Application of Liquid-Liquid Extraction for N-terminal Myristoylation Proteomics.

Proteins can be modified by lipids in various ways, for example, by myristoylation, palmitoylation, farnesylation, and geranylgeranylation-these processes are collectively referred to as lipidation. Current chemical proteomics using alkyne lipids has enabled the identification of lipidated protein candidates but does not identify endogenous lipidation sites and is not readily applicable to in vivo systems. Here, we introduce a proteomic methodology for global analysis of endogenous protein N-terminal myristoylation sites that combines liquid-liquid extraction of hydrophobic lipidated peptides with liquid chromatography-tandem mass spectrometry using a gradient program of acetonitrile in the high concentration range. We applied this method to explore myristoylation sites in HeLa cells and identified a total of 75 protein N-terminal myristoylation sites, which is more than the number of high-confidence myristoylated proteins identified by myristic acid analog-based chemical proteomics. Isolation of myristoylated peptides from HeLa digests prepared with different proteases enabled the identification of different myristoylated sites, extending the coverage of N-myristoylome. Finally, we analyzed in vivo myristoylation sites in mouse tissues and found that the lipidation profile is tissue-specific. This simple method (not requiring chemical labeling or affinity purification) should be a promising tool for global profiling of protein N-terminal myristoylation.

Small-angle x-ray scattering investigation of the integration of free fatty acids in polysorbate 20 micelles.

A critical quality attribute for liquid formulations is the absence of visible particles. Such particles may form upon polysorbate hydrolysis resulting in release of free fatty acids into solution followed by precipitation. Strategies to avoid this effect are of major interest for the pharmaceutical industry. In this context, we investigated the structural organization of polysorbate micelles alone and upon addition of the fatty acid myristic acid (MA) by small-angle x-ray scattering. Two complementary approaches using a model of polydisperse core-shell ellipsoidal micelles and an ensemble of quasiatomistic micelle structures gave consistent results well describing the experimental data. The small-angle x-ray scattering data reveal polydisperse mixtures of ellipsoidal micelles containing about 22-35 molecules per micelle. The addition of MA at concentrations up to 100 µg/mL reveals only marginal effects on the scattering data. At the same time, addition of high amounts of MA (>500 µg/mL) increases the average sizes of the micelles indicating that MA penetrates into the surfactant micelles. These results together with molecular modeling shed light on the polysorbate contribution to fatty acid solubilization preventing or delaying fatty acid particle formation.

Structural and Large-scale Analysis Unveil the Intertwined Paths Promoting NMT-catalyzed Lysine and Glycine Myristoylation.

N-myristoyltransferases (NMTs) catalyze protein myristoylation, a lipid modification crucial for cell survival and a range of pathophysiological processes. Originally thought to modify only N-terminal glycine α-amino groups (G-myristoylation), NMTs were recently shown to also modify lysine ε-amino groups (K-myristoylation). However, the clues ruling NMT-dependent K-myristoylation and the full range of targets are currently unknown. Here we combine mass spectrometry, kinetic studies, in silico analysis, and crystallography to identify the specific features driving each modification. We show that direct interactions between the substrate's reactive amino group and the NMT catalytic base promote K-myristoylation but with poor efficiency compared to G-myristoylation, which instead uses a water-mediated interaction. We provide evidence of depletion of proteins with NMT-dependent K-myristoylation motifs in humans, suggesting evolutionary pressure to prevent this modification in favor of G-myristoylation. In turn, we reveal that K-myristoylation may only result from post-translational events. Our studies finally unravel the respective paths towards K-myristoylation or G-myristoylation, which rely on a very subtle tradeoff embracing the chemical landscape around the reactive group.

A metabolic labeling protocol to enrich myristoylated proteins from Caenorhabditis elegans.

