Palmitoyl Research Articles

Multi-metal tolerance of DHHC palmitoyl transferase-like protein isolated from metal contaminated soil.

The microbiota inhabiting in metal polluted environment develops strong defense mechanisms to combat pollution and sustain life. Investigating the functional genes of the eukaryotic microbiota inhabiting in these environments by using metatranscriptomics approach was the focus of this study. Size fractionated eukaryotic cDNA libraries (library A, < 0.5 kb, library B, 0.5-1.0 kb, and library C, > 1.0 kb) were constructed from RNA isolated from the metal contaminated soil. The library C was screened for Cadmium (Cd) tolerant genes by using Cd sensitive yeast mutant ycf1Δ by functional complementation assay, which yielded various clones capable of growing in Cd amended media. One of the Cd tolerant clones, PLCg39 was selected because of its ability to grow at high concentrations of Cd. Sequence analysis of PLCg39 showed homology with DHHC palmitoyl transferases, which are responsible for addition of palmitoyl groups to proteins and usually possess metal coordination domains. The cDNA PLCg39 was able to confer tolerance to Cd-sensitive (ycf1Δ), Copper-sensitive (cup1Δ) and Zn-sensitive (zrc1Δ) yeast mutants when grown at different concentrations of Cd (40-100 μM), Cu (150-1000 μM) and Zn (10-13 mM), respectively. The DHHC mutant akr1Δ transformed with PLCg39 rescued from the metal sensitivity indicating the role of DHHC palmitoyl transferase in metal tolerance. This study demonstrated that PLCg39 acts as a potential metal tolerant gene which could be used in bioremediation, biosensing and other biotechnological fields.

Increased Cytotoxic Efficacy of Protocatechuic Acid in A549 Human Lung Cancer Delivered via Hydrophobically Modified-Chitosan Nanoparticles As an Anticancer Modality.

The growing incidence of global lung cancer cases against successful treatment modalities has increased the demand for the development of innovative strategies to complement conventional chemotherapy, radiation, and surgery. The substitution of chemotherapeutics by naturally occurring phenolic compounds has been touted as a promising research endeavor, as they sideline the side effects of current chemotherapy drugs. However, the therapeutic efficacy of these compounds is conventionally lower than that of chemotherapeutic agents due to their lower solubility and consequently poor intracellular uptake. Therefore, we report herein a hydrophobically modified chitosan nanoparticle (pCNP) system for the encapsulation of protocatechuic acid (PCA), a naturally occurring but poorly soluble phenolic compound, for increased efficacy and improved intracellular uptake in A549 lung cancer cells. The pCNP system was modified by the inclusion of a palmitoyl group and physico-chemically characterized to assess its particle size, Polydispersity Index (PDI) value, amine group quantification, functional group profiling, and morphological properties. The inclusion of hydrophobic palmitoyl in pCNP-PCA was found to increase the encapsulation of PCA by 54.5% compared to unmodified CNP-PCA samples whilst it only conferred a 23.4% larger particle size. The single-spherical like particles with uniformed dispersity pCNP-PCA exhibited IR bands, suggesting the successful incorporation of PCA within its core, and a hydrophobic layer was elucidated via electron micrographs. The cytotoxic efficacy was then assessed by using an MTT cytotoxicity assay towards A549 human lung cancer cell line and was compared with traditional chitosan nanoparticle system. Fascinatingly, a controlled release delivery and enhanced therapeutic efficacy were observed in pCNP-PCA compared to CNP, which is ascribed to lower IC50 values in the 72-h treatment in the pCNP system. Using the hydrophobic system, efficacy of PCA was significantly increased in 24-, 48-, and 72-h treatments compared to a single administration of the compound, and via the unmodified CNP system. Findings arising from this study exhibit the potential of using such modified nanoparticulate systems in increasing the efficacy of natural phenolic compounds by augmenting their delivery potential for better anti-cancer responses.

Enzyme-Assisted Synthesis of High-Purity, Chain-Deuterated 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine.

