Date of Award

4-18-2008

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Masaru Miyagi

Abstract

Light induced retinal degeneration in animals has been used to study human retinal degenerative disorders. Apoptosis is the final common pathway for photoreceptor degeneration in both the experimental model and human disease but the mechanism is unknown. Oxidative stress plays a role because antioxidants have been shown to be protective against experimental degeneration. Inhibitors of nitric oxide synthases (NOS) are also protective suggesting a role for nitric oxide. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) a key enzyme in glycolysis was identified as one of the proteins undergoing significant nitration in our studies on light induced retinal degeneration. This study focuses on the changes occurring in the rod photoreceptor outer segments, particularly the role of nitration and its effects on GAPDH.Cyclic light-reared rats treated with and without an antioxidant, dimethylthiourea, were exposed to intense green light for 8 h. A subset of these rats was kept in the dark for 24 h after light exposure. Western analyses were performed to examine the amounts of nitrotyrosine in the outer segments and NOS in the retina. 2D-Western followed by liquid chromatography tandem mass spectrometry was used to identify nitrated proteins. Rabbit GAPDH was nitrated with tetranitromethane and the loss of activity and amount of nitration were determined by UV-Visible spectroscopy. The site of nitration was identified by tandem mass spectrometry. The nicotinamide adenine dinucleotide (NAD+) binding affinity was determined by a competitive binding assay using radioactively labeled and unlabeled NAD+. Biophysical characterization of the protein was performed using several methods including gel filtration, fluorescence spectroscopy and circular dichroism spectroscopy. Quantitative changes in the proteome of the photoreceptor outer segments upon light exposure were determined using a proteolytic 18O stable isotope labeling strategy.Proteins in the outer segment show increased nitration, mediated by inducible NOS, in the dark following light exposure. Glycolytic proteins appear to be the major targets of nitration. GAPDH loses activity upon nitration because it loses binding affinity for NAD+ upon nitration. The proteomic study identified several proteins that undergo changes in amount upon light exposure. Bioinformatics analysis suggests the involvement of two major apoptotic pathways, Bcl-2 and c-Myc.

Download

Share

COinS