A5009005262- Retinal Development and Disorders
- Cell Adhesion Molecules Research
- Retinal Diseases and Treatments
- Photoreceptor and optogenetics research
- Glaucoma and retinal disorders
- Neurobiology and Insect Physiology Research
- Cellular transport and secretion
- Protein Kinase Regulation and GTPase Signaling
- Receptor Mechanisms and Signaling
- bioluminescence and chemiluminescence research
- Cellular Mechanics and Interactions
- Phosphodiesterase function and regulation
- Extracellular vesicles in disease
- Connexins and lens biology
- Advanced Proteomics Techniques and Applications
- Neuroscience and Neuropharmacology Research
- Retinal Imaging and Analysis
- Glycosylation and Glycoproteins Research
- RNA Research and Splicing
- Signaling Pathways in Disease
- Microtubule and mitosis dynamics
- Genetic and Kidney Cyst Diseases
- Enzyme Structure and Function
- Ubiquitin and proteasome pathways
- Advanced Glycation End Products research
Duke University
2016-2025
Duke Medical Center
2008-2024
Duke University Hospital
1998-2023
Smith-Kettlewell Eye Research Institute
2007-2021
Ophthalmology Associates (United States)
2008
Harvard University
2001-2007
Massachusetts Eye and Ear Infirmary
2000-2005
Millennium Engineering and Integration (United States)
2003
Northwestern University
1997-2002
Howard Hughes Medical Institute
1998
Both the α and βγ subunits of heterotrimeric guanine nucleotide–binding proteins (G proteins) communicate signals from receptors to effectors. Gβγ can regulate a diverse array effectors, including ion channels enzymes. Gα bound diphosphate (Gα-GDP) inhibit signal transduction through subunits, suggesting common interface on for binding effector interaction. The molecular basis interaction with effectors was characterized by mutational analysis Gβ residues that make contact Gα-GDP. Analysis...
The recognition between G protein and cognate receptor plays a key role in specific cellular responses to environmental stimuli. Here we explore specificity receptor-G coupling by taking advantage of the ability 5-hydroxytryptamine1B (5-HT1B) discriminate heterotrimers containing Galphai1 or Galphat. Gi1 can interact with 5-HT1B stabilize high affinity agonist binding state this receptor, but Gt cannot. A series Galphat/Galphai1 chimeric proteins have been generated Escherichia coli, their...
Abstract Genes encoding cell-surface proteins control nervous system development and are implicated in neurological disorders. These genes produce alternative mRNA isoforms which remain poorly characterized, impeding understanding of how disease-associated mutations cause pathology. Here we introduce a strategy to define complete portfolios full-length encoded by individual genes. Applying this approach neural molecules, identify thousands unannotated expressed retina brain. By mass...
The G protein transducin has been an often-used model for biochemical, structural, and mechanistic studies of function. Experimental have limited, however, by the inability to express quantities mutants in heterologous systems with ease. In this study we made a series alpha t/G i1 chimeras differing at as few 11 positions from native t. Ten are properly folded, contain GDP, can assume A1F4(-)-induced activated conformation, interact beta gamma t light-activated rhodopsin. They differ...
The R7 subfamily of the regulators G protein signaling (RGS) proteins is represented by four members broadly expressed in mammalian nervous system. Here we report that brain all form tight complexes with a previously unidentified protein, which call R7-binding or R7BP. We initially identified R7BP as co-precipitating RGS9, from extracts obtained striatal region brain. further showed forms complex RGS9 vitro and this binding occurs via N-terminal DEP domain RGS9. throughout entire central...
of the sequence; trPDE,,, complex PDE u and p subunits obtained by trypsinization; GTPyS, guanosine 5'-(y-thio)
Inherited retinal degenerations, caused by mutations in over 100 individual genes, affect approximately 2 million people worldwide. Many of the underlying cause protein misfolding or mistargeting affected photoreceptors. This places an increased burden on folding and degradation machinery, which may trigger cell death. We analyzed how these cellular functions are degenerating rods transducin γ-subunit (Gγ 1 ) knockout mouse. These produce large amounts β-subunit (Gβ ), cannot fold without Gγ...
The retinal pigmented epithelium (RPE) forms the outer blood-retinal barrier in eye and its polarity is responsible for directional secretion uptake of proteins, lipoprotein particles extracellular vesicles (EVs). Such a secretional division dictates directed interactions between systemic circulation (basolateral) retina (apical). Our goal to define polarized proteomes physical characteristics EVs released from RPE. Primary cultures porcine RPE cells were differentiated into monolayers on...
Inherited retinal degenerations, affecting more than 2 million people worldwide, are caused by mutations in over 200 genes. This suggests that the most efficient therapeutic strategies would be mutation independent, i.e., targeting common pathological conditions arising from many disease-causing mutations. Previous studies revealed one such condition is an insufficiency of ubiquitin-proteasome system to process misfolded or mistargeted proteins affected photoreceptor cells. We now report...