1-Palmitoyl-d31-2-oleoyl-d32-sn-glycero-3-phosphocholine (POPC-d63) with the palmitoyl and oleoyl chains deuterium-labeled was produced in three steps from 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine, deuterated palmitic acid, and deuterated oleic anhydride. Esterification at the sn-2 position was achieved under standard chemical conditions, using DMAP to catalyze the reaction between the 2-lysolipid and oleic anhydride-d64. Complete regioselective sn-1 acyl substitution was achieved in two steps using operationally simple, enzyme-catalyzed regioselective hydrolysis and esterification to substitute the sn-1 chain for a perdeuterated analogue. This method provides chain-deuterated POPC with high chemical purity (>96%) and complete regiopurity, useful for a variety of experimental techniques. This chemoenzymatic semisynthetic approach is a general, modular method of producing highly pure, mixed-acyl phospholipids, where the advantages of both chemical synthesis (efficiency, high yields) and biocatalytic synthesis (specificity, nontoxicity) are realized.

Interfacial Water Structure at Zwitterionic Membrane/Water Interface: The Importance of Interactions between Water and Lipid Carbonyl Groups.

In this work, atomistic molecular dynamics (MD) simulations of palmitoyl-oleoyl-phosphatidylcholine (POPC) bilayer were carried out to investigate the effect of water models on membrane dipole potential, which is primarily associated with the preferential orientation of molecular dipoles at the membrane–water interface. We discovered that the overestimation of the dipole potential by the TIPS3P water model can be effectively reduced by the TIP4P water model. On the one hand, the TIP4P water model decreases the negative contribution of lipid to the dipole potential through influencing the orientation of lipid headgroups. On the other hand, the TIP4P water model reduces the positive contribution of water to the dipole potential by increasing the preference of H-down orientation (the water dipole orients toward the bilayer center). Interestingly, the TIP4P water model affects the orientation of interfacial water molecules more obviously than that of lipid headgroups, leading to the decrease in the dipole potential. Furthermore, the MD results revealed that the water close to the positively charged choline (namely, N-associated water) prefers the H-down orientation while the water around the negatively charged phosphate (namely, P-associated water) favors the H-up orientation, in support of recent experimental and MD studies. However, interfacial water molecules are more strongly influenced by the phosphate groups than by the choline groups, resulting in the net H-up orientation (the water dipole orients toward the bilayer center) in the region of lipid headgroups. In addition, it is intriguing that the preference of H-up orientation decreases when water molecules penetrate more deeply into the lipid bilayer. This is attributed to the counteracting effect of lipid carbonyl groups, and the effect varies with the lipid chains (oleoyl and palmitoyl chains), suggesting the important role of lipid carbonyl groups.

Palmitoylome profiling indicates that androgens promote the palmitoylation of metabolism-related proteins in prostate cancer-derived LNCaP cells