Myristoylation is a type of lipidation with important functions. Owing to the lack of high-quality antibodies against myristoylation, developing alternative methods for profiling myristoylated proteins is important. Here, we provide a protocol for metabolic labeling using click chemistry to profile myristoylated proteins in C. elegans. Our approach improves the signal/noise ratio by covalently linking the myristoylated proteins to the beads. This protocol provides a highly specific and reproducible way for enriching myristoylated proteins, which could be modified to analyze other types of lipidations. For complete details on the use and execution of this protocol, please refer to Tang et al. (2021).

Protein N-myristoylation: functions and mechanisms in control of innate immunity.

Protein N-myristoylation is an important fatty acylation catalyzed by N-myristoyltransferases (NMTs), which are ubiquitous enzymes in eukaryotes. Specifically, attachment of a myristoyl group is vital for proteins participating in various biological functions, including signal transduction, cellular localization, and oncogenesis. Recent studies have revealed unexpected mechanisms indicating that protein N-myristoylation is involved in host defense against microbial and viral infections. In this review, we describe the current understanding of protein N-myristoylation (mainly focusing on myristoyl switches) and summarize its crucial roles in regulating innate immune responses, including TLR4-dependent inflammatory responses and demyristoylation-induced innate immunosuppression during Shigella flexneri infection. Furthermore, we examine the role of myristoylation in viral assembly, intracellular host interactions, and viral spread during human immunodeficiency virus-1 (HIV-1) infection. Deeper insight into the relationship between protein N-myristoylation and innate immunity might enable us to clarify the pathogenesis of certain infectious diseases and better harness protein N-myristoylation for new therapeutics.

Erythorbyl fatty acid ester as a multi-functional food emulsifier: Enzymatic synthesis, chemical identification, and functional characterization of erythorbyl myristate.

Erythorbyl myristate (EM), a potential multi-functional food emulsifier, was newly synthesized by immobilized lipase-catalyzed esterification between antioxidative erythorbic acid and antibacterial myristic acid. The yield and productivity of EM were 56.13 ± 2.51 mg EM/g myristic acid and 1.76 ± 0.08 mM/h, respectively. The molecular structure of EM was identified as (R)-2-((R)-3,4-dihydroxy-5-oxo-2,5-dihydrofuran-2-yl)-2-hydroxyethyl tetradecanoate using HPLC-ESI/MS and 2D [1H-1H] NMR COSY. The hydrophilic-lipophilic balance of EM was 11.5, suggesting that EM could be proper to stabilize oil-in-water emulsions. Moreover, isothermal titration calorimetry demonstrated the micellar thermodynamic behavior of EM and determined its critical micelle concentration (0.36 mM). In terms of antioxidative property, EM exhibited the radical scavenging activity against DPPH (EC50: 35.47 ± 0.13 µM) and ABTS (EC50: 36.45 ± 1.98 µM) radicals. Finally, EM showed bacteriostatic and bactericidal activities against Gram-positive foodborne pathogens (minimum inhibitory concentration: 0.06-0.60 mM; minimum bactericidal concentration: 0.07-0.93 mM).

Power function setting in charged aerosol detection for the linearization of detector response - optimization strategies and their application.

Charged aerosol detection (CAD) is an universal technique in liquid chromatography that is increasingly used for the quality control of drugs. Consequently, it has found its way into compendial monographs promoted by its simple and robust application. However, the response of CAD is inherently nonlinear due to its principle of function. Thus, easy and rapid linearization procedures, in particular regarding compendial applications, are highly desirable. One effective approach to linearize the detector's signal makes use of the built-in power function value (PFV) setting of the instrument. The PFV is basically a multiplication factor to the power law exponent of the equation describing the CAD's response, thereby altering the detector's signal output to optimize the quasi-linear range of the response curve. The experimental optimization of the PFV for a series of analytes is a time-consuming process, limiting the practicability of this approach. Here, two independent approaches for the determination of the optimal PFV based on an empirical model and a mathematical transformation in each case, are evaluated. Both approaches can be utilized to predict the optimal PFV for each analyte solely based on the experimental results of a series of calibration standards obtained at a single PFV. The approaches were applied to the HPLC-UV-CAD impurity analysis of the drug gabapentin to improve the observed nonlinear response of the impurities in the range of interest. The predicted optimal PFV of both approaches were in good agreement with the experimentally obtained optimal PFV of the analytes. As a result, the accuracy of the method was significantly improved when using the optimal PFV (90 - 105% versus 81 - 115% recovery rate for quantitation by either single-point calibration or linear regression) for the majority of the analytes. The final method with a PFV adjusted to 1.30 was validated with respect to ICH guideline Q2(R1).

