Farnesylation and lipid unsaturation are critical for the membrane binding of the C-terminal segment of G-Protein Receptor Kinase 1.

Many proteins are modified by the covalent addition of different types of lipids, such as myristoylation, palmitoylation and prenylation. Lipidation is expected to promote membrane association of proteins. Visual phototransduction involves many lipid-modified proteins. The G-Protein-coupled receptor of rod photoreceptors, rhodopsin, is inactivated by G-Protein-coupled Receptor Kinase 1 (GRK1). The C-terminus of GRK1 is farnesylated and its truncation has been shown to result in a very high decrease of its enzymatic activity, most likely because of the loss of its membrane localization. Little information is available on the membrane binding of GRK1 as well as of most prenylated proteins. Measurements of the membrane binding of the non-farnesylated and farnesylated C-terminal segment of GRK1 were thus performed using lipids typical of those found in rod outer segment disk membranes. Their random coil secondary structure was determined using circular dichroism and infrared spectroscopy. The non-farnesylated C-terminal segment of GRK1 has no surface activity. In contrast, the farnesylated C-terminal segment of GRK1 shows a particularly strong binding to lipid monolayers bearing at least one unsaturated fatty acyl chain. No binding is observed in the presence of monolayers of saturated phospholipids, in agreement with the low affinity of farnesylated Ras proteins for lipids in the liquid-ordered state. Altogether, these data demonstrate that the farnesyl group of the C-terminal segment of GRK1 is mandatory for its membrane binding, which is favored by particular lipids or lipid mixtures. This information will also be useful for the understanding of the membrane binding of other prenylated proteins.

Membrane binding properties of the C-terminal segment of retinol dehydrogenase 8.

Light absorption by rhodopsin leads to the release of all-trans retinal (ATRal) in the lipid phase of photoreceptor disc membranes. Retinol dehydrogenase 8 (RDH8) then reduces ATRal into all-trans retinol, which is the first step of the visual cycle. The membrane binding of RDH8 has been postulated to be mediated by one or more palmitoylated cysteines located in its C-terminus. Different peptide variants of the C-terminus of RDH8 were thus used to obtain information on the mechanism of membrane binding of this enzyme. Steady-state and time-resolved fluorescence measurements were performed using short and long C-terminal segments of bovine RDH8, comprising one or two tryptophan residues. The data demonstrate that the amphipathic alpha helical structure of the first portion of the C-terminus of RDH8 strongly contributes to its membrane binding, which is also favored by palmitoylation of at least one of the cysteines located in the last portion of the C-terminus.

Dengue fusion peptide in Langmuir monolayers: A binding parameter study.

Membrane fusion is known to be the primary mechanism of entry of flaviviruses into host cells. Several studies reported the investigation of the membrane fusion mechanism mediated by the fusion peptide, a component of the membrane protein surrounding the flaviviruses. In this study, we investigated the interaction of Dengue fusion peptide (FLAg) with Langmuir monolayers to uncover the role of membrane charges and organization in its membrane binding. Binding parameters of FLAg were obtained by measuring its adsorption onto Langmuir monolayers of different types of individual lipids, as well as their mixtures. Specific peptide binding was observed in the presence of charged lipid monolayers at different pHs, revealing that the lipid composition of the membrane modulates peptide interaction, and the preference of the peptide for negatively charged lipids.

Structural information and membrane binding of truncated RGS9-1 Anchor Protein and its C-terminal hydrophobic segment.

