Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Not So Hot Rods: Mutations of Rhodopsin Kinase in Regards to Oguchi Disease J. Bade, C. Czerniak, S. Daley, A. Kellicut, J. Killoren, E. Krupski, L. Semler, C. Wilkins Teacher: Mark Arnholt Mentor: Melissa Wilk, B.S., Medical College of Wisconsin Abstract The average person’s eyes adapt to darkness within minutes. For those with Oguchi disease, adaptation can be slowed to several hours. Oguchi disease is an autosomal recessive disorder that results in greatly slowed phototransduction. Phototransduction is a cascade reaction beginning with a photon activating rhodopsin in the rod and leading to hyperpolarization of the cell. Oguchi disease is caused by mutations in rhodopsin kinase which prevent the phosphorylation of rhodopsin, lowering rhodopsin’s affinity for arrestin. This reduced ability to bind arrestin decreases the speed in which rhodopsin is deactivated and prepped to reactivate. After a long period in a dark environment, the rhodopsin is eventually deactivated by arrestin, allowing it to be recycled. The Hartford Union High School SMART (Students Modelling a Research Topic) Team has designed a model of rhodopsin kinase to investigate its structure-function relationship. Oguchi disease can be caused by two different mutations in rhodopsin kinase: large deletion or point mutation. In our 3D model, we will highlight the complete deletion of exon five, the partial deletion at the C-terminus, and point mutations in the catalytic domain (Val380Asp and Pro391His) that cause Oguchi disease. Understanding the structure-function relationships of rhodopsin kinase could shed more light on night blindness. This program is supported by a grant from NIH and CTSA. Function of Rhodopsin Kinase When a photon of light enters the eye, phototransduction, the process enabling us to see, begins. Within the rod photoreceptors, the cells responsible for night vision, the photon activates rhodopsin, stimulating a cascade of neural events resulting in the recognition of light. Following activation of rhodospin, a protein called rhodopsin kinase is responsible for deactivating rhodopsin so that it can be recycled for another round of activation. During phototransduction, calcium levels decrease, allowing recoverin, a calcium-binding protein, to release rhodopsin kinase. Rhodopsin kinase then uses ATP to phosphorylate the activated rhodopsin. Once phosphorylated, arrestin binds the rhodopsin, fully deactivating it and allowing for the recycling of retinal within the rhodopsin. Without the rhodopsin kinase, the rhodopsin cannot be recycled properly, resulting in slowed dark adaptation. Structure of Rhodopsin Kinase Oguchi Disease Oguchi Disease is caused by mutations in rhodopsin kinase which result in greatly slowed adaptation of the rods of the retina. For a person living with this disease, the adaption to the dark will take several hours, whereas a person without the disease, adaption will only take several minutes. This makes everyday tasks such as driving at night, navigating a dark room, and going into a dimly lit restaurant difficult. Patients with Oguchi Disease have a distinct fundus appearance, with a golden sheen under light adapted states (left) that disappears with dark adaption (right). Model of Rhodopsin Kinase based on file 3C51.pdb ● A phosphate binding loop is formed by the Glycine-rich β1-β2 turn. It directly interacts with the triphosphate tail of ATP and helps stabilize the phosphorylation transition state. ● The N-terminal amino acid residues 8-17 of rhodopsin kinase appear to be critical for rhodopsin phosphorylation and recoverin binding. ● Point mutations Val380Asp and Pro391His in catalytic domain, as well as a deletion of exon 5, disrupt the normal function of rhodopsin kinase, resulting in slowed dark adaptation. Mutations in Rhodopsin Kinase Summary One of three mutations in the rhodopsin kinase gene cause Oguchi disease: a point mutation, two point mutations in the catalytic domain, or a complete deletion of exon 5. PCR can be used to identify the exon 5 deletion of this protein. Rhodopsin kinase is involved in the process of phototransduction in the rods of the retina where it works to prepare the rod to accept another photon of light. The specific mutations alter the shape of rhodopsin kinase and ultimately slow down the process of adaptation to a lack of light by nine times[1]. Oguchi disease is the condition of slowed adaptation to darkness. Understanding the underlying cause of Oguchi disease may help in the development of future treatments for this debilitating disease. References Oguchi disease is typically caused by one of three mutations in the rhodopsin kinase gene: point mutations in the Val380Asp or Pro391His, or a total deletion of exon 5. Cideciyan and other used polymerase chain reaction (PCR) to amplify the rhodopsin kinase gene with and without an exon 5 deletion. Different primers were used to pinpoint the exons, then the DNA fragments were run through the gel. In gel electrophoresis, strands that travel further toward the negative end are shorter, and strands that stay closer to the positive end are longer. The results of the gel electrophoresis show a complete absence of the exon 5 sequence in lane one. The largest mutation that causes Oguchi disease is the complete deletion of exon 5, a 1.7 kb deletion that removes 42 amino acids from the protein. This greatly alters the shape of the protein, making it inert and therefore slowing down the rate of rhodopsin deactivation. There are also two point mutations in the catalytic domain that can cause Oguchi disease, Val380Asp and Pro391His. These mutations reside in exon 5, and are involved in phosphorylating rhodopsin to help deactivate it. Another point mutation, at amino acid 334, not in the catalytic domain, has been known to also cause Oguchi disease. 1. Cideyciyan, A.V., Zhao, X., Nielsen, L., Khani, S.C., Jacobson, S.G., Palcaewski, K. (1997). Null mutation in the rhodopsin kinase gene slows recovery kinetics of rod and cone phototransduction in man. Proc. Natl. Acad. Sci. USA, 95(1): 328-333. 1. Baylor, D.A., Burns, M.E. (1998). Control of rhodopsin activity in vision. Eye, 12(Pt 3b): 521-525. 1. Singh, P., Wang, B., Maeda, T., Palczewski, K., Tesmer, J.J.G. (2008). Structures of rhodopsin kinase in different ligand states reveal key elements involved in G protein-coupled receptor kinase activation. J Biol Chem, 283(20): 14053-14062. SMART Teams are supported by the National Center for Advancing Translational Sciences, National Institutes of Health, through Grant Number 8UL1TR0C0055. Its contents are solely the responsibility of the author and do not necessarily represent the official views of the NIH.