Project

Back to overview

The role of CRALBP in the chemistry of vision

English title The role of CRALBP in the chemistry of vision
Applicant Stocker Achim
Number 156419
Funding scheme Project funding (Div. I-III)
Research institution Departement für Chemie und Biochemie Universität Bern
Institution of higher education University of Berne - BE
Main discipline Biochemistry
Start/End 01.11.2014 - 31.10.2018
Approved amount 397'000.00
Show all

All Disciplines (4)

Discipline
Biochemistry
Structural Research
Molecular Biology
Nutritional Research, Vitaminology

Keywords (6)

vitamin A; 11-cis-retinal; crystal structure; RLBP1; visual cycle; retinitis pigmentosa

Lay Summary (German)

Lead
Ziel dieses Projektes ist es, die Mechanismen des sog. Visuellen Zyklus zur Erhaltung der chemischen Reinheit des Sehprozesses zu untersuchen.
Lay summary

Kontinuierliches Sehen beginnt mit der photoinduzierten Isomerisierung von lichtsensitivem 11-cis-Retinal zu all-trans-Retinal. All-trans-Retinal ist nicht mehr photosensitiv, und es muss daher in einer Reihe von biochemischen Reaktionen in 11-cis-Retinal zurückverwandelt werden. Hierbei erweist sich der Schritt der trans-zu-cis Rückisomerisierung als erstaunlich unselektiv, liefert er doch neben 11-cis auch 9-cis und 13-cis-Retinoide.

Weitere Faktoren müssen also im gesunden Auge dafür sorgen, dass die chemische Reinheit des Sehprozesses erhalten bleibt. In diesem Zusammenhang hat sich gezeigt, dass u.a. das zelluläre retinalbindende Protein (CRALBP) frischgebildetes 11-cis-Retinal einkapselt und so vor vorzeitiger Photoisomerisierung schützt. Zudem scheint CRALBP auf bisher unbekannten Wegen dazu beizutragen, die Bildung der 9- und 13-cis Isomeren im Auge zu unterdrücken. Diese Mechanismen sollen hier untersucht werden.

Das Projekt befasst sich mit Grundlagenforschung. Es konnte in den letzten Jahren gezeigt werden, dass 9-cis-Retinal als Surrogat von 11-cis-Retinal vom Körper im normalen Sehprozess verwendet werden kann und sich somit in der Substitutionstherapie zur partiellen Wiederherstellung des Sehprozesses eignet. Die Rolle des Trägerproteins CRALBP soll in diesem Zusammenhang gezielt untersucht werden.

Direct link to Lay Summary Last update: 29.09.2014

Responsible applicant and co-applicants

Employees

Name Institute

Publications

Publication
Self-Assembly of α-Tocopherol Transfer Protein Nanoparticles: A Patchy Protein Model
Peltzer Raphael Mathias, Kolli Hima Bindu, Stocker Achim, Cascella Michele (2018), Self-Assembly of α-Tocopherol Transfer Protein Nanoparticles: A Patchy Protein Model, in The Journal of Physical Chemistry B, 122(28), 7066-7072.
Self-assembled α-Tocopherol Transfer Protein Nanoparticles Promote Vitamin E Delivery Across an Endothelial Barrier
Aeschimann Walter, Staats Stefanie, Kammer Stephan, Olieric Natacha, Jeckelmann Jean-Marc, Fotiadis Dimitrios, Netscher Thomas, Rimbach Gerald, Cascella Michele, Stocker Achim (2017), Self-assembled α-Tocopherol Transfer Protein Nanoparticles Promote Vitamin E Delivery Across an Endothelial Barrier, in Scientific Reports, 7(1), 4970-4970.
Structural insights on cholesterol endosynthesis: Binding of squalene and 2,3-oxidosqualene to supernatant protein factor
Christen Monika, Marcaida Maria J., Lamprakis Christos, Aeschimann Walter, Vaithilingam Jathana, Schneider Petra, Hilbert Manuel, Schneider Gisbert, Cascella Michele, Stocker Achim (2015), Structural insights on cholesterol endosynthesis: Binding of squalene and 2,3-oxidosqualene to supernatant protein factor, in Journal of Structural Biology, 190(3), 261-270.
Mechanisms of recognition and binding of α-TTP to the plasma membrane by multi-scale molecular dynamics simulations.
Lamprakis Christos, Stocker Achim, Cascella Michele (2015), Mechanisms of recognition and binding of α-TTP to the plasma membrane by multi-scale molecular dynamics simulations., in Frontiers in molecular biosciences, 36-36.
Mechanisms of Ligand–Protein Interaction in Sec-14-like Transporters Investigated by Computer Simulations
Helbling Rachel E., Lamprakis Christos, Aeschimann Walter, Bolze Cristin S., Stocker Achim, Cascella Michele (2014), Mechanisms of Ligand–Protein Interaction in Sec-14-like Transporters Investigated by Computer Simulations, in CHIMIA International Journal for Chemistry, 68(9), 615-619.
Human cellular retinaldehyde-binding protein has secondary thermal 9-cis-retinal isomerase activity.
Bolze Christin S, Helbling Rachel E, Owen Robin L, Pearson Arwen R, Pompidor Guillaume, Dworkowski Florian, Fuchs Martin R, Furrer Julien, Golczak Marcin, Palczewski Krzysztof, Cascella Michele, Stocker Achim (2014), Human cellular retinaldehyde-binding protein has secondary thermal 9-cis-retinal isomerase activity., in Journal of the American Chemical Society, 136(1), 137-46.