Visual phototransduction takes place in photoreceptor cells. Light absorption by rhodopsin leads to the activation of transducin as a result of the exchange of its GDP for GTP. The GTP-bound ⍺-subunit of transducin then activates phosphodiesterase (PDE), which in turn hydrolyzes cGMP leading to photoreceptor hyperpolarization. Photoreceptors return to the dark state upon inactivation of these proteins. In particular, PDE is inactivated by the protein complex R9AP/RGS9-1/Gß5. R9AP (RGS9-1 anchor protein) is responsible for the membrane anchoring of this protein complex to photoreceptor outer segment disk membranes most likely by the combined involvement of its C-terminal hydrophobic domain as well as other types of interactions. This study thus aimed to gather information on the structure and membrane binding of the C-terminal hydrophobic segment of R9AP as well as of truncated R9AP (without its C-terminal domain, R9AP∆TM). Circular dichroism and infrared spectroscopic measurements revealed that the secondary structure of R9AP∆TM mainly includes ⍺-helical structural elements. Moreover, intrinsic fluorescence measurements of native R9AP∆TM and individual mutants lacking one tryptophan demonstrated that W79 is more buried than W173 but that they are both located in a hydrophobic environment. This method also revealed that membrane binding of R9AP∆TM does not involve regions near its tryptophan residues, while infrared spectroscopy validated its binding to lipid vesicles. Additional fluorescence measurements showed that the C-terminal segment of R9AP is membrane embedded. Maximum insertion pressure and synergy data using Langmuir monolayers suggest that interactions with specific phospholipids could be involved in the membrane binding of R9AP∆TM.

Comparison between the enzymatic activity, structure and substrate binding of mouse and human lecithin retinol acyltransferase.

Lecithin retinol acyltransferase (LRAT) is involved in the visual cycle where it catalyzes the formation of all-trans retinyl ester. The mouse animal model has been widely used to study LRAT. Primary sequence alignment shows 80% identity and 90% similarity between human and mouse LRAT. However, human LRAT has a proline at position 173 (hLRAT (P173)) while an arginine can be found at this position for the mouse protein (mLRAT (R173)). Moreover, residue 173 is important for the human protein since a substitution mutation of this residue to a leucine (P173L-hLRAT) caused night blindness in a patient. The present study was thus undertaken to determine whether mouse and human LRAT have a similar enzymatic activity, structure and substrate binding affinity using a truncated form of LRAT (tLRAT). The enzymatic activity and binding affinity to the substrate, all-trans retinol, of mtLRAT (R173) were found to be 2.7- and 3.9-fold lower, respectively, than that of htLRAT (P173). Moreover, the enzymatic activity of P173L-htLRAT is 6.3-fold lower compared to that of htLRAT (P173). Furthermore, a significant difference was observed between the intrinsic fluorescence emission as well as between the circular dichroism spectra of mtLRAT (R173) and htLRAT (P173). In addition, mtLRAT proteins are less thermostable than htLRAT proteins, which suggests that structural differences exist between the mouse and human proteins. Altogether, these data strongly suggest that the much lower catalytic activity of mtLRAT (R173) compared to that of htLRAT (P173) mostly results from differences between their structure, predominantly revealed by their dissimilar thermal stability, as well as their efficiency to bind all-trans retinol. Therefore, conclusions regarding the behavior of human LRAT based on measurements performed with mouse LRAT must be made with caution. Also, the much lower enzymatic activity of P173L-htLRAT compared to that of htLRAT (P173) might explain the night blindness of a patient carrying this mutation.

Identification of an alternative translation initiation site in the sequence of the commonly used Glutathione S-Transferase tag.

A large number of proteins are expressed in fusion with a tag to perform their purification. Glutathione-S-Transferase (GST) is a widely used tag to achieve passenger protein purification. Accordingly, commonly used commercial expression vectors contain the coding sequence of GST in order to express fusion proteins. However, fusion proteins are sometimes expressed in a truncated form that may result from an incomplete synthesis, proteolytic cleavage or the presence of a functional alternative translation initiation site. In particular, a truncated as well as a full-length fusion protein were observed when expressing RGS9-1 Anchor Protein without its C-terminal segment (bR9AP) in fusion with GST. Moreover, this truncated protein was found to be purified together with the full-length fusion protein. Here, we identified for the first time an alternative translation initiation site within the sequence of GST that likely becomes accessible for translation only when it is fused with a passenger protein. Indeed, bioinformatics analyses suggest that the secondary structure of the mRNA of the GST-bR9AP fusion protein is different from that of GST alone, which likely allows accessibility of an alternative Shine-Dalgarno sequence coupled with an additional initiation codon within the sequence of GST. The functionality of this alternative translation initiation site was confirmed by site-directed mutagenesis, which resulted in the absence of a truncated fusion protein and, consequently, only a purified full-length fusion protein. This is an extremely important finding in view of the wide use of GST as a purification and solubility-enhancing tag.