To explore potential therapeutic targets other than androgen-deprivation treatment for prostate cancer by screening the proteins induced by androgen at palmitoylation modification level in LNCaP cells. The LNCaP cells were treated with androgen (Methyltrienolone, R1881, 5 nmol/L) or dimethyl sulfoxide (DMSO) for 24 h, and then labeled with alkynyl palmitic acid Alk-C16 (100 μmol/L). After that, the cells were collected, lysed, the total protein was extracted, agarose beads labeled with azide (1 mmol/L) were added, and the click-chemistry reaction was carried out at room temperature for 1 h. The covalent bond formed by click-chemistry reaction of azide and alkynyl group was used to enrich the palmitoylated proteins on agarose beads. Label-free quantitation (LFQ) was used to compare the protein palmitoylation level of R1881 treated and untreated cells to screen the proteins induced by androgen at palmitoylation modification level. In this experiment, 907 potential palmitoylated proteins (mascot score>2, P<0.05) were identified, among which 430 proteins had LFQ values not zero at least twice. Among the 430 proteins, the palmitoylation levels of 92 candidates were increased by androgen treatment, and their LFQ values were significantly upregulated (>1.5-fold, P<0.05) in ≥2 samples of androgen-treated vs. untreated LNCaP cells. We also used the software of cytoscape to classify the 92 proteins, and found that the known functional proteins of them could be divided into three categories: metabolism related, protein folding related and translation initiation related. Among them, metabolism related proteins included lipid metabolism (6), glucose metabolism (7) and respiratory electron transport chain (8), and a small amount of amino acid metabolism (2) and other metabolism related proteins (2). Notably, the ratio of LFQ of cytochrome b-c1 complex subunit 2 (UQCRC2) was significantly (>3-fold, P<0.05) higher in androgen-treated cells compared with untreated cells, indicating that the palmitoylation level of UQCRC2 was enhanced by androgen most significantly than that of others. The second was long-chain acyl CoA dehydrogenase (ACADVL) related to lipid metabolism and glucose 6-phosphate dehydrogenase (PGD) related to glucose metabolism, but the LFQ ratio of them was less than 3-fold. The research on palmitoylation mechanism of metabolism, especially the proteins related to respiratory electron transport chain, will provide a new guidance for the diagnosis and treatment of prostate cancer and the development of targeted drugs.

Supramolecular Organogelation Directed by Weak Noncovalent Interactions in Palmitoylated 1,5-Anhydro-d-Glucitol Derivatives.

We synthesized a series of novel alicyclic compounds by modifying 1,5-anhydro-d-glucitol with two to four palmitoyl chains, and we explored their self-assembly and gelation behaviors in paraffin. The obtained organogels were studied by field emission scanning electron microscopy, atomic force microscopy, variable-temperature Fourier transform IR spectroscopy, X-ray diffraction analysis, polarized optical microscopy, and transmission spectroscopy. While all the palmitoylated derivatives spontaneously formed fibrous networks and gelated the paraffin, an acetylated derivative of 1,5-anhydro-d-glucitol did not gelatinize the solvent, thus indicating the importance of aliphatic chains for gelation. Interestingly, α- and β- d-glucopyranose with five palmitoyl chains neither gelatinized the solvent nor formed fibrous networks, thus suggesting that the absence of C-1 substitution in 1,5-anhydro-d-glucitol is important for gelation. Fourier transform IR spectroscopy suggested that the formation of weak hydrogen bonds between the carbonyl groups and the C-H groups was the driving force for formation of the supramolecular fibers and for gelation of the solvent.

Direct Synthesis of Rhenium and Technetium-99m Metallosurfactants by a Transmetallation Reaction of Lipophilic Groups: Potential Applications in the Radiolabeling of Liposomes.

A new zinc dithiocarbamate functionalized with palmitoyl groups is described as a useful tool for the preparation of metallosurfactants through a transmetallation reaction with the transition metals rhenium and technetium. An amphiphilic rhenium complex is synthesized by a transmetallation reaction with the zinc complex in presence of the polar phosphine sodium triphenylphosphine trisulfonate, which leads to a rhenium complex with a lipophilic dithiocarbamate and a polar phosphine ligand. The study of this rhenium complex has shown that it self-aggregates, leading to the formation of aggregates that have been analyzed by dynamic light scattering and cryotransmission electron microscopy (cryo-TEM). In addition, this amphiphilic rhenium complex is incorporated into soy phosphatidylcholine liposomes, whether liposomes are prepared by mixing phospholipid and the rhenium complex or by the incorporation of the rhenium complex into preformed liposomes. The one-pot reaction of the radiocompound [99mTc(H2O)3(CO)3]+ with the above-mentioned zinc dithiocarbamate, the phosphine sodium triphenylphosphine trisulfonate and the phospholipid soy phosphatidylcholine, leads to liposomes labeled with a Tc-99m homologous complex of the rhenium complex, in accordance with the high-performance liquid chromatography (HPLC) data.

