The phototransduction messenger, cGMP, mediates rod and cone response to light. The synthesis of cGMP by retinal guanylyl (guanylate) cyclase (RetGC), controlled by calcium through guanylyl cyclase activating proteins (GCAPs), is one of the most critical steps in photoresponse recovery. The defects in its regulation cause multiple forms of congenital human blindness. While the general importance and basic principles of the RetGC regulation have been established, some of the key mechanistic aspects remain poorly understood, especially how protein-protein interactions in GCAP and RetGC result in the cyclase activation and inhibition or triggering retinal diseases. Seeking answers to the questions addressed in this proposal conforms to the NEI mission to "conduct research and disseminate information with respect to blinding eye diseases, mechanisms of visual function and preservation of sight". This proposal is based on characterization of new RetGC mutations causing Leber congenital amaurosis (LCA) and novel findings about the RetGC regulation: 1) that GCAP1 desensitized by disease-causing mutations preferentially targets RetGC1 isozyme in vivo;2) that GCAP1 acts as the 'first-response'Ca2+ sensor activating RetGC1 early in photoresponse;3) that N-fatty acylation in GCAP1 affects its function as a calcium sensor for RetGC1 via an intramolecular 'tug'action;4) that two regions in GCAP1 molecule emerge as a likely cyclase-binding interface;5) that a photoreceptor protein, RD3, acts as a potent inhibitor of RetGC activity, but fails to inhibit the cyclase when affected by LCA-related mutations. We propose a broad integrated approach to verify new hypotheses and delineate mechanisms in RetGC/GCAP regulatory pathways using a combination of protein biochemistry, molecular biology, and molecular genetics.
Aim 1 will address the molecular structure of GCAP1 with the emphasis on establishing the key to the conformational transition of GCAP1 into its RetGC activator state, elucidating the protein architecture for the disease-causing constitutive active GCAP1 mutants, and testing a hypothesis of "Ca2+-myristoyl tug" - across-the-molecule action of the N-fatty acyl group that controls Ca2+ sensitivity of GCAP1.
Aim 2 will investigate how the molecular mechanisms of RetGC catalytic activity and regulation become altered by newly characterized mutations causing LCA1 blindness.
Aim 3 will seek understanding of biological role of RD3 as a novel, linked to LCA and cone-rod degeneration, negative regulator of the RetGC/GCAP pathway. By completing these specific aims, we expect to overcome some critical barriers in understanding of RetGC regulation and its role in normal photoreceptor physiology and in retinal diseases.

Public Health Relevance

Mutations altering cGMP synthesis in photoreceptors cause severe congenital blinding diseases including cone dystrophy, cone-rod dystrophy (CORD), and Leber's congenital amaurosis (LCA). In this application we seek better understanding of the molecular mechanisms underlying regulation of cGMP synthesis in rods and cones and expect that this new knowledge will help better design strategies for treatment of such diseases.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01EY011522-19
Application #
8698752
Study Section
(BVS)
Program Officer
Neuhold, Lisa
Project Start
Project End
Budget Start
Budget End
Support Year
19
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Salus University
Department
None
Type
Schools of Optometry/Opht Tech
DUNS #
City
Elkins Park
State
PA
Country
United States
Zip Code
19027
Lim, Sunghyuk; Dizhoor, Alexander M; Ames, James B (2014) Structural diversity of neuronal calcium sensor proteins and insights for activation of retinal guanylyl cyclase by GCAP1. Front Mol Neurosci 7:19
Peshenko, Igor V; Olshevskaya, Elena V; Lim, Sunghyuk et al. (2014) Identification of target binding site in photoreceptor guanylyl cyclase-activating protein 1 (GCAP1). J Biol Chem 289:10140-54
Wen, Xiao-Hong; Dizhoor, Alexander M; Makino, Clint L (2014) Membrane guanylyl cyclase complexes shape the photoresponses of retinal rods and cones. Front Mol Neurosci 7:45
Boye, Sanford L; Peshenko, Igor V; Huang, Wei Chieh et al. (2013) AAV-mediated gene therapy in the guanylate cyclase (RetGC1/RetGC2) double knockout mouse model of Leber congenital amaurosis. Hum Gene Ther 24:189-202
Lim, Sunghyuk; Peshenko, Igor V; Dizhoor, Alexander M et al. (2013) Backbone (1)H, (13)C, and (15)N resonance assignments of guanylyl cyclase activating protein-1, GCAP1. Biomol NMR Assign 7:39-42
Jacobson, Samuel G; Cideciyan, Artur V; Peshenko, Igor V et al. (2013) Determining consequences of retinal membrane guanylyl cyclase (RetGC1) deficiency in human Leber congenital amaurosis en route to therapy: residual cone-photoreceptor vision correlates with biochemical properties of the mutants. Hum Mol Genet 22:168-83
Xu, Jianhua; Morris, Lynsie; Thapa, Arjun et al. (2013) cGMP accumulation causes photoreceptor degeneration in CNG channel deficiency: evidence of cGMP cytotoxicity independently of enhanced CNG channel function. J Neurosci 33:14939-48
Peshenko, Igor V; Olshevskaya, Elena V; Dizhoor, Alexander M (2012) Interaction of GCAP1 with retinal guanylyl cyclase and calcium: sensitivity to fatty acylation. Front Mol Neurosci 5:19
Peshenko, Igor V; Olshevskaya, Elena V; Lim, Sunghyuk et al. (2012) Calcium-myristoyl Tug is a new mechanism for intramolecular tuning of calcium sensitivity and target enzyme interaction for guanylyl cyclase-activating protein 1: dynamic connection between N-fatty acyl group and EF-hand controls calcium sensitivity. J Biol Chem 287:13972-84
Jiang, Li; Zhang, Houbin; Dizhoor, Alexander M et al. (2011) Long-term RNA interference gene therapy in a dominant retinitis pigmentosa mouse model. Proc Natl Acad Sci U S A 108:18476-81

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