Thiol-ene enabled preparation of S-lipidated anti-HBV peptides.

Despite significant efforts made towards treatments for Hepatitis B virus (HBV), a long-term curative treatment has thus far eluded scientists. Recently, the Sodium Taurocholate Co-Transporting Polypeptide (NTCP) receptor has been identified as the entry pathway of HBV into hepatocytes. Myrcludex B, an N-terminally myristoylated 47-mer peptide mimic of the preS1 domain of the Hepatitis B virion, was identified as a potent protein-protein interaction (PPI) inhibitor blocking HBV fusion (IC50 = 140 pM). Herein we report an optimised chemical synthesis of Myrcludex B and a series of novel analogues. Employing a small modification to the Cysteine Lipidation of a Peptide or Amino acid (CLipPA) thiol-ene reaction, a library of S-lipidated Myrcludex B and truncated (21-mer) analogues were prepared, providing novel chemical space to probe for the discovery of novel anti-HBV peptides. The S-lipidated analogues showed an equivalent or a slight decrease (∼2-fold) in binding effectiveness to NTCP expressing hepatocytes compared to Myrcludex B. Three S-lipidated analogues were highly potent HBV inhibitors (IC50 0.97-3.32 nM). These results demonstrate that incorporation of heteroatoms into the lipid 'anchor' is tolerated by this antiviral scaffold and to the best of our knowledge constitutes the first report of potent S-lipidated antiviral peptides. Interestingly, despite only moderate reductions in binding effectiveness, truncated analogues possessed dramatically reduced inhibitory activity thus providing new insights into the structure activity relationship of these hitherto unreported antiviral S-lipopeptides.

Green synthesis, antimicrobial, antibiofilm and antitumor activities of superparamagnetic γ-Fe2O3 NPs and their molecular docking study with cell wall mannoproteins and peptidoglycan.

Fatty acids-assisted superparamagnetic maghemite (γ-Fe2O3) NPs was biologically synthesized using extract of polyherbal drug Liv52 (L52E). The NPs were characterized by UV-vis spectroscopy, FT-IR, SEM, TEM, EDX, XRD and VSM. The major biological molecules present in L52E analysed by GC-MS were saturated fatty acids (palmitic acid 21.95%; stearic acid 13.99%; myristic acid 1.14%), monounsaturated fatty acid (oleic acid 18.43%), polyunsaturated fatty acid (linoleic acid 20.45%), and aromatic phenol (cardanol monoene 11.92%) that could imply in bio-fabrication and stabilization of γ-Fe2O3 NPs. The FT-IR spectra revealed involvement of carboxylic group of fatty acids, amide group of proteins and hydroxyl group of phenolic compounds that acts as reducing and capping agents. The synthesized NPs were used to investigate their antimicrobial, antibiofilm activity against P. aeruginosa, MRSA and C. albicans and anticancer activity on colon cancer cells (HCT-116) for biomedical applications. Further, molecular docking study was performed to explore the interaction of Fe2O3 NPs with major cell wall components i.e., peptidoglycan and mannoproteins. The docking studies revealed that Fe2O3 interacted efficiently with peptidoglycan and mannoproteins and Fe2O3 get accommodated into catalytic cleft of mannoprotein. Due to magnetic property, the biological activity of γ-Fe2O3 can be further enhanced by applying external magnetic field alone or in amalgamation with other therapeutics drugs.

The Effect of Olive Fruit Fly Bactrocera oleae (Rossi) Infestation on Certain Chemical Parameters of Produced Olive Oils.