A Trifunctional Linker for Palmitoylation and Peptide and Protein Localization in Biological Membranes.

Attachment of lipophilic groups is an important post‐translational modification of proteins, which involves the coupling of one or more anchors such as fatty acids, isoprenoids, phospholipids, or glycosylphosphatidyl inositols. To study its impact on the membrane partitioning of hydrophobic peptides or proteins, we designed a tyrosine‐based trifunctional linker. The linker allows the facile incorporation of two different functionalities at a cysteine residue in a single step. We determined the effect of the lipid modification on the membrane partitioning of the synthetic α‐helical model peptide WALP with or without here and in all cases below; palmitoyl groups in giant unilamellar vesicles that contain a liquid‐ordered (Lo) and liquid‐disordered (Ld) phase. Introduction of two palmitoyl groups did not alter the localization of the membrane peptides, nor did the membrane thickness or lipid composition. In all cases, the peptide was retained in the Ld phase. These data demonstrate that the Lo domain in model membranes is highly unfavorable for a single membrane‐spanning peptide.

Lipidomics Identified Lysophosphatidylcholine&amp;nbsp;and Phosphatidylethanolamine&amp;nbsp;as Novel&amp;nbsp;Biomarkers for Diagnosis of Laryngeal&amp;nbsp;Cancer

Background: Laryngeal cancer (LaC) remains one of the most common tumors of the respiratory tract with higher incidence in men than in women. The larynx is a small but vital organ on the neck. The dysfunction of the larynx can cause serious health problems such as hoarseness, respiratory distress, and dysphonia. Lipid has been recognized as a crucial role in tumorigenesis. However, the lipid biomarkers are lacking and the lipid molecular pathogenesis of LaC has remained obscure to date. Methods: To identify new LaC-related lipid biomarkers used for the diagnosis or early diagnosis of LaC and to uncover their molecular characteristics, we conducted serum lipidomics study from LaC patients (n = 29) and normal controls (NC) (n = 36) using nontargeted lipidomics profiling based on ultrahigh-performance liquid chromatography (UHPLC)/Q-TOF 5600 Plus mass spectrometry. Multivariate and univariate statistics analyses were used to discriminate LaC patients from NC. Findings: As expected, a lipid panel including LPC (16:0) and PE (18:0p_20:4) was found to distinguish the LaC patients from healthy individuals with very high diagnosis performance (area under the curve (AUC) = 1.000, sensitivity = 1.000, and specificity = 1.000). In addition, the levels of Cer, CerG1, SM, PC, PC-O, PE, PI, PS, and ChE in the LaC group significantly increased as compared with the NC group. However, the levels of LPC, LPC-O, LPE, LPE-p, and DG in the LaC group significantly deceased when the one was compared with the NC group. Among significantly changed lipid species, lysophospholipids containing a palmitoyl chain or an arachidonic acid acyl chain remarkably decreased and phospholipids including a palmitoyl chain or an arachidonic acid acyl chain increased in the LaC patients. Interpretation: Our results not only indicate that lipidomics is powerful tool to explore abnormal lipid metabolism for the laC, but suggest that lysophospholipids and phospholipids may serve as potential biomarkers for diagnosis of laC. Funding Statement: This work was supported by the National Natural Science Foundation of China Grants (No. 11772087), and the Joint Fund Project of Collaborative Innovation Center for Individualized Diagnosis and Treatment (dy2yhws201805). Declaration of Interests: The authors declare no competing financial interest. Ethics Approval Statement: This study was approved by the Second Affiliated Hospital of Dalian Medical University Institutional Ethics Review Board, and all participants provided written informed consent.

Preclinical evaluation of cationic DOTA-triarginine-lipid conjugates for theranostic liquid brachytherapy.

