Research in my laboratory is directed toward determining the molecular structure and function of membrane proteins and elucidating their role in cell physiology and disease. Currently, a significant part of our research is focused on vertebrate retinal photoreceptor proteins and their role in 1) phototransduction and other signaling pathways, 2) rod and cone photoreceptor cell structure and morphogenesis, 3) lipid transport across membranes; 4) protein and vesicle trafficking; and 5) inherited retinal degenerative diseases which are a significant cause of blindness in the world. We are using this information to develop novel therapeutic approaches including drug discovery, gene supplemental therapy, and DNA base editing as potential treatments for inherited retinal degenerative diseases including macular degeneration and retinitis pigmentosa. Research projects include the structure and functional characterization of ABCA4, an ATP-binding cassette protein involved in the clearance of retinal from photoreceptors and linked to Stargardt disease, peripherin2-rom1 complex involved in photoreceptor disc membrane structure and curvature and associated with retinitis pigmentosa and macular dystrophy, RD3 and guanylate cyclase involved in production and regulation of cGMP and its trafficking to outer segments and linked to Lebers Congenital Amaurosis (LCA1 and LCA12), and P4-ATPases involved in the transport of phospholipids across membranes and associated the generation of phospholipid asymmetry across membranes and many inherited and complex neurological diseases.
To accomplish these goals, we are developing and applying a wide variety of current and emerging biochemical, biophysical, immunochemical, molecular and cell biology approaches to 1) purify and reconstitute membrane proteins into lipid vesicles for structure-function analyses; 2) determine the high resolution structure of proteins and their structural, functional and regulatory domains; 3) identify specific protein-protein interactions responsible for the formation of molecular assemblies; 4) determine the molecular and cellular mechanisms underlying retinal degenerative diseases; 5) develop novel reagents and cell and animal models for analysis of retinal degenerative diseases; and 6) investigate therapeutic interventions for selective retinal degenerative diseases. Some of the techniques currently being employed include:
• Generation and characterization of monoclonal antibodies
• cDNA cloning and sequencing
• Heterologous protein expression in bacteria, yeast, insect and mammalian cells
• Generation of mutant and chimeric proteins for mechanistic studies
• Immunoaffinity/ immunoprecipitation of proteins
• HPLC and FPLC for analysis of proteins and small molecules
• Mass spectroscopy for proteomic studies
• SDS gel electrophoresis and Western blotting for protein analysis
• Immunofluoresecence confocal scanning microscopy of labeled cells
• Electron microscopy for morphological and immunogold labeling studies
• Lipid and drug binding and transport assays
• Subcellular fractionation techniques
• Cell and tissue culture techniques
• Generation and characterization of knockout and transgenic mice
• Gene therapy and drug discovery
• Enzyme assays
• Cryo-electron microscopy
Selected Research Projects:
Rod and Cone Photoreceptor Cells.
The outer segment is a specialized compartment of rod and cone photoreceptor cells where light is captured and converted into an electrical signal in a signaling process known as phototransduction. The rod outer segment consists of a stack of over 1000 discs surrounded by a plasma membrane. We are identifying and characterizing novel membrane and cytoskeletal proteins important in phototransduction, generation and renewal of outer segments, protein trafficking from the inner to the outer segment, and
photoreceptor cell survival.
ABCA4 and Stargardt Macular Degeneration.
ABCA4 is a member of the family of ATP-binding cassette (ABC) transporters that resides in outer segment disc membranes. Over 800 mutations in ABCA4 are responsible for a relatively common early onset retinal degenerative disease known as Stargardt macular degeneration. We have recently shown that ABCA4 functions as retinoid-lipid transporter which plays a key role in the removal of potentially toxic retinoids from photoreceptors. We are currently studying the structure, function and mechanism of transport of ABCA4 and disease-associated mutants in order to determine how selected mutations in ABCA4 cause Stargardt disease. We are also exploring the use of drug and gene therapy as potential treatments for Stargardt disease.
RD3, GC, and Leber Congenital Amaurosis.
