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