R., Vuong, L., Brobst, D., Fliesler, S.J., Peachey, N.S., Gorbatyuk, M.S., Naash, M.I. and Al-Ubaidi, M.R. (2014) Rhodopsin’s glycosylation is necessary for its stability and incorporation into photoreceptor outer segment discs (In revision for Human Molecular Genetics).
University of Oklahoma Health Sciences
Department of Cell Biology
940 Stanton L. Young Blvd.
Oklahoma City, OK 73104
Phone: (405) 271-8001 ext. 47979
Fax: (405) 271-3548