Photoreceptors are compartmentalized neurons in which all proteins responsible for evoking visual signals confined to the outer segment. Yet, mechanisms establishing and maintaining photoreceptor compartmentalization poorly understood. Here we investigated targeting of two related membrane proteins, R9AP syntaxin 3, one residing within other excluded from Surprisingly, have found that only 3 has information encoded its sequence removal redirects this protein Furthermore, endoplasmic...
Light causes massive translocation of G-protein transducin from the light-sensitive outer segment compartment rod photoreceptor cell. Remarkably, significant is observed only when light intensity exceeds a critical threshold level. We addressed nature this using series mutant mice and found that can be shifted to either lower or higher intensity, dependent on whether ability GTPase-activating complex inactivate GTP-bound decreased increased. also demonstrated not cellular signaling...
The retina is a highly ordered tissue whose outermost layers are formed by subcellular compartments of photoreceptors generating light-evoked electrical responses. We studied protein distributions among individual photoreceptor separating the entire layer flat-mounted frozen into series thin tangential cryosections and analyzing compositions each section label-free quantitative mass spectrometry. Based on 5038 confidently identified peptides assigned to 896 database entries, we generated...
Visual signal transduction takes place on the surface of flat membrane vesicles called photoreceptor discs, which reside inside light-sensitive outer segment organelle vertebrate cells. Although biochemical studies have indicated that discs are built with a handful highly specialized proteins, proteomic yielded databases consisting hundreds entries. We addressed this controversy by employing protein correlation profiling, allows identification unique components organelles can be fractionated...
Purpose: Lysophosphatidic acid (LPA), a bioactive lipid, has been shown to increase resistance aqueous humor outflow (AH) through the trabecular meshwork (TM). The molecular basis for this response of TM LPA, however, is not completely understood. In study, we explored possible involvement mechanosensitive Yes-associated protein (YAP) and its paralog, transcriptional coactivator with PDZ-binding domain (TAZ), activation in extracellular matrix (ECM) production by LPA-induced contractile...
The light-sensitive outer segment of the vertebrate photoreceptor is a highly modified primary cilium filled with disc-shaped membranes that provide vast surface for efficient photon capture. formation each disc initiated by ciliary membrane evagination driven an unknown molecular mechanism reportedly requiring actin polymerization. Since distinct F-actin network resides precisely at site morphogenesis, we employed unique proteomic approach to identify components this potentially driving...
Strong evidence suggests that dysregulated lipid metabolism involving dysfunction of the retinal pigmented epithelium (RPE) underlies pathogenesis age-related macular degeneration (AMD), leading cause irreversible blindness in elderly. A hallmark AMD is overproduction lipid- and protein-rich extracellular deposits accumulate matrix (Bruch's membrane (BrM)) adjacent to RPE. We analyzed apolipoprotein A-1 (ApoA-1)-containing lipoproteins isolated from BrM elderly human donor eyes found a...
Photoreceptor cells generate neuronal signals in response to capturing light. This process, called phototransduction, takes place a highly specialized outer segment organelle. There are significant discrepancies the reported amounts of many proteins supporting this particularly those low abundance, which limits our understanding their molecular organization and function. In study, we used quantitative mass spectrometry simultaneously determine abundances 20 key structural functional residing...
The photoreceptor outer segment is a modified cilium filled with hundreds of flattened "disc" membranes responsible for efficient light capture. To maintain health and functionality, segments are continuously renewed through the addition new discs at their base. This process driven by branched actin polymerization nucleated Arp2/3 complex. induce polymerization, requires nucleation promoting factor. Here, we show that factor driving disc morphogenesis pentameric WAVE complex identify all...
In photoreceptor cells of vertebrates light activates a series protein-protein interactions resulting in activation cGMP-phosphodiesterase (PDE). Interaction between the GTP-bound form rod G-protein alpha-subunit (alpha t) and PDE inhibitory gamma-subunit (P gamma) is key event for effector enzyme activation. This interaction has been studied using P gamma labeled with fluorescent probe, lucifer yellow vinyl sulfone, at Cys-68 LY) sites on alpha t have investigated. Addition tGTP S to LY...
The remarkable ability of our vision to function under ever-changing conditions ambient illumination is mediated by multiple molecular mechanisms regulating the light sensitivity rods and cones. One such mechanism involves massive translocation signaling proteins, including G-protein transducin, into out light-sensitive photoreceptor outer segment compartment. Transducin extends operating range rods, but in cones transducin never translocates, which puzzling because typically much brighter...
The retinal pigmented epithelium (RPE) constitutes the outer blood-retinal barrier, enables photoreceptor function of eye, and is constantly exposed to oxidative stress. As such, dysfunction RPE underlies pathology leading development age-related macular degeneration (AMD), cause vision loss among elderly in industrialized nations. A major responsibility process segments, which relies on proper functioning its endocytic pathways endosomal trafficking. Exosomes other extracellular vesicles...