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Retinoic Acid Synthesis from Holo-Cellular Retinol Binding Protein in the Somatic Cells of Rat Testis.
No full textProbing the positioning of CRBPs upon phospholipid bilayers interaction by a suite of NMR experiments
No full textVitamin A must be adequately distributed within the mammalian body to produce visual chromophore in the eyes and all-trans-retinoic acid in other tissues. The cellular trafficking and metabolism of vitamin A are regulated primarily by specific high-affinity carriers called CRBP-I and CRBP-II. Both proteins deliver retinol to microsomal membrane-bound enzymes, either for esterification with fatty acids (LRAT) [1, 2] or for oxidation to retinaldehyde (RDH) [3]. Our current understanding of these processes remains incomplete, but there is evidence that the membrane microenvironment plays a role in the interactions of holo CRBPs with enzymes [3].
To address this hypothesis, we have performed a suite of NMR experiments with retinol-bound CRBP-I and CRBP-II in the presence of model membranes composed of either anionic or zwitterionic phospholipids, at varying protein:lipid molar ratios and ionic strength. Besides NMR, other biophysical techniques were employed to achieve a better understanding of the ongoing processes.
TROSY spectra provided insights into the involved protein residues and conformational dynamics. The interaction with the liposomes differs significantly between the two homologous proteins; moreover, it depends upon the phospholipid charge and ionic strength, suggesting an electrostatic nature of the binding.
The data indicate that holo CRBP-I, in contrast to CRBP-II, interacts more strongly with the anionic lipid vesicles and some conformational changes are observed in the ligand entry portal. Nevertheless, there is apparently no protein region embedded inside the bilayer, as judged by H/D exchange measurements.
This study may help to understand certain aspects of the mechanisms of ligand delivery by CRBPs.
References
[1] J. Amengual, M. Golczak, K. Palczewski, and J.von Lintig J. Biol. Chem. 287, 24216-24227 (2012).
[2] W. Jiang and J.L. Napoli Biochim. Biophys. Acta 1820, 859-869 (2012).
[3] J.L. Napoli Biochim. Biophys. Acta 1821, 152-167 (2012)
Interactions of cytoplasmic retinol-binding proteins with phospholipid vesicles: insights into the physiological functions
No full textVitamin A plays a key role in vision, cell growth and differentiation. In the cell, retinol has several fates: (a) it can be stored as retinyl ester of long chain fatty acids through the action of lecithin-retinol acyl transferase (LRAT) [1, 2]; (b) most non-esterified retinol is probably bound to cellular retinol-binding protein I (CRBP-I); (c) retinol can enter the oxidative pathway for the synthesis of retinoic acid, through the action of retinol dehydrogenases (RDH) [3]. CRBP-I is ubiquitous, whereas the homologous CRBP-II is expressed solely in the enterocytes. Our current understanding of these processes remains largely incomplete, but there is evidence that while LRAT is able to access CRBP-bound retinol, the membrane-bound RDH are inactive towards the protein/ligand complex, suggesting that the membrane microenvironment may trigger retinol transfer from the holo protein to the oxidative enzymes.
To address this hypothesis we have performed a suite of NMR experiments with retinol-bound CRBP-I and CRBP-II in the presence of model membranes composed of either anionic or zwitterionic phospholipids, at varying protein:lipid molar ratios and ionic strength. Besides NMR, other biophysical techniques were employed to achieve a better understanding of the ongoing processes.
The results in the presence of phospholipid bilayers will be discussed, in comparison with our previous data collected in buffer [4, 5]. All these studies may help to understand certain aspects of the distinct physiological functions of CRBPs.
References
[1] J. Amengual et al. J. Biol. Chem. 287, 24216-24227 (2012).
[2] W. Jiang and J.L. Napoli Biochim. Biophys. Acta 1820, 859-869 (2012).
[3] S. Portè et al. Chemico-Biological Interactions 202, 186-194 (2013).
[4] T. Mittag, L. Franzoni et al. J. Am. Chem. Soc. 128, 9844-9848 (2006).
[5] L. Franzoni et al. J. Lipid Res. 51, 1332-1343 (2010)
New insights on the protein-ligand interaction differences between the two primary cellular retinol carriers
No full textThe main retinol carriers in the cytosol are the cellular retinol-binding proteins types I and II (CRBP-I, CRBP-II), which exhibit distinct tissue distributions. They play different roles in the maintenance of vitamin A homeostasis and feature a 100-fold difference in retinol affinity whose origin has not been described in detail. NMR-based hydrogen/deuterium exchange measurements show that, while retinol binding endows both proteins with a more rigid structure, many amide protons exchange much faster in CRBP-II than in CRBP-I in both apo and holo form, despite the conserved three-dimensional fold. The remarkable difference in intrinsic stability between the two homologues appears to modulate their binding properties: the stronger retinol-binder CRBP-I displays a reduced flexibility of the backbone structure with respect to CRBP-II. This difference must derive from specific evolution-based amino acid substitutions, resulting in additional stabilization of the CRBP-I scaffold: in fact, we have identified a number of potential salt-bridges on the protein surface as well as several key interactions inside the binding cavity. Furthermore, our NMR data demonstrate that helix alpha-II of the characteristic helix-turn-helix motif in the ligand portal region exists in both apo and holo CRBP-II. As a consequence, the previously proposed model of retinol binding requires revision
Structural stability modulates ligand affinity in cellular retinol-binding proteins types I and II
No full textLigand-binding is modulated by structural stability in the case of the closely homologous cellular retinol-binding proteins type I and II
No full textNew insights on the protein-ligand interaction differences between the two primary cellular retinol carriers
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