Systematic analysis of the expression, solubility and purification of a passenger protein in fusion with different tags.

Purification of recombinant proteins is often achieved using a purification tag which can be located either at the N- or C-terminus of a passenger protein of interest. Many purification tags exist and their advantages and limitations are well documented. However, designing fusion proteins can be a challenging task to get a fully expressed, soluble and highly purified passenger protein. Besides, there is a lack of systematic studies on the use of a single tag versus combined tags and on the effect of the position of the tags in the construct. In the present study, 9 different fusion proteins were expressed in Escherichia coli using some of the most commonly used purification tags: maltose-binding protein (MBP), glutathione S-transferase (GST) and polyHis tag. The expression and purification of N-terminus single-tagged fusion proteins (MBP, GST and polyHis) and fusion proteins with combined tags at different positions have been tested. Both the identity of the tag(s) and its position were found to have a strong effect on the expression, solubility and purification yields of the fusion proteins. Consequently, the different fusion proteins assayed have shown varying expression, solubility and purification yields, which were also dependent on the passenger protein. Therefore, there is a compelling need to design various fusion proteins with different single or combined tags to identify optimized constructions allowing to achieve high levels of expression, solubility and purification of the passenger protein.

Novel approaches to probe the binding of recoverin to membranes.

Recoverin is a protein involved in the phototransduction cascade by regulating the activity of rhodopsin kinase through a calcium-dependent binding process at the surface of rod outer segment disk membranes. We have investigated the interaction of recoverin with zwitterionic phosphatidylcholine bilayers, the major lipid component of the rod outer segment disk membranes, using both 31P and 19F solid-state nuclear magnetic resonance (NMR) and infrared spectroscopy. In particular, several novel approaches have been used, such as the centerband-only detection of exchange (CODEX) technique to investigate lipid lateral diffusion and 19F NMR to probe the environment of the recoverin myristoyl group. The results reveal that the lipid bilayer organization is not disturbed by recoverin. Non-myristoylated recoverin induces a small increase in lipid hydration that appears to be correlated with an increased lipid lateral diffusion. The thermal stability of recoverin remains similar in the absence or presence of lipids and Ca2+. Fluorine atoms have been strategically introduced at positions 4 or 12 on the myristoyl moiety of recoverin to, respectively, probe its behavior in the interfacial and more hydrophobic regions of the membrane. 19F NMR results allow the observation of the calcium-myristoyl switch, the myristoyl group experiencing two different environments in the absence of Ca2+ and the immobilization of the recoverin myristoyl moiety in phosphatidylcholine membranes in the presence of Ca2+.

The hydrophobic region of the Leishmania peroxin 14: requirements for association with a glycosome mimetic membrane.

Protein import into the Leishmania glycosome requires docking of the cargo-loaded peroxin 5 (PEX5) receptor to the peroxin 14 (PEX14) bound to the glycosome surface. To examine the LdPEX14-membrane interaction, we purified L. donovani promastigote glycosomes and determined the phospholipid and fatty acid composition. These membranes contained predominately phosphatidylethanolamine, phosphatidylcholine, and phosphatidylglycerol (PG) modified primarily with C18 and C22 unsaturated fatty acid. Using large unilamellar vesicles (LUVs) with a lipid composition mimicking the glycosomal membrane in combination with sucrose density centrifugation and fluorescence-activated cell sorting technique, we established that the LdPEX14 membrane-binding activity was dependent on a predicted transmembrane helix found within residues 149-179. Monolayer experiments showed that the incorporation of PG and phospholipids with unsaturated fatty acids, which increase membrane fluidity and favor a liquid expanded phase, facilitated the penetration of LdPEX14 into biological membranes. Moreover, we demonstrated that the binding of LdPEX5 receptor or LdPEX5-PTS1 receptor-cargo complex was contingent on the presence of LdPEX14 at the surface of LUVs.

Membrane fluidity is a driving force for recoverin myristoyl immobilization in zwitterionic lipids.

