James P. Dillon

Non‑enzymatic glycation of α‑crystallin as an in vitro model for aging, diabetes and degenerative diseases

Alpha crystallin, a small heat-shock protein, has been studied extensively for its chaperone function. Alpha crystallin subunits are expressed in stress conditions and have been found to prevent apoptosis by inhibiting the activation of caspase pathway. Non-enzymatic glycation of protein leads to the formation of advanced glycation end-products (AGEs). These AGEs bind to receptors and lead to blocking the signaling pathways or cause protein precipitation as observed in aggregation-related diseases. Methylglyoxal (MGO) is one of the major glycating agents expressed in pathological conditions due to defective glycolysis pathway. MGO reacts rapidly with proteins, forms AGEs and finally leads to aggregation. The goal of this study was to understand the non-enzymatic glycation-induced structural damage in alpha crystallin using biophysical and spectroscopic characterization. This will help to develop better disease models for understanding the biochemical pathways and also in drug discovery.

Early Diagnosis of Diabetes through the Eye.

Diabetes mellitus is a metabolic disorder characterized by high blood sugar levels which give rise to complications in the eye, kidneys and the brain. Diabetes triggers the development of ocular diseases like diabetic retinopathy and cataracts which are the leading cause of blindness around the world. The most common method for the diagnosis of diabetes involves measuring the blood sugar levels in the body. One major disadvantage of this method is the fluctuating blood sugar levels which contribute to false negative results. This leads to delay in treatment, eventually causing permanent damage to the organs. Therefore, diagnosis of diabetes at an early stage is very crucial. One biomarker for diabetes related diseases is the formation of Advanced Glycation End‐products (AGEs) that result from the Maillard reaction of proteins with glucose. α‐crystallin in the ocular lens is a small heat shock protein with no protein turnover and hence acts as a record for post‐translational modifications especially glycation which forms AGEs. We have used steady state and time resolved fluorescence measurements to study the spectroscopic changes in α‐crystallin with increase in time of glycation and the intact lenses from diabetic and nondiabetic donors. Overall, this study was focused on developing a noninvasive diagnostic tool for early detection of diabetes mellitus.

A2E-Mediated Photochemical Modification to Fibronectin and its Implications to Age-Related Changes in Bruch's Membrane†

Lipofuscin accumulates normally with age and is more pronounced in retinal dystrophies such as age‐related macular degeneration. The major bis‐retinoid component of lipofuscin is A2E. In addition to cell damage effects by A2E, we have previously demonstrated that blue‐light–mediated A2E leads to modifications in the basement membrane protein laminin. Therefore, the purpose of this study was to advance the understanding of A2E photooxidation effects on fibronectin, the major glycoprotein of Bruchs membrane. In this study, A2E was irradiated with blue light in the presence of a fibronectin peptide consisting of amino acids from the integrin binding region. The modification sites were identified via LC/MS. Our research indicated that blue light irradiation caused cleavage throughout the A2E molecule closest to the pyridinium ring, and attached to the fibronectin peptide preferentially at lysine and arginine residues. All of these reactions are similar to the Maillard reaction. Altogether this study suggests that blue‐light–irradiated A2E modifies peptides and forms advance glycation endproducts. Furthermore, these results can be used to identify modifications that occur in Bruchs membrane in vivo.