Olives affected by active and damaging infestation (olive fruit fly Bactrocera oleae (Rossi)) were assayed for their chemical composition. Biophenols were determined by HPLC, sterols, triterpenic dialcohols, and fatty acids by gas chromatography analysis. The acquired data were statistically analyzed. Oils produced from "Istrska belica" fruit affected by active infestation compared to the oils made from fruit affected by damaging infestation showed higher amounts of total oleuropein biofenols (377.3 versus (vs.) 106.6 mg/kg), total biophenols (755 vs. 377 mg/kg), lignans (85.3 vs. 32.9 mg/kg), the dialdehydic form of decarboxymethyl oleuropein aglycone (DMO-Agl-dA) (148.3 vs. 49.0 mg/kg), its oxidized form (DMO-Agl-dA)ox (35.2 vs. 8.5 mg/kg), the dialdehydic form of oleuropein aglycone (O-Agl-dA) (61.1 vs. 8.0 mg/kg), the dialdehydic form of ligstroside aglycone (L-Agl-dA) (63.5 vs. 28.0 mg/kg), the aldehydic form of oleuropein aglycone (O-Agl-A) (40.6 vs. 8.4 mg/kg), and lower amounts of tyrosol (Tyr) (6.0 vs. 13. 9 mg/kg) and the aldehydic form of ligstroside aglycone (L-Agl-A) (13.8 vs. 40.3 mg/kg). Higher values of stigmasterol (2.99%) and lower values of campesterol (2.25%) were determined in oils affected by damaging infestation; an increase in triterpenic dialcohols was also observed (3.04% for damaging and 1.62% for active infestation). Oils affected by damaging infestation, compared to active infestation, showed lower amounts of oleic acid (73.89 vs. 75.15%) and higher amounts of myristic (0.013 vs. 0.011%), linoleic (7.27 vs. 6.48%), and linolenic (0.74 vs. 0.61%) acids.

Aggregation Behavior, Antibacterial Activity and Biocompatibility of Catanionic Assemblies Based on Amino Acid-Derived Surfactants.

The surface activity, aggregates morphology, size and charge characteristics of binary catanionic mixtures containing a cationic amino acid-derived surfactant N(π), N(τ)-bis(methyl)-L-Histidine tetradecyl amide (DMHNHC14) and an anionic surfactant (the lysine-based surfactant Nα-lauroyl-Nεacetyl lysine (C12C3L) or sodium myristate) were investigated for the first time. The cationic surfactant has an acid proton which shows a strong pKa shift irrespective of aggregation. The resulting catanionic mixtures exhibited high surface activity and low critical aggregation concentration as compared with the pure constituents. Catanionic vesicles based on DMHNHC14/sodium myristate showed a monodisperse population of medium-size aggregates and good storage stability. According to Small-Angle X-Ray Scattering (SAXS), the characteristics of the bilayers did not depend strongly on the system composition for the positively charged vesicles. Negatively charged vesicles (cationic surfactant:myristate ratio below 1:2) had similar bilayer composition but tended to aggregate. The DMHNHC14-rich vesicles exhibited good antibacterial activity against Gram-positive bacteria and their bactericidal effectivity declined with the decrease of the cationic surfactant content in the mixtures. The hemolytic activity and cytotoxicity of these catanionic formulations against non-tumoral (3T3, HaCaT) and tumoral (HeLa, A431) cell lines also improved by increasing the ratio of cationic surfactant in the mixture. These results indicate that the biological activity of these systems is mainly governed by the cationic charge density, which can be modulated by changing the cationic/anionic surfactant ratio in the mixtures. Remarkably, the incorporation of cholesterol in those catanionic vesicles reduces their cytotoxicity and increases the safety of future biomedical applications of these systems.

Impact of Silicone Oil on Free Fatty Acid Particle Formation due to Polysorbate 20 Degradation.