Liquid brachytherapy is an emerging technology for internal radiation therapy where liquids containing radionuclides are administered directly into solid tumors. These technologies are less invasive than conventional brachytherapy, and can potentially improve the dose coverage and homogeneity of the radioactivity distribution within the tumor. For this purpose, we have developed a novel cationic micelle system for delivery of a range of radionuclides. The system is applicable for emitters of alpha, beta or photon radiation, and enables dose-mapping via theranostic nuclear imaging.Methods: The cationic micelles were developed as linear surfactants comprising the chelator DOTA, a triarginine sequence and a palmitoyl or stearoyl fatty acid chain. The critical micelle concentration of the surfactants was determined, and the micelles were radiolabelled with 64Cu or 177Lu in high radiochemical purity (>95%). The tumor retention and biodistribution of the 64Cu-radiolabeled surfactants, administered as micelles or formulated in liposomes, were investigated in vivo by PET/CT in a tumor bearing mouse model.Results: The interaction of the micelles with anionic lipid membranes was demonstrated to be favourable, using a liposome partition assay. In vivo, the surfactants formulated both as cationic micelles and liposomes displayed the best intratumoral retention, with micelles providing more homogeneous activity distribution.Conclusion: A cationic, surfactant-based drug delivery system was developed and demonstrated promise as a vehicle for liquid brachytherapy when formulated as micelles or in liposomes. The system enables accurate dosimetry due to the flexible radiochemistry of DOTA.

Functionalized Lipopeptide Micelles as Highly Efficient NMR Depolarization Seed Points for Targeted Cell Labelling in Xenon MRI.

Improving diagnostic imaging and therapy by targeted compound delivery to pathological areas and across biological barriers is of urgent need. A lipopeptide, P-CrA-A2, composed of a highly cationic peptide sequence (A2), an N-terminally attached palmitoyl chain (P) and cryptophane molecule (CrA) for preferred uptake into blood-brain barrier (BBB) capillary endothelial cells, was generated. CrA allows reversible binding of Xe for NMR detection with hyperpolarized nuclei. The lipopeptide forms size-optimized micelles with a diameter of about 11 nm at low micromolar concentration. Their high local CrA payload has a strong and switchable impact on the bulk magnetization through Hyper-CEST detection. Covalent fixation of CrA does not impede micelle formation and does not hamper its host functionality but simplifies Xe access to hosts for inducing saturation transfer. Xe Hyper-CEST magnetic resonance imaging (MRI) allows for distinguishing BBB endothelial cells from control aortic endothelial cells, and the small micelle volume with a sevenfold improved CrA-loading density compared to liposomal carriers allows preferred cell labelling with a minimally invasive volume (≈16 000-fold more efficient than 19 F cell labelling). Thus, these nanoscopic particles combine selectivity for human brain capillary endothelial cells with great sensitivity of Xe Hyper-CEST MRI and might be a potential MRI tool in brain diagnostics.

Hydrophobic agave fructans for sustained drug delivery to the human colon

Given their glycosidic β 2−1 and β 2−6 linkages, fructans from Agave tequilana Weber var. azul (AtF) may be employed for the design of drug carriers targeting the human colon. However, the high water solubility of AtF prevents their use for such a purpose. In this work, AtF were esterified with acetyl, lauroyl and palmitoyl groups with the aim to encapsulate ibuprofen as a model drug and characterize its enzymatic release in simulated colon conditions. It was found that efficiency of encapsulation increased from 0.8% (unmodified fructan) to 17 or 21.5% when AtF was esterified with acetyl groups at high degree of substitution (2.75 DS) or with a long acyl chain such as palmitoyl at low DS (0.18) respectively. Hydrophilic fructan based microspheres (AtF, and lauroyl fructan, 46 and 61.3° contact angle respectively) had poor encapsulation efficiency (0.8–1.5%) and lost most of their cargo (85%) at gastric conditions. In contrast, hydrophobic fructan based microspheres (acetyl and palmytoyl, 91–98° contact angle) showed high stability at gastric pH and were able to release the drug for long periods. These characteristics make hydrophobic fructans appropriate vehicles that show great potential for the delivery of drugs to the colon, allowing long drug bioavailability during its delivery and release.

