ABSTRACT
INTRODUCTION: Phosphatidylinositol-4,5-bisphosphate (PIP2) is a cofactor necessary for the activity of KCNQ1 channels. Some Long QT mutations of KCNQ1, including R243H, R539W and R555C have been shown to decrease KCNQ1 interaction with PIP2. A previous study suggested that R539W is paradoxically less sensitive to intracellular magnesium inhibition than the WT channel, despite a decreased interaction with PIP2. In the present study, we confirm this peculiar behavior of R539W and suggest a molecular mechanism underlying it. METHODS AND RESULTS: COS-7 cells were transfected with WT or mutated KCNE1-KCNQ1 channel, and patch-clamp recordings were performed in giant-patch, permeabilized-patch or ruptured-patch configuration. Similar to other channels with a decreased PIP2 affinity, we observed that the R243H and R555C mutations lead to an accelerated current rundown when membrane PIP2 levels are decreasing. As opposed to R243H and R555C mutants, R539W is not more but rather less sensitive to PIP2 decrease than the WT channel. A molecular model of a fragment of the KCNQ1 C-terminus and the membrane bilayer suggested that a potential novel interaction of R539W with cholesterol stabilizes the channel opening and hence prevents rundown upon PIP2 depletion. We then carried out the same rundown experiments under cholesterol depletion and observed an accelerated R539W rundown that is consistent with this model. CONCLUSIONS: We show for the first time that a mutation may shift the channel interaction with PIP2 to a preference for cholesterol. This de novo interaction wanes the sensitivity to PIP2 variations, showing that a mutated channel with a decreased affinity to PIP2 could paradoxically present a slowed current rundown compared to the WT channel. This suggests that caution is required when using measurements of current rundown as an indicator to compare WT and mutant channel PIP2 sensitivity.
Subject(s)
Cholesterol/metabolism , KCNQ1 Potassium Channel/metabolism , Long QT Syndrome/genetics , Mutation/genetics , Phosphatidylinositol 4,5-Diphosphate/metabolism , Animals , Arrhythmias, Cardiac/genetics , Arrhythmias, Cardiac/metabolism , Brugada Syndrome , COS Cells , Cardiac Conduction System Disease , Cell Line , Chlorocebus aethiops , Cholesterol/genetics , Heart Conduction System/abnormalities , Heart Conduction System/metabolism , KCNQ1 Potassium Channel/genetics , Long QT Syndrome/metabolism , Magnesium/metabolism , Phosphatidylinositol 4,5-Diphosphate/geneticsABSTRACT
Diffuse bronchiectasis is a common problem in respiratory clinics. We hypothesized that mutations in the solute carrier 26A9 (SLC26A9) gene, encoding for a chloride (Cl(-)) transporter mainly expressed in lungs, may lead to defects in mucociliary clearance. We describe two missense variants in the SLC26A9 gene in heterozygote patients presenting with diffuse idiopathic bronchiectasis : p.Arg575Trp, identified in a patient also heterozygote for p.Phe508del in the CFTR gene; and p.Val486Ile. Expression of both mutants in Xenopus laevis oocytes abolished SLC26A9-mediated Cl(-) conductance without decreasing protein membrane expression. Coexpression of CFTR with SLC26A9-p.Val486Ile resulted in a significant increase in the Cl(-) current induced by PKA stimulation, similar to that obtained in oocytes expressing CFTR and SLC26A9-WT. In contrast, coexpression of CFTR with SLC26A9-p.Arg575Trp inhibited SLC26A9-enhanced CFTR activation upon PKA. Further structure-function analyses led us to propose a site encompassing Arg575 in the SLC26A9-STAS domain for CFTR-SLC26A9 interaction. We hypothesize that SLC26A9-p.Arg575Trp prevented SLC26A9-mediated functional activation of CFTR by altering SLC26A9-CFTR interaction. Although we cannot confirm that these mutations by themselves are deleterious, we propose that they trigger the pathogenic role of a single CFTR mutation and provide insight into a novel mechanism of Cl(-) transport alteration across the respiratory mucosa, based on functional inhibition of CFTR.
