Research in my lab is directed toward identifying and characterizing membrane proteins and defining their role in cellular processes and disease.
Our current interest focuses on vertebrate retinal rod and cone proteins with the goal of elucidating their role in 1) phototransduction and other signaling pathways; 2) photoreceptor cell structure and outer segment morphogenesis; 3) lipid transport across membranes; 4) protein and vesicle trafficking within cells; and 5) inherited retinal degenerative diseases which comprise a leading cause of blindness in the world. We are using this information to design and develop novel treatments for retinal degenerative diseases.
Retina is composed of 5 main neural cells (photoreceptors, horizontal cells, bipolar cells, amacrine cells, and ganglion cells), 1 main glial cell (Muellar cell) and 2 synaptic layers (outer plexiform layer (OPL) and inner plexiform layer (IPL). There are two principal types of photoreceptor cells (rod and cone cells). These cells are structurally and functionally separated into sections (outer segment involved in phototransduction, inner segment containing the metabolic and synthetic machinery found in eukaryotic cells, cell body containing the nucleus, and the synaptic region for transmission of an electrical signal from photoreceptors to secondary neurons). Over 300 genes have been linked to inherited retinal degenerative diseases with most genes highly expressed in photoreceptors and adjacent retinal pigment epithelial (RPE) cells where they encode proteins crucial for the survival and function of photoreceptors and vision.
The outer segment of rod photoreceptor cells consists of a stack of over 500 closed discs enclosed by a plasma membrane. The discs have one or more incisures that penetrate toward the center of the disc. The flat membranes are densely packed with rhodopsin, the photoreceptor protein that captures light. Additional proteins are found along the rim and incisures (peripherin2:Rom1 complex, ABCA4, guanylate cyclase). A different set of proteins is localized along the plasma membrane (cyclic nucleotide-gated channel, sodium-calcium-potassium exchanger, embigin, others). Rhodopsin is an exception as it is present both in the disc and plasma membrane.
Phototransduction is initiated when a photon of light converts 11-cis retinal within rhodopsin to all-trans retinal leading to the activation of rhodopsin. Activated rhodopsin catalyzes the conversion of the G-protein transducin from its inactive state (transducin-GDP) to its active state (transducin-GTP). Activated transducin binds to and activates phosphodiesterase resulting in a decrease in the second messenger cGMP and its dissociation from the cyclic nucleotide channel (CNGC) in the plasma membrane. This results in the closure of CNGC, a hyperpolarization of cell, and transmission of the signal from photoreceptors to secondary neurons at the synapse. The photoreceptor is returned to its dark state through the inactivation of rhodopsin by a kinase catalyzed phosphorylation reaction, and the binding of arrestin, the activation of guanylate cyclase via it regulatory protein GCAP, and inctivation of phosphodiesterase through the hydrolysis of GTP to GDP bound to transducin. These reactions lead to the restoration of cGMP levels, reopening of the CNG channels, and a return of the cell to its depolarized state.
All-trans retinal released from rhodopsin as part of phototransduction has to be converted back to 11-cis retinal for the regeneration of rhodopsin. This is carried out by the visual cycle, a series of enzymes present in photoreceptor outer segments and retinal pigment epithelial cells. A key protein involved in the clearance of retinal from photoreceptor outer segment disc is ABCA4.
ABCA4 is a 250 kDa ABC transporter localized along the rim region of rod and cone photoreceptor outer segments discs. It uses the energy from ATP binding and hydrolysis to transport N-retinylidene-phosphatidylethanolamine (N-Ret-PE), the Schiff base adduct of retinal and phosphatidylethanolamine, across disc membranes. This facilitates the clearance of retinal from disc membranes through the visual cycle. We and other groups have recently determined the structure of ABCA4 by cryo-electron microscopy.
Loss of function mutations in ABCA4 result in the accumulation of
N-Ret-PE in disc membranes. This compound reacts with another molecule of all-trans retinal or 11-cis retinal to form bis-retinoids which accumulate in RPE cells following phagocytosis of photoreceptor outer segments. Loss of function mutations in ABCA4 are known to cause Stargardt disease, a relatively common retinal degenerative disease characterized by the accumulation of lipofuscin and bis-retinoids in RPE cells, photoreceptor degeneration and a loss in vision.
Current research is directed toward defining the mechanism of ABCA4-mediated N-Ret-PE transport across membranes, elucidating molecular and cellular mechanisms underlying Stargardt disease, and developing drug and gene based treatments which can slow or prevent visual loss in Stargardt patients.
P4-ATPases, Energy -dependent Phospholipid flippases
P4-ATPases comprise a subfamily of P-type ATPases that use the energy from ATP hydrolysis to transport or flip specific lipids across the membrane bilayer. This generates transmembrane lipid asymmetry, a property important for a number of cellular processes including vesicular transport, apoptosis, phagocytosis, blood clotting, and regulation of protein activity. There are 14 P4-ATPases encoded by the human genome. Several are known to specifically flip phosphatidylserine and phosphatidylethanolamine across membranes. Most P4-ATPases consist of a catalytic subunit (nominally defined as the P4-ATPase) and an accessory subunit (referred to as CDC50 or TMEM30).
Current research is focused on analysis of the structure, function and regulation of specific P4-ATPases and their role in cellular processes and diseases.