Collaboration

Group / person Country
Types of collaboration
Prof. Christian Grimm/Lab of Retinal Cell Biology, Ophtalmology, USZ, University of Zürich Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure
Prof. Michele Cascella/Department of Chemistry (CTCC), University of Oslo Norway (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Prof. Krzysztof Palczewski/ Department of Pharmacology, Case Western University, Cleveland United States of America (North America)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
Prof. Dimitrios Fotiadis/Institut für Biochemie und Molekulare Medizin, Bühlstrasse 28, CH-3012 Bern Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
Invited Lecture Individual talk Native Human Protein Nanoconstructs with Acquired Transcytocic Properties 29.06.2018 Department of Pharmacology - Western Case University in Cleveland, United States of America Stocker Achim;
Invited lecture Individual talk Native Human Protein Nanoconstructs with Acquired Transcytocic Properties 28.06.2018 WUSTL Ophtalmology - Washington University in St. Louis, United States of America Stocker Achim;
Invited lecture - BioTechMed-Graz Individual talk Native Human Protein Nanoconstructs with Acquired Transcytocic Properties through Endothelial Barriers 08.05.2018 Technische Universität Graz, Austria Stocker Achim;
5th NANO TODAY Conference Talk given at a conference Self-Assembled Alpha-Tocopherol Transfer Protein Nanoparticles Promote Vitamin E Delivery across an Endothelial Barrier 06.12.2017 Hawaii, United States of America Stocker Achim;
Seminar am Institute of Human Nutrition and Food Science Individual talk Homeostasis of α-Tocopherol and of 11-cis-Retinal is Regulated by Proteins of the Sec14 Superfamily 07.11.2016 Kiel, Germany Stocker Achim;


Communication with the public

Communication Title Media Place Year
Talks/events/exhibitions Retinitis Pigmentosa: Was kann Strukturbiologie zur Aufklärung der primären Ursachen beitragen Western Switzerland German-speaking Switzerland 2015
Talks/events/exhibitions Vortragsreihe BioChemie am Samstag: Im Maschinenraum des Lebens German-speaking Switzerland 2014

Patents

Title Date Number Inventor Owner

Use-inspired outputs

Associated projects

Number Title Start Funding scheme
157704 Direct electron detector and phase plate for cryo-transmission electron microscopy of biological samples 01.12.2014 R'EQUIP
130497 Structural and functional characterization of the retinoid visual cycle in the vertebrate eye 01.06.2010 Project funding (Div. I-III)