Subject(s)
Antiporters/genetics , Lung Diseases/diagnosis , Lung Diseases/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Animals , Antiporters/chemistry , Antiporters/metabolism , Case-Control Studies , Child , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Exons , Female , Gene Expression , Humans , Lung Diseases/pathology , Male , Middle Aged , Mutation , Oocytes/metabolism , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Phenotype , Protein Binding , Protein Interaction Domains and Motifs , Sulfate Transporters , Tomography, X-Ray Computed , Xenopus laevis , Young AdultABSTRACT
CADY is a cell-penetrating peptide spontaneously making non-covalent complexes with Short interfering RNAs (siRNAs) in water. Neither the structure of CADY nor that of the complexes is resolved. We have calculated and analyzed 3D models of CADY and of the non-covalent CADY-siRNA complexes in order to understand their formation and stabilization. Data from the ab initio calculations and molecular dynamics support that, in agreement with the experimental data, CADY is a polymorphic peptide partly helical. Taking into consideration the polymorphism of CADY, we calculated and compared several complexes with peptide/siRNA ratios of up to 40. Four complexes were run by using molecular dynamics. The initial binding of CADYs is essentially due to the electrostatic interactions of the arginines with siRNA phosphates. Due to a repetitive arginine motif (XLWR(K)) in CADY and to the numerous phosphate moieties in the siRNA, CADYs can adopt multiple positions at the siRNA surface leading to numerous possibilities of complexes. Nevertheless, several complex properties are common: an average of 14±1 CADYs is required to saturate a siRNA as compared to the 12±2 CADYs experimentally described. The 40 CADYs/siRNA that is the optimal ratio for vector stability always corresponds to two layers of CADYs per siRNA. When siRNA is covered by the first layer of CADYs, the peptides still bind despite the electrostatic repulsion. The peptide cage is stabilized by hydrophobic CADY-CADY contacts thanks to CADY polymorphism. The analysis demonstrates that the hydrophobicity, the presence of several positive charges and the disorder of CADY are mandatory to make stable the CADY-siRNA complexes.
Subject(s)
Cell-Penetrating Peptides/chemistry , Peptides/chemistry , RNA, Small Interfering/metabolism , Amino Acid Motifs , Arginine/chemistry , Genetic Vectors , Hydrophobic and Hydrophilic Interactions , Models, Molecular , Molecular Conformation , Molecular Dynamics Simulation , Protein Binding , Protein Structure, Secondary , Static Electricity , Thermodynamics , Time FactorsSubject(s)
Cell-Penetrating Peptides/chemistry , Drug Carriers/chemistry , Nanoparticles/chemistry , Peptides/chemistry , RNA, Small Interfering/chemistry , Animals , Cell Line , Cell Membrane Permeability , Humans , Jurkat Cells , Nanoparticles/administration & dosage , RNA, Small Interfering/administration & dosageABSTRACT
We extended the use of Peplook, an in silico procedure for the prediction of three-dimensional (3D) models of linear peptides to the prediction of 3D models of cyclic peptides and thanks to the ab initio calculation procedure, to the calculation of peptides with non-proteinogenic amino acids. Indeed, such peptides cannot be predicted by homology or threading. We compare the calculated models with NMR and X-ray models and for the cyclic peptides, with models predicted by other in silico procedures (Pep-Fold and I-Tasser). For cyclic peptides, on a set of 38 peptides, average root mean square deviation of backbone atoms (BB-RMSD) was 3.8 and 4.1 Å for Peplook and Pep-Fold, respectively. The best results are obtained with I-Tasser (2.5 Å) although evaluations were biased by the fact that the resolved Protein Data Bank models could be used as template by the server. Peplook and Pep-Fold give similar results, better for short (up to 20 residues) than for longer peptides. For peptides with non-proteinogenic residues, performances of Peplook are sound with an average BB-RMSD of 3.6 Å for 'non-natural peptides' and 3.4 Å for peptides combining non-proteinogenic residues and cyclic structure. These results open interesting possibilities for the design of peptidic drugs.