Full-Length P2X7 Structures Reveal How Palmitoylation Prevents Channel Desensitization

P2X receptors are trimeric, non-selective cation channels activated by extracellular ATP. The P2X7 receptor subtype is a pharmacological target because of involvement in apoptotic, inflammatory, and tumor progression pathways. It is the most structurally and functionally distinct P2X subtype, containing a unique cytoplasmic domain critical for the receptor to initiate apoptosis and not undergo desensitization. However, lack of structural information about the cytoplasmic domain has hindered understanding of the molecular mechanisms underlying these processes. We report cryoelectron microscopy structures of full-length rat P2X7 receptor in apo and ATP-bound states. These structures reveal how one cytoplasmic element, the C-cys anchor, prevents desensitization by anchoring the pore-lining helix to the membrane with palmitoyl groups. They show a second cytoplasmic element with a unique fold, the cytoplasmic ballast, which unexpectedly contains a zinc ion complex and a guanosine nucleotide binding site. Our structures provide first insights into the architecture and function of a P2X receptor cytoplasmic domain.

Engineering the stereoisomeric structure of seed oil to mimic human milk fat

Human milk fat substitute (HMFS) is a class of structured lipid that is widely used as an ingredient in infant formulas. Like human milk fat, HMFS is characterized by enrichment of palmitoyl (C16:0) groups specifically at the middle (sn-2 or β) position on the glycerol backbone, and there is evidence that triacylglycerol (TAG) with this unusual stereoisomeric structure provides nutritional benefits. HMFS is currently made by in vitro enzyme-based catalysis because there is no appropriate biological alternative to human milk fat. Most of the fat currently used in infant formulas is obtained from plants, which exclude C16:0 from the middle position. In this study, we have modified the metabolic pathway for TAG biosynthesis in the model oilseed Arabidopsis thaliana to increase the percentage of C16:0 at the middle (vs. outer) positions by more than 20-fold (i.e., from ∼3% in wild type to >70% in our final iteration). This level of C16:0 enrichment is comparable to human milk fat. We achieved this by relocating the C16:0-specific chloroplast isoform of the enzyme lysophosphatidic acid acyltransferase (LPAT) to the endoplasmic reticulum so that it functions within the cytosolic glycerolipid biosynthetic pathway to esterify C16:0 to the middle position. We then suppressed endogenous LPAT activity to relieve competition and knocked out phosphatidylcholine:diacylglycerol cholinephosphotransferase activity to promote the flux of newly made diacylglycerol directly into TAG. Applying this technology to oilseed crops might provide a source of HMFS for infant formula.

Structure, Topology, and Dynamics of Membrane-Inserted Polypeptides and Lipids by Solid-State NMR Spectroscopy: Investigations of the Transmembrane Domains of the DQ Beta-1 Subunit of the MHC II Receptor and of the COP I Protein p24.

MHC class II receptors carry important function in adaptive immunity and their malfunctioning is associated with diabetes type I, chronic inflammatory diseases and other autoimmune diseases. The protein assembles from the DQ alpha-1 and DQ beta-1 subunits where the transmembrane domains of these type I membrane proteins have been shown to be involved in homo- and heterodimer formation. Furthermore, the DQ alpha 1 chain carries a sequence motif that has been first identified in the context of p24, a protein involved in the formation of COPI vesicles of the intracellular transport machinery, to specifically interact with sphingomyelin-C18 (SM-C18). Here we investigated the membrane interactions and dynamics of DQ beta-1 in liquid crystalline POPC phospholipid bilayers by oriented 15N solid-state NMR spectroscopy. The 15N resonances are indicative of a helical tilt angle of the membrane anchor sequence around 20°. Two populations can be distinguished by their differential dynamics probably corresponding the DQ beta-1 mono- and homodimer. Whereas, this equilibrium is hardly affected by the addition of 5 mole% SM-C18 a single population is visible in DMPC lipid bilayers suggesting that the lipid saturation is an important parameter. Furthermore, the DQ alpha-1, DQ beta-1 and p24 transmembrane helical domains were reconstituted into POPC or POPC/SM-C18 lipid bilayers where the fatty acyl chain of either the phosphatidylcholine or of the sphingolipid have been deuterated. Interestingly in the presence of both sphingolipid and polypeptide a strong decrease in the innermost membrane order of the POPC palmitoyl chain is observed, an effect that is strongest for DQ beta-1. In contrast, for the first time the polypeptide interactions were monitored by deuteration of the stearoyl chain of SM-C18. The resulting 2H solid-state NMR spectra show an increase in order for p24 and DQ alpha-1 which both carry the SM recognition motif. Thereby the data are suggestive that SM-C18 together with the transmembrane domains form structures imposing positive curvature strain on the surrounding POPC lipids. This effect is attenuated when SM-C18 is recognized by the protein.