Recoverin is the only protein for which the phenomenon of calcium-myristoyl switch has been demonstrated without ambiguity. It is located in rod disk membranes where the highest content in polyunsaturated lipid acyl chains can be found. However, although essential to better understand the inactivation of the phototransduction process, the role of membrane fluidity on recoverin recruitment is unclear. We have therefore investigated the immobilization of the recoverin myristoyl moiety in the presence of phosphocholine bilayers using 2H solid-state NMR spectroscopy. Several lipids with different acyl chains were selected to investigate model membranes characterized by different fluidity. Immobilization of the recoverin myristoyl moiety was successfully observed but only in the presence of calcium and in specific lipid disordered states, showing that an optimal fluidity is required for recoverin immobilization.

How to gather useful and valuable information from protein binding measurements using Langmuir lipid monolayers.

This review presents data on the influence of various experimental parameters on the binding of proteins onto Langmuir lipid monolayers. The users of the Langmuir methodology are often unaware of the importance of choosing appropriate experimental conditions to validate the data acquired with this method. The protein Retinitis pigmentosa 2 (RP2) has been used throughout this review to illustrate the influence of these experimental parameters on the data gathered with Langmuir monolayers. The methods detailed in this review include the determination of protein binding parameters from the measurement of adsorption isotherms, infrared spectra of the protein in solution and in monolayers, ellipsometric isotherms and fluorescence micrographs.

Phosphatidylserine Allows Observation of the Calcium-Myristoyl Switch of Recoverin and Its Preferential Binding.

Recoverin undergoes a calcium-myristoyl switch during visual phototransduction. Indeed, calcium binding by recoverin results in the extrusion of its myristoyl group, which allows its membrane binding. However, the contribution of particular lipids and of specific amino acids of recoverin in its membrane binding has not yet been demonstrated. In the present work, the affinity of recoverin for the negatively charged phosphatidylserine has been clearly shown to be governed by a cluster of positively charged residues located in its N-terminal segment. Moreover, the calcium-myristoyl switch of recoverin was only observed upon binding onto monolayers of phosphatidylserine and not in the case of other anionic phospholipids. Fluorescence microscopy experiments with mixed lipid monolayers allowed confirmation of the specific binding of myristoylated recoverin to phosphatidylserine, whereas the extent of penetration of recoverin in phosphatidylserine monolayers was estimated by ellipsometry. A model has thus been proposed for the membrane binding of myristoylated recoverin in the presence of calcium.

Discriminating Lipid- from Protein-Calcium Binding To Understand the Interaction between Recoverin and Phosphatidylglycerol Model Membranes.

Recoverin is a protein involved in the phototransduction cascade by regulating the activity of rhodopsin kinase through a calcium-dependent binding process at the surface of rod outer segment disk membranes. Understanding how calcium modulates these interactions and how it interacts with anionic lipid membranes is necessary to gain insights into the function of recoverin. In this work, infrared spectroscopy allowed us to show that the availability of calcium to recoverin is modulated by the presence of complexes involving phosphatidylglycerol (PG), which in turn regulates its interactions with this negatively charged lipid. Calcium can indeed be sequestered into strongly bound complexes with PG and is thus sparingly available to recoverin. The thermal stability of recoverin then decreases, which results in weakened interactions with PG. By contrast, when calcium is fully available to recoverin, the protein is thermally stable, indicating that it binds two calcium ions, which results in favorable interactions with negatively charged lipids. Consequently, the protein induces an increase in the chain-melting phase transition temperature of PG, which is indicative of an enhanced lipid chain packing resulting from the peripheral location of the protein. The secondary structure of recoverin is not affected by its interactions with anionic membrane lipids. Similar results have been obtained with saturated and unsaturated anionic lipids. This work shows that the recruitment of recoverin at the surface of anionic lipid membranes is dependent on the availability of calcium.

Functional Impact of Collagens on the Activity Directed by the Promoter of the α5 Integrin Subunit Gene in Corneal Epithelial Cells.