Subject(s)
Computer Simulation , Models, Molecular , Peptides, Cyclic/chemistry , Software , Algorithms , Amino Acid Sequence , Drug Design , Drug Discovery , Molecular Sequence Data , Protein Folding , Protein Structure, SecondaryABSTRACT
Caveolin-1 has a segment of hydrophobic amino acids comprising approximately residues 103-122. We have performed an in silico analysis of the conformational preference of this segment of caveolin-1 using PepLook. We find that there is one main group of stable conformations corresponding to a hydrophobic U bent model that would not traverse the membrane. Furthermore, the calculations predict that substituting the Pro(110) residue with an Ala will change the conformation to a straight hydrophobic helix that would traverse the membrane. We have expressed the P110A mutant of caveolin-1, with a FLAG tag at the N terminus, in HEK 293 cells. We evaluate the topology of the proteins with confocal immunofluorescence microscopy in these cells. We find that FLAG tag at the N terminus of the wild type caveolin-1 is not reactive with antibodies unless the cell membrane is permeabilized with detergent. This indicates that in these cells, the hydrophobic segment of this protein is not transmembrane but takes up a bent conformation, making the protein monotopic. In contrast, the FLAG tag at the N terminus of the P110A mutant is equally exposed to antibodies, before and after membrane permeabilization. We also find that the P110A mutation causes a large reduction of endocytosis of caveolae, cellular lipid accumulation, and lipid droplet formulation. In addition, we find that this mutation markedly reduces the ability of caveolin-1 to form structures with the characteristic morphology of caveolae or to partition into the detergent-resistant membranes of these cells. Thus, the single Pro residue in the membrane-inserting segment of caveolin-1 plays an important role in both the membrane topology and localization of the protein as well as its functions.
Subject(s)
Caveolae/metabolism , Caveolin 1/metabolism , Endocytosis/physiology , Proline/metabolism , Amino Acid Substitution , Animals , Caveolin 1/genetics , Cell Line , Humans , Mice , Mutation, Missense , Proline/genetics , Protein Structure, SecondaryABSTRACT
Delivery of siRNA remains a major limitation to their clinical application, and several technologies have been proposed to improve their cellular uptake. We recently described a peptide-based nanoparticle system for efficient delivery of siRNA into primary cell lines: CADY. CADY is a secondary amphipathic peptide that forms stable complexes with siRNA and improves their cellular uptake independently of the endosomal pathway. In the present work, we have combined molecular modeling, spectroscopy, and membrane interaction approaches in order to gain further insight into CADY/siRNA particle mechanism of interaction with biological membrane. We demonstrate that CADY forms stable complexes with siRNA and binds phospholipids tightly, mainly through electrostatic interactions. Binding to siRNA or phospholipids triggers a conformational transition of CADY from an unfolded state to an alpha-helical structure, thereby stabilizing CADY/siRNA complexes and improving their interactions with cell membranes. Therefore, we propose that CADY cellular membrane interaction is driven by its structural polymorphism which enables stabilization of both electrostatic and hydrophobic contacts with surface membrane proteoglycan and phospholipids.
Subject(s)
Peptides/chemistry , RNA, Small Interfering/genetics , Amino Acid Sequence , Animals , Base Sequence , Biological Transport , Cell Line , Cell Membrane/metabolism , Circular Dichroism , Glycosaminoglycans/chemistry , Glycosaminoglycans/metabolism , Liposomes/chemistry , Liposomes/metabolism , Micelles , Models, Molecular , Molecular Sequence Data , Normal Distribution , Oligoribonucleotides/chemistry , Peptides/chemical synthesis , Peptides/metabolism , Phospholipids/chemistry , Phospholipids/metabolism , Protein Conformation , Protein Structure, Secondary/genetics , RNA, Small Interfering/metabolismABSTRACT
Biological membranes are complex structures composed largely of proteins and lipids. These components have very different structural and physical properties and consequently they do not form a single homogeneous mixture. Rather components of the mixture are more enriched in some regions than in others. This can be demonstrated with simple lipid mixtures that spontaneously segregate components so as to form different lipid phases that are immiscible with one another. The segregation of molecular components of biological membranes also involves proteins. One driving force that would promote the segregation of membrane components is the preferential interaction between a protein and certain lipid components. Among the varied lipid components of mammalian membranes, the structure and physical properties of cholesterol is quite different from that of other major membrane lipids. It would therefore be expected that in many cases proteins would have very different energies of interaction with cholesterol vs. those of other membrane lipids. This would be sufficient to cause segregation of components in membranes. The factors that facilitate the interaction of proteins with cholesterol are varied and are not yet completely understood. However, there are certain groups that are present in some proteins that facilitate interaction of the protein with cholesterol. These groups include saturated acyl chains of lipidated proteins, as well as certain amino acid sequences. Although there is some understanding as to why these particular groups favour interaction with cholesterol, our knowledge of these molecular features is not sufficiently developed to allow for the design of agents that will modify such binding.
