Visual Cycle Structural Biology and Biochemistry
We study a process known as the visual cycle that occurs within the retina and the retinal pigment epithelium and is responsible for maintaining the light sensitivity of the retinal photoreceptor cells, as required for the initiation of phototransduction1. The principal task of the visual cycle is to transform all-trans-retinal released from rod and cone opsins back into 11-cis-retinal, which serves as the universal visual chromophore of vertebrate visual pigments. This chemically simple change (isomerization of a single double bond), is accomplished in vivo through numerous catalytic and transport processes occurring in the photoreceptors, the retinal pigment epithelium, and the Müller glia. We investigate this pathway on the molecular level using X-ray crystallography and other structural biology methods together with complementary biochemical approaches. In particular, we have led efforts to structurally characterize the key retinoid isomerase of the visual cycle2, known as RPE65, and have contributed to the structural characterization of several other visual cycle and phototransduction proteins including LRAT, rhodopsin, transducin, visual arrestin, and retinoid-binding proteins3,4.
Visual Cycle Physiology, Pathophysiology, and Therapeutics
We investigate visual cycle physiology through the use of mouse models in which the gene encoding the protein of interest is deleted (i.e. knock-out mice). In particular, we are interested in the contributions of proteins expressed in both the retinal pigment epithelium and the Müller glia to the production of visual chromophore for rod and cone photoreceptors, for example, CRALBP, RGR, and DES1. To facilitate this effort, we recently developed an inducible Cre mouse line (Rpe65CreERT2) that allows for temporally controlled, high-efficiency excision of floxed DNA specifically within the retinal pigment epithelium. We are also interested in the pathophysiology of diseases related to the dysfunction of visual cycle proteins such as retinitis pigmentosa and Stargardt macular dystrophy. Questions related to these diseases are addressed through animal models, biochemical analyses, and structural biology. In addition to providing insights into retinal disease, our studies also aim to elucidate approaches for treating currently incurable retinopathies. For example, in collaboration with the Gandhi laboratory at UCI we have shown that artificial visual chromophore prodrug (9-cis-retinyl acetate) can rescue visual function at all levels of the visual circuit in adult Lrat-/- mice, a finding suggesting that the visual circuit can adapt and recover even in the post-critical period.
Visual Cycle Modulation
We have contributed to efforts to develop and improve a class of small molecules known as visual cycle modulators that are predicted to have therapeutic effects in retinal diseases including Stargardt disease and diabetic retinopathy. These compounds act as inhibitors of RPE65 and thereby produce an overall slowed rate of visual cycle flux and diminished rate of visual opsin recovery. This action may be therapeutically beneficial by reducing the levels of all-trans-retinal, which can have toxic effects itself or form adducts such as A2E which are also believed to be toxic. Another mechanism, relevant to diabetic retinopathy, is the modulation of overall photoreceptor metabolism, which can have a beneficial effect on reducing the oxidative stress associated with hyperglycemia. Using a structure-guided drug design approach, we have generated a series of compounds related to emixustat (a visual cycle modulator studied in clinical trials) that have modified RPE65 active site binding affinity, pharmacokinetic stability, or selective mechanisms of action. In addition to potential therapeutic applications, these molecules have proven valuable as tool compounds to study visual cycle physiology.
Carotenoid cleavage dioxygenases
The laboratory has a major research interest in a superfamily of enzymes known as carotenoid cleavage dioxygenases that are involved in numerous of biological functions ranging from hormone production in animals and plants, coloration, generation of chemoattractants, and degradation of environmental phenylpropanoids. Our interest in these enzymes mainly lies in their function in the production of retinaldehyde required for vertebrate vision. We also employ stilbene-cleaving members of the superfamily as model systems owing to their facile ability to be produced in large quantities and the more favorable solubility characteristics of their hydroxylated stilbenoid substrates. The lab has pioneered methods to express and purify these enzymes in quantities needed for high-resolution structural studies and sophisticated spectroscopic analysis. We have performed key studies demonstrating that CCDs are in fact dioxygenases instead of monooxygenases, as originally believed. We have also led efforts to resolve the three-dimensional structures of these enzymes alone and in complex with substrates, products, and inhibitors. We collaborate with spectroscopists, including the Solomon lab at Stanford University and the Hendrich lab at Carnegie Mellon University, to elucidate catalytically important details regarding the iron centers of these enzymes. Our current efforts are focused on understanding the basis of secondary activities in certain CCDs, particularly double bond isomerization, and resolving the mechanistic basis for initiation and control of dioxygenase reactivity. Our research on CCDs is currently funded by the National Science Foundation.