PURPOSE: Polysorbate 20 (PS20), a commonly used surfactant in biopharmaceutical formulations, can undergo hydrolytic degradation resulting in free fatty acids (FFAs) that precipitate to form particles. This work investigates the ability for silicone oil (si-oil) coated on the interior walls of prefilled syringes (PFSs) to act as a sink for FFAs and potentially delay FFA particle formation. METHODS: Myristic acid distribution coefficient was measured in a two-phase system containing si-oil and formulation buffer at a range of aqueous conditions. An empirical model was built from these data to predict distribution coefficient based on aqueous conditions. To verify the model, PS20 was degraded using model lipases side-by-side in glass vials and PFSs while monitoring sub-visible particles. RESULTS: The empirical model demonstrates that the partitioning of myristic acid into si-oil is maximized at low pH and low PS20 concentration. The model predicts that the presence of si-oil at levels typical in PFSs provides at most an 8.5% increase in the total carrying capacity for myristic acid compared to a non-coated glass vial. The time to onset of FFA particles was equivalent between degradations performed in two PFS models coated with differing levels of silicone oil and in non-coated glass vials. CONCLUSION: Herein, we demonstrate that FFAs partition from aqueous solution into si-oil. However, the extent of the partitioning effect is not large enough to delay PS20-related FFA particle formation at typical formulation conditions (pH 5.0-7.5, 0.01% - 0.1% w/v PS20) filled in typical PFSs (<1.0 mg si-oil/mL aqueous fill).

Improving Prediction of Free Fatty Acid Particle Formation in Biopharmaceutical Drug Products: Incorporating Ester Distribution during Polysorbate 20 Degradation.

Polysorbate 20 (PS20) is a commonly used surfactant in biopharmaceutical formulations. It is a heterogeneous surfactant containing a distribution of fatty acid esters, which are subject to hydrolytic degradation, generating free fatty acids (FFAs). The FFAs can form visible or subvisible particles in drug product on stability. A previous FFA solubility model, developed by our group, predicts solubility limits for the three most prevalent FFA degradation products of PS20: lauric, myristic, and palmitic acid. The model takes into account two formulation parameters, pH and PS20 concentration, and their effect on FFA solubility. This work identifies a third parameter that has an impact on FFA solubility: PS20 ester distribution. When PS20 is hydrolytically degraded, the ester distribution of the remaining surfactant changes on stability. Ester distribution is known to influence the critical micelle concentration (CMC) of PS20 such that the monoesters have a much higher CMC compared to the higher-order esters (HOE). We hypothesize that as PS20 degrades, the CMC changes, affecting the proportion of PS20 that is present in micelles and capable of sequestering and solubilizing FFAs in these micelles. Here, PS20 was separated into monoester, HOE, and polyol fractions. The monoester and HOE fractions were mixed together to generate the mock degradation profiles of hydrolytically degraded PS20. FFA solubility was measured as a function of the concentration of these mock-degraded (MD) PS20s. The results indicate that ester distribution does have an impact on FFA solubility, especially at higher MD PS20 concentrations. HOEs solubilize up to 30 µg/mL more lauric acid than an equivalent amount of monoesters at a MD PS20 level of 0.06% w/v. With the addition of % HOE peak area fraction as a third parameter representing the ester distribution of PS20, the refined FFA solubility model more accurately predicts FFA solubility in protein formulations at 5 °C. The refined model suggests that drug products containing trace levels of host cell proteins (HCPs) that preferentially degrade HOEs of PS20 are at a higher risk of particle formation.

Preformulation Studies of a Stable PTEN-PDZ Lipopeptide Able to Cross an In Vitro Blood-Brain-Barrier Model as a Potential Therapy for Alzheimer's Disease.