Subject(s)
Cholesterol/metabolism , Membrane Microdomains/metabolism , Membrane Proteins/metabolism , Animals , Humans , Intracellular Signaling Peptides and Proteins/metabolism , Intracellular Signaling Peptides and Proteins/physiology , Lipid Bilayers/chemistry , Membrane Lipids/metabolism , Membranes, Artificial , Models, MolecularABSTRACT
Transfers of cargoes into cells by means of carrier peptides are multi-steps biological phenomenon the mechanisms of which are unclear. We here discuss bases of realistic in silico molecular modeling approaches of the formation of non-covalent complexes considering CPPs and cargo diversities.
Subject(s)
Cell-Penetrating Peptides/chemistry , Computer Simulation , Models, Molecular , Animals , Humans , Structure-Activity RelationshipABSTRACT
We compare mean force potential values of a large series of PDB models of proteins and peptides and find that, either as monomers or polymers, proteins longer than 200-250 residues have equivalent MFP values that are averaged to -65+/-3 kcal/aa. This value is named the standard or stability value. The standard value is reached irrespective of sequences and 3D folds. Peptides are too short to follow the rule and frequently exist as populations of conformers; one exception is peptides in amyloid fibrils. Fibrils surpass the standard value in accordance with their uppermost stability. In parallel, we calculate median MFP values of amino acids in stably folded PDB models of proteins: median values vary from -25 for Gly to -115 kcal/aa for Trp. These median values are used to score primary sequences of proteins: all sequences converge to a mean value of -63.5+/-2.5 kcal/aa, i.e., only 1.5 kcal less than the folded model standard. Sequences from unfolded proteins have lower values. This supports the conclusion that sequences carry in an important message and more specifically that diversity of amino acids in sequences is mandatory for stability. We also use the median amino acid MFP to score residue stability in 3D folds. This demonstrates that 3D folds are compromises between fragments of high and fragments of low scores and that functional residues are often but not always in the extreme score values. The approach opens to possibilities of evaluating any 3D model and of detecting functional residues and should help in conducting mutation assays.
Subject(s)
Models, Molecular , Protein Folding , Protein Stability , Proteins/chemistry , Amino Acid Sequence , Databases, Protein , Mutagenesis , Mutation , Protein Structure, Quaternary , Protein Structure, Tertiary , Proteins/geneticsABSTRACT
Blends of anhydrous milk fat (AMF) and linseed oil (70:30) and of AMF, rapeseed oil (RO), and linseed oil (LO) (70:20:10) were submitted to enzymatic interesterification. The oxidative stabilities of the blends, the interesterified (IE) blends, and IE blends with 50 ppm of alpha-tocopherol added as antioxidant were studied. Samples were stored in open flasks at 60, 25, and 4 degrees C and periodically submitted to peroxide, p-anisidine, and TBA value determinations and UV measurement at 232 and 268 nm. The analysis of volatile compounds was carried out by SPME for the samples stored at 60 degrees C. Peroxides appeared to be the only significant oxidation products after 12 weeks of storage at 4 degrees C. As expected, the binary blends (BB) were more sensitive to oxidation than the ternary blends (TB). The BB were associated with increased volatile emission compared to the TB. Interesterification led to variable effects on the oxidation of fat mixtures, depending on composition and temperature (beneficial effect on BB, at both 25 and 60 degrees C, and a rather neutral effect on TB). The IE blends exhibited higher volatile release prior to aging. A pro-oxidant effect of alpha-tocopherol addition was observed at 25 degrees C on both BB and TB. At 60 degrees C, an antioxidant effect was observed on TB.