Abstract

Background: Continuous vision in vertebrates critically depends on two highly evolved regenerative reaction cycles for the transformation of “inactive” all-trans-retinal towards “active” 11-cis-retinal or 11-cis-retinol within the retinal pigment epithelium (RPE) and the Müller cells respectively. The light sensitive visual chromophores 11-cis-retinal/ol are subsequently chaperoned back into rod- and cone-receptor cells by the cellular retinaldehyde binding protein (CRALBP). In mammals, 11-cis-retinal and 11-cis-retinol are the predominant photoactive pigments though thermodynamically least favorable, compared to the other cis-isomers. This dilemma exists throughout rod and also cone-dominated pigment regeneration cycles, both inherently lacking isomerase activities with chemical selectivity. So far, only CRALBP has been reported to provide selectivity by geometric and kinetic restriction of substrate access for 9-cis- and 11-cis-retinoids. Its essential role for chemical purity in cis-retinoid re-flux to opsin receptors is evidenced in the RLBP-/- knockout mouse model accumulating mostly trans-retinoids and suffering from profound delay in regeneration of rhodopsin. Characterization of known clinical gene mutations of human CRALBP have revealed its association with severe phenotypes of retinitis pigmentosa (RP).Working Hypothesis: CRALBP possesses a hydrophobic binding pocket restricting substrate access by shape complementarity towards 9-cis- and 11-cis-retinoids. It is proposed that binding of 11-cis-retinoids to the pocket is governed by opposing functional requirements including photo-protection and geometric selectivity on one hand and efficient substrate release on the other. High affinity binding of 9-cis-retinal to CRALBP affords its aldehyde tail to accommodate in an alternate niche and is compensated for by residual micro-solvation. In the absence of light, bound 9-cis-retinal is quantitatively isomerized in CRALBP to 9,13-dicis-retinal by thermally driven, reversible protonation. Strong modulation of isomeric purity of this isomerase reaction is observed for the Bothnia disease mutant R234W. It is proposed based on these data and the reported expression of CRALBP in non-image-forming tissues that thermal 9-cis-retinal isomerase activity in CRALBP may be association with 9-cis-retinoid specific transport and chemistry. Experiments carried out in our lab have evidenced the formation of CRALBP oligomeric particles bound to 9-cis-retinal with highly increased photo-resistance. In addition, disease-associated RP mutations of CRALBP structurally map onto ataxia (AVED) mutations in the homologous a-TTP, which our group has shown to form 16nm spheres as evidenced by crystallography. In both proteins, these point mutations would disrupt important oligomerization sites within the spheres. The high degree of functional and structural conservation of ataxia and RP mutations strongly suggests that oligomerization may be a conserved functional feature of these proteins and possibly of other SEC14-like lipid carriers.Specific Aims: We aim to understand on a functional level alternate binding of 11-cis- and 9-cis-retinoids by CRALBP and its role in deselecting specific isomers in cyclic pigment regeneration. Retinoid regeneration of the eye has ultimate selectivity for 11-cis-retinoids. Its proper function affords specific retinoid interaction modes of CRALBP and possibly, not yet well understood protein-protein interactions including oligomerization of the protein. We aim to conduct in vitro and in vivo studies complemented by computational methods for: (I) In vitro functional characterization of disease linked CRALBP mutations. (II) The role of CRALBP in the eye’s selectivity for 11-cis-retinoids. (iii) CRALBP’s function as binder and thermal isomerase of 9-cis-retinal. (iv) Biological implications of nano-spherical CRALBP oligomers.Experimental Design: (i) Interactions with retinoid substrates will be assayed in clinically relevant point mutants of CRALBP for photo-resistance and lipid-transfer efficiency. Thermodynamic parameters will be assessed by differential scanning fluorimetry, circular dichroism and complemented by computational studies. (ii) Chemical selectivity of the visual cycle will be studied in vivo in a minimal isomerization cell culture model provided by Prof. Krzysztof Palczewski (Department of Pharmacology, Case Western Reserve University, Cleveland, USA). The cell line will be transiently transfected with CRALBP or its point mutants in combination with the dehydrogenase RDH5. The cellular retinoid isomeric content will be analyzed by HPLC (iii) Polyclonal CRALBP antibodies will be produced and used with magnetic beads to trap and analyze native CRALBP in human and bovine tissues of non- image forming origin. Protein probes will be analyzed by gel-electrophoresis and Western blotting, and the retinoids after extraction by HPLC. Transmission electron microscopy (TEM) imaging of the samples will be carried out in the group of Prof. Dimitrios Fotiadis (IBMM, Bern, Switzerland). (iv) The in vivo role of CRALBP oligomerization and its association with clinical mutants will be studied by the methods highlighted in (i-iii). Complementary in vivo studies using the RPE65-/- mouse model will be carried out in the group of Prof. Christian Grimm (Laboratory of Retinal Cell Biology, Schlieren, Switzerland).Expected Value of Proposed Project: Cis-retinoid specificity in the visual context is highly important from a biomedical point of view, e.g. point mutations in the cis-specific retinoid carrier CRALBP may cause primary inherited retinal disorders and blindness through loss of geometric specificity. Thus, the in vitro and in vivo functions of CRALBP and specifically the role of self induced oligomerization of CRALBP are of high value to the scientific community in order to understand retinoid disorders and, importantly, also for structure-based drug development.
-