RD3 is a 23 kDa protein encoded by a gene associated with Leber Congenital Amaurosis Type 12 (LCA12), a inherited retinal dystrophy which causes severe vision loss in infants. Mutations in RD3 also cause rod and cone photoreceptor degeneration in the rd3 mouse and rcd2 collie which serve as valuable animal models for LCA12. We have recently shown that RD3 plays a crucial role in the trafficking of guanylate cyclase (GC) to rod and cone outer segments. GC is a a key protein involved in the production of cGMP, the second messenger for phototransduction, and associated with Leber Congenital Amaurosis Type 1 (LCA1). Ongoing studies are directed toward elucidating the mechanism by which RD3 facilitates vesicle trafficking of GC to outer segments. We are also carrying out studies to determine if the delivery of the Rd3 gene to photoreceptors using AAV vectors can restore vision and maintain photoreceptor cell survival in the rd3 mouse as a proof-of-concept for the application of gene therapy as a possible treatment for LCA12.
Phospholipid Transport across Membranes.
P4-ATPases and ABCA Transporters function in the transport of phospholipids to generate lipid asymmetry across biological membranes and facilitate lipid homeostasis. We have recently purified and characterized a P4-ATPase known as ATP8A2 and have shown that it is localized in rod and cone photoreceptors where it transports or flips phosphatidylserine and phosphatidylethanolamine across cell membranes, a process that is important in vesicle trafficking and phototransduction. We have also shown that both ABCA4 and ABCA1 function as phospholipid transporters, but transport phospholipids in opposite directions. Current research is focused on analysis of the structure and function of various P4-ATPases and ABCA transporters in order to define in detail their mechanism of action and role in biological processes and disease.
|Current Members of the Laboratory|
|Postdocs/Research Associates||Graduate/Undergraduate||Research Assistants|
- Scortecci JF, Molday LL, Curtis SB, Garces FA, Panwar P, Van Petegem F, Molday RS. Cryo-EM structures of the ABCA4 importer reveal mechanisms underlying substrate binding and Stargardt disease. Nat Commun. 2021 12(1):5902. PMID: 34625547.
- Molday RS, Garces FA, Scortecci JF, Molday LL. Structure and function of ABCA4 and its role in the visual cycle and Stargardt macular degeneration. Prog Retin Eye Res. 2021 Dec 23:101036. Epub ahead of print. PMID: 34954332.
- Garces FA, Scortecci JF, Molday RS. Functional Characterization of ABCA4 Missense Variants Linked to Stargardt Macular Degeneration. Int J Mol Sci. 2020 22(1):185. PMID: 33375396.
- Choi H, Andersen JP, Molday RS. Expression and functional characterization of missense mutations in ATP8A2 linked to severe neurological disorders. Hum Mutat. 2019 40(12):2353-2364. PMID: 31397519.
- Wang J, Molday LL, Hii T, Coleman JA, Wen T, Andersen JP, Molday RS. Proteomic Analysis and Functional Characterization of P4-ATPase Phospholipid Flippases from Murine Tissues. Sci Rep. 2018 8(1):10795. PMID: 30018401.
- Andersen JP, Vestergaard AL, Mikkelsen SA, Mogensen LS, Chalat M, Molday RS. P4-ATPases as Phospholipid Flippases-Structure, Function, and Enigmas. Front Physiol. 2016 7:275. PMID: 27458383.
- Molday RS, Moritz OL. Photoreceptors at a Glance. J Cell Sci. 2015 128(22):4039-45. doi: 10.1242/jcs.175687. PMID: 26574505.
- Molday LL, Djajadi H, Yan P, Szczygiel L, Boye SL, Chiodo VA, Gregory-Evans K, Sarunic MV, Hauswirth WW, Molday RS. RD3 gene delivery restores guanylate cyclase localization and rescues photoreceptors in the Rd3 mouse model of Lebers congenital amaurosis 12. Hum Mol Genet. 2013 22(19):3894-905. PMID: 23740938.
- Quazi F, Lenevich S, Molday RS. ABCA4 is an N-retinylidene-phosphatidylethanolamine and phosphatidylethanolamine importer. Nat Commun. 2012 3:925. PMID: 22735453.
- Zhong H, Molday LL, Molday RS, Yau KW. The heteromeric cyclic nucleotide-gated channel adopts a 3A:1B stoichiometry. Nature. 2002 420(6912):193-8 PMID: 12432397