Subject(s)
Fats/chemistry , Linseed Oil/chemistry , Lipase/chemistry , Milk/chemistry , Plant Oils/chemistry , Animals , Brassica rapa/chemistry , Esterification , Fatty Acids, Monounsaturated , Oxidation-Reduction , Rapeseed Oil , VolatilizationABSTRACT
The membrane-proximal external region (MPER) of the gp41 fusion protein of HIV is highly conserved among isolates of this virus and is considered a target for vaccine development. This region also appears to play a role in membrane fusion as well as localization of the virus to cholesterol-rich domains in membranes. The carboxyl terminus of MPER has the sequence LWYIK and appears to have an important role in cholesterol interactions. We have tested how amino acid substitutions that would affect the conformational flexibility of this segment could alter its interaction with cholesterol. We studied a family of peptides (all peptides as N-acetyl-peptide amides) with P, G, or A substituting for W and I of the LWYIK sequence. The peptide having the greatest effect on cholesterol distribution in membranes was the most flexible one, LGYGK. The corresponding mutation in gp41 resulted in a protein retaining 72% of the fusion activity of the wild-type protein. Two other peptides were synthesized, also containing two Gly residues, GWGIK and LWGIG, and did not have the ability to sequester cholesterol as efficiently as LGYGK did. Making the corresponding mutants of gp41 showed that these other two double Gly substitutions resulted in proteins that were much less fusogenic, although they were equally well expressed at the cell surface. The study demonstrates that drastic changes can be made in the LWYIK segment with the retention of a significant fraction of the fusogenic activity, as long as the mutant proteins interact with cholesterol.
Subject(s)
Cholesterol/metabolism , HIV Envelope Protein gp41/metabolism , Membrane Fusion , Amino Acid Sequence , Animals , COS Cells , Calorimetry, Differential Scanning , Cell Line , Chlorocebus aethiops , Cholesterol/chemistry , HIV Envelope Protein gp41/chemistry , HIV Envelope Protein gp41/genetics , Humans , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/metabolismABSTRACT
The epsilon isoform of diacylglycerol kinase (DGKepsilon) is unique among mammalian DGKs in having a segment of hydrophobic amino acids comprising approximately residues 20 to 41. Several algorithms predict this segment to be a transmembrane (TM) helix. Using PepLook, we have performed an in silico analysis of the conformational preference of the segment in a hydrophobic environment comprising residues 18 to 42 of DGKepsilon. We find that there are two distinct groups of stable conformations, one corresponding to a straight helix that would traverse the membrane and the second corresponding to a bent helix that would enter and leave the same side of the membrane. Furthermore, the calculations predict that substituting the Pro32 residue in the hydrophobic segment with an Ala will cause the hydrophobic segment to favor a TM orientation. We have expressed the P32A mutant of DGKepsilon, with a FLAG tag (an N-terminal 3xFLAG epitope tag) at the amino terminus, in COS-7 cells. We find that this mutation causes a large reduction in both k(cat) and K(m) while maintaining k(cat)/K(m) constant. Specificity of the P32A mutant for substrates with polyunsaturated acyl chains is retained. The P32A mutant also has higher affinity for membranes since it is more difficult to extract from the membrane with high salt concentration or high pH compared with the wild-type DGKepsilon. We also evaluated the topology of the proteins with confocal immunofluorescence microscopy using NIH 3T3 cells. We find that the FLAG tag at the amino terminus of the wild-type enzyme is not reactive with antibodies unless the cell membrane is permeabilized with detergent. We also demonstrate that at least a fraction of the wild-type DGKepsilon is present in the plasma membrane and that comparable amounts of the wild-type and P32A mutant proteins are in the plasma membrane fraction. This indicates that in these cells the hydrophobic segment of the wild-type DGKepsilon is not TM but takes up a bent conformation. In contrast, the FLAG tag at the amino terminus of the P32A mutant is exposed to antibody both before and after membrane permeabilization. This modeling approach thus provides an explanation, not provided by simple predictive algorithms, for the observed topology of this protein in cell membranes. The work also demonstrates that the wild-type DGKepsilon is a monotopic protein.
Subject(s)
Cell Membrane/enzymology , Diacylglycerol Kinase/chemistry , Diacylglycerol Kinase/metabolism , Isoenzymes/chemistry , Isoenzymes/metabolism , Protein Conformation , Amino Acid Sequence , Animals , COS Cells , Chlorocebus aethiops , Diacylglycerol Kinase/genetics , Diglycerides/chemistry , Diglycerides/metabolism , Enzyme Stability , Epitopes , Isoenzymes/genetics , Mice , Models, Molecular , Molecular Sequence Data , Mutation , NIH 3T3 Cells , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Substrate SpecificityABSTRACT
Colipase is a key element in the lipase-catalyzed hydrolysis of dietary lipids. Although devoid of enzymatic activity, colipase promotes the pancreatic lipase activity in physiological intestinal conditions by anchoring the enzyme at the surface of lipid droplets. Analysis of structures of NMR colipase models and simulations of their interactions with various lipid aggregates, lipid droplet, and bile salt micelle, were carried out to determine and to map the lipid binding sites on colipase. We show that the micelle and the oil droplet bind to the same side of colipase 3D structure, mainly the hydrophobic fingers. Moreover, it appears that, although colipase has a single direction of interaction with a lipid interface, it does not bind in a specific way but rather oscillates between different positions. Indeed, different NMR models of colipase insert different fragments of sequence in the interface, either simultaneously or independently. This supports the idea that colipase finger plasticity may be crucial to adapt the lipase activity to different lipid aggregates.