PURPOSE: The early step of corneal wound healing is characterized by the massive production of fibronectin (FN), whose secretion is progressively replaced by collagens from the basal membrane as wound healing proceeds. Here, we examined whether expression of the gene encoding the α5 subunit from the FN-binding integrin α5ß1 changes as corneal epithelial cells (CECs) are cultured in the presence of collagen type I (CI) or type IV (CIV). METHODS: Responsiveness of the α5 gene toward collagen was determined by transfection of α5 promoter/chloramphenicol acetyltransferase (CAT) plasmids into rabbit and human CECs cultured on BSA or collagens. Electrophoretic mobility shift assays and Western blots were used to monitor the transcription factors required for basal α5 gene transcription in the presence of collagens. Gene profiling on microarrays was used to determine the impact of collagens on the patterns of genes expressed by CECs. RESULTS: All collagen types repressed the full-length α5/CAT promoter activity in confluent CECs. A moderate increase was observed in subconfluent rabbit CECs grown on CIV but not on CI. These collagen-dependent regulatory influences also correlated with alterations in the transcription factors Sp1/Sp3, NFI, and AP-1 that ensure α5 gene basal transcription. Microarray analyses revealed that CI more profoundly altered the pattern of genes expressed by human CECs than CIV. CONCLUSIONS: Collagens considerably suppressed α5 gene expression in CECs, suggesting that during wound healing, they may interfere with the influence FN exerts on CECs by altering their adhesive and migratory properties through a mechanism involving a reduction in α5 gene expression.

Lipid Selectivity, Orientation, and Extent of Membrane Binding of Nonacylated RP2.

Retinitis pigmentosa 2 (RP2) is an ubiquitary protein of 350 residues. The N-terminus of RP2 contains putative sites of myristoylation and palmitoylation. The dually acylated protein is predominantly localized to the plasma membrane. However, clinically occurring substitution mutations of RP2 in photoreceptors lead to the expression of a nonacylated protein, which was shown to be misrouted to intracellular organelles using different cell lines. However, the parameters responsible for the modulation of the membrane binding of nonacylated RP2 (naRP2) are still largely unknown. The maximal insertion pressure of naRP2 has thus been determined after its injection into the subphase underneath monolayers of phospholipids, which are typical of photoreceptor membranes. These data demonstrated that naRP2 shows a preferential binding to saturated phospholipid monolayers. Moreover, polarization modulation infrared reflection absorption spectroscopy has allowed comparison of the secondary structure of this protein in solution and upon binding to phospholipid monolayers. In addition, simulations of these spectra have allowed to determine that the ß-helix of naRP2 has an orientation of 60° with respect to the normal, which remains unchanged regardless of the type of phospholipid. Finally, ellipsometric measurements of naRP2 demonstrated that its particular affinity for saturated phospholipids can be explained by its larger extent of insertion in this phospholipid monolayer compared to that in polyunsaturated phospholipid monolayers.

Characterization of the human α9 integrin subunit gene: Promoter analysis and transcriptional regulation in ocular cells.

α9ß1 is the most recent addition to the integrin family of membrane receptors and consequently remains the one that is the least characterized. To better understand how transcription of the human gene encoding the α9 subunit is regulated, we cloned the α9 promoter and characterized the regulatory elements that are required to ensure its transcription. Transfection of α9 promoter/CAT plasmids in primary cultured human corneal epithelial cells (HCECs) and uveal melanoma cell lines demonstrated the presence of both negative and positive regulatory elements along the α9 promoter and positioned the basal α9 promoter to within 118 bp from the α9 mRNA start site. In vitro DNaseI footprinting and in vivo ChIP analyses demonstrated the binding of the transcription factors Sp1, c-Myb and NFI to the most upstream α9 negative regulatory element. The transcription factors Sp1 and NFI were found to bind the basal α9 promoter individually but Sp1 binding clearly predominates when both transcription factors are present in the same extract. Suppression of Sp1 expression through RNAi also caused a dramatic reduction in the expression of the α9 gene. Most of all, addition of tenascin-C (TNC), the ligand of α9ß1, to the tissue culture plates prior to seeding HCECs increased α9 transcription whereas it simultaneously decreased expression of the α5 integrin subunit gene. This dual regulatory action of TNC on the transcription of the α9 and α5 genes suggests that both these integrins must work together to appropriately regulate cell adhesion, migration and differentiation that are hallmarks of tissue wound healing.

Structure and binding of the C-terminal segment of R9AP to lipid monolayers.

