The goal of this proposed study is to quantitatively characterize the lysine N?-acetylation (LysAc) events (acetylome) in the retinal nuclear fraction from diabetic animals, and determine how the individual LysAc sites are modulated by histone acetyltransferase, p300. Diabetic retinopathy is the most common diabetic eye disease and a leading cause of blindness in American adults. It is caused by changes in the blood vessels of the retina, which eventually leads to the progressive loss of ganglion cells, horizontal cells, amacrine cells and photoreceptors. Unfortunately, currently no effective therapies to prevent or slow the progress of the disease are available. Therefore, we must continue searching new therapeutic targets. Recent proteomic studies on LysAc have revealed that LysAc is a common and dynamic post-translational modification present in both histone and non-histone proteins, suggesting that LysAc is used as a regulatory modification similar to that of protein phosphorylation. Interestingly, modulation of lysine acetylation events in diabetic animals by either inhibiting histone acetyltransferase (p300) or activating NAD+-dependent protein deacetylase (Sirt1) has been shown to have beneficial effect on the development of retinopathy. Thus, we hypothesize that dysregulation of lysine acetylation events are involved in the development of diabetic retinopathy. In this R21 proposal, we will quantitatively characterize the acetylome in the retinal nuclear fraction. We will employ cutting-edge mass spectrometry based quantitative proteomic techniques, in conjunction with an affinity enrichment technique for acetyl lysine containing peptides. The obtained acetylome data set will be analyzed using bioinformatics-based tools to map the biological processes that are involved in the development of diabetic retinopathy. We will also verify the identified LysAc sites and biological processes by mass spectrometry as well as using biochemical approaches. The identifications of differentially acetylated sites will provide significant impact in the field, because such knowledge is essential to understand the pathophysiology of the disease and provide a foundation for the development of novel therapeutic strategies for diabetic retinopathy. The following specific aims are proposed.
Specific Aim 1 : To identify LysAc sites and biological processes/pathways that are involved in the development of diabetic retinopathy and Specific Aim 2: To verify the LysAc sites and biological processes/pathways identified in Aim 1.
Diabetes is the leading cause of new cases of blindness among adults aged 20-74 years. However, no effective therapies to prevent or slow the progress of the disease are available. The identifications of differentially acetylated sites will provide a foundation for the development of novel therapeutic strategies for diabetic retinopathy.
| Mullangi, Vennela; Mamillapalli, Sireesha; Anderson, David J et al. (2014) Long-range stabilization of anthrax protective antigen upon binding to CMG2. Biochemistry 53:6084-91 |