Subject(s)
Bile Acids and Salts/metabolism , Colipases/metabolism , Computational Biology , Lipid Metabolism , Micelles , Amino Acid Sequence , Animals , Bile Acids and Salts/chemistry , Colipases/chemistry , Enzyme Stability , Lipids/chemistry , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Sequence Data , SwineABSTRACT
Despite numerous investigations, the important structural features of Cell Penetrating Peptides (CPPs) remain unclear as demonstrated by the difficulties encountered in designing new molecules. In this study, we focused our interest on Penetratin and Transportan and several of their variants. Penetratin W48F and Penetratin W48F/W56F exhibit a reduced and a complete lack of cellular uptake, respectively; TP07 and TP10 present a similar cellular uptake as Transportan and TP08, TP13 and TP15 display no or weak internalization capacity. We applied the algorithmic method named PepLook to analyze the peptide polymorphism. The study reveals common conformational characteristics for the CPPs and their permeable variants: they all are polymorphic. Negative, non permeable, mutants share the opposite feature since they are monomorphic. Finally, we support the hypothesis that structural polymorphism may be crucial since it provides peptides with the possibility of adapting their conformation to medium hydrophobicity and or to partner diversity.
Subject(s)
Carrier Proteins/chemistry , Galanin/chemistry , Polymorphism, Genetic , Recombinant Fusion Proteins/chemistry , Wasp Venoms/chemistry , Amino Acid Sequence , Cell-Penetrating Peptides , Models, Molecular , Molecular Sequence Data , Protein ConformationABSTRACT
Lipozyme TL IM was used in a solvent-free batch and microaqueous system for enzymatic interesterification of anhydrous milkfat (AMF) with linseed oil (LO) in binary blends and with rapeseed oil (RO) in one ternary blend. The aim was to obtain and characterize physicochemically fats enriched with unsaturated C 18 fatty acids (oleic, linoleic, and, especially, linolenic acids) from natural vegetable oils. Binary blends of AMF/LO 100/0, 90/10, 80/20, 70/30, and 60/40 (w/w) were interesterified. The change in triacylglycerol (TAG) profiles showed that quasi-equilibrium conditions were reached after 4-6 h of reaction. Free fatty acid contents <1%. The decrease in solid fat content and in dropping point temperature obtained with increasing content of LO and interesterification resulted in good plastic properties for the products originating from the blends 70/30 and 60/40. This was confirmed by textural measurements. Melting profiles determined by differential scanning calorimetry showed complete disappearance of low-melting TAGs from LO and the formation of intermediary species with a lower melting temperature. Oxidative stability of the interesterified products was diminished with increasing LO content, resulting in low oxidation induction times. A ternary blend composed of AMF/RO/LO 70/20/10 gave satisfactory rheological and oxidative properties, fulfilling the requirements for a marketable spread and, moreover, offering increased potential health benefits due to the enriched content in polyunsaturated fatty acid residues.