The morphogen Sonic hedgehog inhibits its receptor Patched by a pincer grasp mechanism.

Hedgehog (HH) ligands, classical morphogens that pattern embryonic tissues in all animals, are covalently coupled to two lipids-- a palmitoyl group at the N-terminus and a cholesteroyl group at the C-terminus. While the palmitoyl group binds and inactivates Patched 1 (PTCH1), the main receptor for HH ligands, the function of the cholesterol modification has remained mysterious. Using structural and biochemical studies, along with the re-assessment of prior cryo-electron microscopy structures, we find that the C-terminal cholesterol attached to Sonic Hedgehog (SHH) binds the first extracellular domain of PTCH1 and promotes its inactivation, thus triggering HH signalling. Molecular dynamics simulations show that this interaction leads to the closure of a tunnel through PTCH1 that serves as the putative conduit for sterol transport. Thus, SHH inactivates PTCH1 by grasping its extracellular domain with two lipidic pincers, the N-terminal palmitate and the C-terminal cholesterol, which are both inserted into the PTCH1 protein core.

A Convenient Synthetic Method to Improve Immunogenicity of Mycobacterium tuberculosis Related T-Cell Epitope Peptides.

Epitopes from different proteins expressed by Mycobacterium tuberculosis (Rv1886c, Rv0341, Rv3873) were selected based on previously reported antigenic properties. Relatively short linear T-cell epitope peptides generally have unordered structure, limited immunogenicity, and low in vivo stability. Therefore, they rely on proper formulation and on the addition of adjuvants. Here we report a convenient synthetic route to induce a more potent immune response by the formation of a trivalent conjugate in spatial arrangement. Chemical and structural characterization of the vaccine conjugates was followed by the study of cellular uptake and localization. Immune response was assayed by the measurement of splenocyte proliferation and cytokine production, while vaccine efficacy was studied in a murine model of tuberculosis. The conjugate showed higher tendency to fold and increased internalization rate into professional antigen presenting cells compared to free epitopes. Cellular uptake was further improved by the incorporation of a palmitoyl group to the conjugate and the resulted pal-A(P)I derivative possessed an internalization rate 10 times higher than the free epitope peptides. Vaccination of CB6F1 mice with free peptides resulted in low T-cell response. In contrast, significantly higher T-cell proliferation with prominent expression of IFN-γ, IL-2, and IL-10 cytokines was measured for the palmitoylated conjugate. Furthermore, the pal-A(P)I conjugate showed relevant vaccine efficacy against Mycobacterium tuberculosis infection.