Phototransduction cascade takes place in disc membranes of photoreceptor cells. Following its activation by light, rhodopsin activates the G-protein transducin causing the dissociation of its GTP-bound α-subunit, which in turn activates phosphodiesterase 6 (PDE6) leading to the hyperpolarization of photoreceptor cells. PDE6 must then be inactivated to return to the dark state. This is achieved by a protein complex which is presumably anchored to photoreceptor disc membranes by means of the transmembrane C-terminal segment of RGS9-1-Anchor Protein (R9AP). Information on the secondary structure and membrane binding properties of the C-terminal segment of R9AP is not yet available to further support its role in the membrane anchoring of this protein. In the present study, circular dichroism and infrared spectroscopy measurements have allowed us to determine that the C-terminal segment of human and bovine R9AP adopts an α-helical structure in solution. Moreover, this C-terminal segment has shown affinity for most of the phospholipids typical of photoreceptor membranes. In fact, the physical state and the type of phospholipid as well as electrostatic interactions influence the binding of the human and bovine peptides to phospholipid monolayers. In addition, these measurements revealed that the human peptide has a high affinity for saturated phosphocholine, which may suggest a possible localization of R9AP in photoreceptor microdomains. Accordingly, infrared spectroscopy measurements have allowed determining that the C-terminal segment of R9AP adopts an ordered α-helical structure in the presence of saturated phospholipid monolayers. Altogether, these data are consistent with the typical α-helical secondary structure and behavior observed for transmembrane segments and with the proposed role of membrane anchoring of the C-terminal segment of human and bovine R9AP.

Structure of the N-terminal segment of human retinol dehydrogenase 11 and its preferential lipid binding using model membranes.

Retinol dehydrogenase 11 (RDH11) has been postulated to be anchored to membranes by means of its N-terminal segment in retinal pigment epithelial (RPE) cells where it participates to the visual cycle. The analysis of the primary sequence of RDH11 revealed that its N-terminal hydrophobic segment could be involved in the anchoring of this enzyme to membranes. However, no information is yet available on the properties of this N-terminal segment to support this role. The secondary structure and membrane binding of two N-terminal peptides of RDH11 with different lengths have thus been investigated to provide this information. Online tools allowed predicting an α-helical secondary structure for both peptides. Infrared spectroscopy and circular dichroism have shown that the α-helix of the Long-peptide (35 amino acids) is longer and more rigid than that of the Short-peptide (25 amino acids) regardless of the type of solvent. Langmuir monolayers have been used as a model membrane to study lipid-peptide interactions. Values of maximum insertion pressure and synergy suggested a preferential binding of the Long-peptide to lipids with a phosphoethanolamine polar head group, which are abundant in the RPE. Furthermore, infrared spectroscopy in monolayers has shown that the α-helical structure of the Long-peptide is more stable in the presence of saturated phospholipids whereas the structure of the Short-peptide is mainly disordered. Altogether, the present data demonstrate that the α-helical hydrophobic core of the N-terminal segment of RDH11 displays properties typical of transmembrane domains, in agreement with its postulated role in the membrane anchoring of this protein.

Retinol dehydrogenases: membrane-bound enzymes for the visual function.

Retinoid metabolism is important for many physiological functions, such as differenciation, growth, and vision. In the visual context, after the absorption of light in rod photoreceptors by the visual pigment rhodopsin, 11-cis retinal is isomerized to all-trans retinal. This retinoid subsequently undergoes a series of modifications during the visual cycle through a cascade of reactions occurring in photoreceptors and in the retinal pigment epithelium. Retinol dehydrogenases (RDHs) are enzymes responsible for crucial steps of this visual cycle. They belong to a large family of proteins designated as short-chain dehydrogenases/reductases. The structure of these RDHs has been predicted using modern bioinformatics tools, which allowed to propose models with similar structures including a common Rossman fold. These enzymes undergo oxidoreduction reactions, whose direction is dictated by the preference and concentration of their individual cofactor (NAD(H)/NADP(H)). This review presents the current state of knowledge on functional and structural features of RDHs involved in the visual cycle as well as knockout models. RDHs are described as integral or peripheral enzymes. A topology model of the membrane binding of these RDHs via their N- and (or) C-terminal domain has been proposed on the basis of their individual properties. Membrane binding is a crucial issue for these enzymes because of the high hydrophobicity of their retinoid substrates.