Subject(s)
Fatty Acids, Unsaturated/analysis , Linseed Oil/chemistry , Lipase/metabolism , Milk/chemistry , Plant Oils/chemistry , Triglycerides/metabolism , Animals , Calorimetry, Differential Scanning , Esterification , Fatty Acids, Monounsaturated , Humans , Oxidation-Reduction , Rapeseed Oil , Rheology , Temperature , Triglycerides/analysisABSTRACT
We investigated the peptides N-acetyl-AWYIK-amide and N-acetyl-VWYIK-amide corresponding to single amino acid substitutions in LWYIK, a segment found in the gp41 protein of HIV and believed to play a role in sequestering this protein to a cholesterol-rich domain in the membrane. The effects of these peptides on the thermotropic phase transitions of 1-stearoyl-2-oleoylphosphatidylcholine (SOPC) and mixtures of SOPC and cholesterol were intermediate between that having the wild-type sequence (LWYIK) and another (IWYIK), the least active peptide previously studied. This correlated with results from studies of single mutations in the gp41 protein of HIV-1, in which L679 of the LWYIK segment is replaced with either A or V, measuring the capability of TZM-BL HeLa-based HIV-1 indicator cells to form syncytia. The peptides were also comparatively analyzed in silico. All together, the results suggest that the mode of interaction of this region of gp41 with the polar heads of membrane lipids contributes to its cholesterol selectivity and that this is somehow related to the biological activity of the viral glycoprotein.
Subject(s)
HIV Envelope Protein gp41/chemistry , Oligopeptides/chemistry , Amino Acid Sequence , Calorimetry, Differential Scanning , Cell Fusion , Cell Line , Cholesterol/chemistry , HIV Envelope Protein gp41/genetics , HIV Envelope Protein gp41/metabolism , HeLa Cells , Humans , Hydrophobic and Hydrophilic Interactions , Membrane Microdomains/chemistry , Membrane Microdomains/metabolism , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Mutagenesis , Mutation , Oligopeptides/chemical synthesis , Oligopeptides/metabolism , Phosphatidylcholines/chemistry , Protein BindingABSTRACT
The TRE2 oncoprotein is structurally related to the RabGAP (GTPase-activating protein) family. However, TRE2 seems enzymatically inactive. Two regions are important for its lack of GAP activity. First, the TBC domain, forming the catalytically active domain of RabGAPs, is non-functional in the oncoprotein. Also involved in TRE2 inactivity is the 93-aa region flanking the TBC domain on the C-terminal side. In order to identify the residues responsible for non-functionality, we performed hydrophobic cluster analysis of the oncoprotein sequence, combined with secondary structure prediction, receptor-binding domain analysis, and a tilted peptide calculation. These analyses were complemented with site-directed and random mutagenesis experiments. On the basis of our data, we hypothesize that the lack of secondary structure of the region flanking the TBC domain in TRE2 may explain why this region plays a role in the lack of GAP activity, even when a potentially functional TBC domain is present.
Subject(s)
GTPase-Activating Proteins/metabolism , Microtubule-Associated Proteins/metabolism , Oncogenes , Proto-Oncogene Proteins/genetics , Ubiquitin Thiolesterase/genetics , Amino Acid Sequence , Computational Biology , DNA Mutational Analysis , Molecular Sequence Data , Mutagenesis , Oncogene Proteins/chemistry , Protein Structure, Tertiary , Proto-Oncogene Proteins/chemistry , Saccharomyces cerevisiae/cytology , Ubiquitin Thiolesterase/chemistryABSTRACT
Model peptides composed of alanine and leucine residues are often used to mimic single helical transmembrane domains. Many studies have been carried out to determine how they interact with membranes. However, few studies have investigated their lipid-destabilizing effect. We designed three peptides designated KALRs containing a hydrophobic stretch of 14, 18, or 22 alanines/leucines surrounded by charged amino acids. Molecular modeling simulations in an implicit membrane model as well as attenuated total reflection-Fourier transform infrared analyses show that KALR is a good model of a transmembrane helix. However, tryptophan fluorescence and attenuated total reflection-Fourier transform infrared spectroscopy indicate that the extent of binding and insertion into lipids increases with the length of the peptide hydrophobic core. Although binding can be directly correlated to peptide hydrophobicity, we show that insertion of peptides into a membrane is determined by the length of the peptide hydrophobic core. Functional studies were performed by measuring the ability of peptides to induce lipid mixing and leakage of liposomes. The data reveal that whereas KALR14 does not destabilize liposomal membranes, KALR18 and KALR22 induce 40 and 50% of lipid-mixing, and 65 and 80% of leakage, respectively. These results indicate that a transmembrane model peptide can induce liposome fusion in vitro if it is long enough. The reasons for the link between length and fusogenicity are discussed in relation to studies of transmembrane domains of viral fusion proteins. We propose that fusogenicity depends not only on peptide insertion but also on the ability of peptides to destabilize the two leaflets of the liposome membrane.