Lamellar Phases Composed of Phospholipid, Cholesterol, and Ceramide, as Studied by 2H NMR

Sphingolipids constitute a significant fraction of cellular plasma membrane lipid content. Among sphingolipids, ceramide levels are usually very low. However, in some cell processes like apoptosis, cell membrane ceramide levels increase markedly because of the activation of enzymes like acid sphingomyelinase. This increase can change the physical state of the membrane by promoting molecular order and inducing solid-ordered (So) phase domains. This effect has been observed in a previous 2H NMR study on membranes consisting of palmitoyl sphingomyelin (PSM) and palmitoyl ceramide (PCer). Cholesterol (Chol), too, is present at high concentrations in mammalian plasma membranes and has a favorable interaction with sphingomyelin (SM), together forming domains in the liquid-ordered phase in model membranes. There are reports that Chol is able to displace ceramide (Cer) in SM bilayers and abolish the So phase domains formed by SM:Cer. This ability of Chol appears to be concentration dependent; in membranes with low Chol and high Cer contents, So phase domains rich in Cer coexist with the continuous fluid phase of the membrane. Here, we studied the effect of increasing PCer concentration in PSM:Chol bilayers, using 2H NMR. Chol:PCer mole ratios were 3:1, 3:2, and 3:3, at a fixed 7:3 phospholipid:cholesterol mol ratio. Both PSM and PCer were monitored in separate samples for changes in their physical state by introducing a perdeuterated palmitoyl chain in either molecule. Moreover, the effect of replacing PSM with DPPC was investigated to test the impact on membrane phase behavior of replacing the sphingosine with a palmitoylated glycerol backbone. We found that PCer can increase acyl chain order in both PSM:Chol and DPPC:Chol bilayers. Especially in bilayers with Chol:PCer 1:1 molar ratios, PCer induces highly stable So phase domains in both PSM and DPPC bilayers near 37°C. However, PCer has a more pronounced ordering effect on PSM compared to DPPC bilayers.

Solid-State NMR Investigations of the MHC II Transmembrane Domains: Topological Equilibria and Lipid Interactions

The major histocompatibility complex class II (MHC II) membrane proteins are key players in the adaptive immune response. An aberrant function of these molecules is associated with a large number of autoimmune diseases such as diabetes type I and chronic inflammatory diseases. The MHC class II is assembled from DQ alpha 1 and DQ beta 1 which come together as a heterodimer through GXXXG-mediated protein-protein interactions and a highly specific protein-sphingomyelin-C18 interaction motif located on DQA1. This association can have important consequences in regulating the function of these membrane proteins. Here, we investigated the structure and topology of the DQA1 and DQB1 transmembrane helical domains by CD-, oriented 2H and 15N solid-state NMR spectroscopies. The spectra at peptide-to-lipid ratios of 0.5 to 2mol% are indicative of a topological equilibrium involving a helix crossing the membrane with a tilt angle of about 20° and another transmembrane topology with around 30° tilt. The latter is probably representing a dimer. Furthermore, at the lowest peptide-to-lipid ratio, a third polypeptide population becomes obvious. Interestingly, the DQB1 and to a lesser extent the DQA1 transmembrane helical domains exhibit a strong fatty acyl chain disordering effect on the inner segments of the 2H-labelled palmitoyl chain of POPC bilayers. This phosphatidylcholine disordering requires the presence of sphingomyelin-C18 suggesting that the ensemble of transmembrane polypeptide and sphingolipid exerts positive curvature strain.

Synthesis of oxytocin derivatives lipidated via a carbonate or carbamate linkage as a long-acting therapeutic agent for social impairment-like behaviors

In the course of our studies of hydrophobic oxytocin (OT) analogues, we newly synthesized lipidated OT (LOT-4a-c and LOT-5a-c), in which a long alkyl chain (C14-C16) is conjugated via a carbonate or carbamate linkage at the Tyr-2 phenolic hydroxy group and a palmitoyl group at the terminal amino group of Cys-1. These LOTs did not activate OT and vasopressin receptors. Among the LOTs, however, LOT-4c, having a C16-chain via a carbonate linkage at the phenolic hydroxyl group of the Tyr-2, showed very long-lasting action for the recovery of impaired social behavior in CD38 knockout mice, a rodent model of autistic phenotypes, whereas the effect of OT itself rapidly diminished. These results indicate that LOT-4c may serve as a potential prodrug in mice.