PURPOSE: Amyloid ß (Aß) drives the accumulation of excess Phosphatase and Tensin Homolog Deleted on Chromosome 10 (PTEN) at synapses, inducing synaptic depression and perturbing memory. This recruitment of PTEN to synapses in response to Aß drives its interaction with PSD95/Disc large/Zonula occludens-1 (PDZ) proteins and, indeed, we previously showed that an oligo lipopeptide (PTEN-PDZ) capable of blocking such PTEN:PDZ interactions rescues the synaptic and cognitive deficits in a mouse model of Alzheimer's disease. Hence, the PTEN:PDZ interaction appears to be crucial for Aß-induced synaptic and cognitive impairment. Here we have evaluated the feasibility of using PTEN-PDZ lipopeptides based on the human/mouse PTEN C-terminal sequence, testing their stability in biological fluids, their cytotoxicity, their ability to self-assemble and their in vitro blood-brain barrier (BBB) permeability. Myristoyl or Lauryl tails were added to the peptides to enhance their cell permeability. METHODS: Lipopeptides self assembly was assessed using electron microscopy and the thioflavin T assay. Stability studies in mouse plasma (50%), intestinal washing, brain and liver homogenates as well as permeability studies across an all human 2D blood-brain barrier model prepared with human cerebral endothelial cells (hCMEC/D3) and human astrocytes (SC-1800) were undertaken. RESULTS: The mouse lauryl peptide displayed enhanced overall stability in plasma, ensuring a longer half-life in circulation that meant there were larger amounts available for transport across the BBB (Papp0-4h: 6.28 ± 1.85 × 10-6 cm s-1). CONCLUSION: This increased availability, coupled to adequate BBB permeability, makes this peptide a good candidate for therapeutic parenteral (intravenous, intramuscular) administration and nose-to-brain delivery. Graphical Abstract.

A Novel Approach to Bacterial Expression and Purification of Myristoylated Forms of Neuronal Calcium Sensor Proteins.

N-terminal myristoylation is a common co-and post-translational modification of numerous eukaryotic and viral proteins, which affects their interaction with lipids and partner proteins, thereby modulating various cellular processes. Among those are neuronal calcium sensor (NCS) proteins, mediating transduction of calcium signals in a wide range of regulatory cascades, including reception, neurotransmission, neuronal growth and survival. The details of NCSs functioning are of special interest due to their involvement in the progression of ophthalmological and neurodegenerative diseases and their role in cancer. The well-established procedures for preparation of native-like myristoylated forms of recombinant NCSs via their bacterial co-expression with N-myristoyl transferase from Saccharomyces cerevisiae often yield a mixture of the myristoylated and non-myristoylated forms. Here, we report a novel approach to preparation of several NCSs, including recoverin, GCAP1, GCAP2, neurocalcin δ and NCS-1, ensuring their nearly complete N-myristoylation. The optimized bacterial expression and myristoylation of the NCSs is followed by a set of procedures for separation of their myristoylated and non-myristoylated forms using a combination of hydrophobic interaction chromatography steps. We demonstrate that the refolded and further purified myristoylated NCS-1 maintains its Са2+-binding ability and stability of tertiary structure. The developed approach is generally suited for preparation of other myristoylated proteins.

Insulin-Sensitizing Activity of Sub-Nanoscaled Polyalkoxyvanadate Clusters.

Sub-nanoscaled polyalkoxyvanadates (PAOVs) functionalized with various aliphatic acids are evaluated for their insulin-sensitizing activity in lowering the blood glucose levels of diabetic mice in typical glucose tolerance tests. All the PAOVs can restore the blood glucose to normal levels after a single oral administration of PAOVs. Among them, the myristic acid-modified PAOVs enable the response of insulin to the repeated glucose challenges, lasting for up to 13 h. The combined administration of PAOVs exerts better glucose control over insulin alone, while the capric acid- and myristic acid-modified ones can enhance the responsiveness of insulin to glucose challenge and is comparable to a clinical-used derivative of insulin. Interestingly, continuous glucose monitoring shows that myristic acid-modified PAOV derivatives sensitize the responsiveness of insulin, almost matching with that of a healthy pancreas. These discoveries open up new opportunities for the application of PAOVs to promote glucose-responsive and long-lasting activity of insulin, which are expected to aid the accurate blood glucose control in insulin therapy while reducing the number of insulin administrations.

In Vitro Investigations of Acetohexamide Binding to Glycated Serum Albumin in the Presence of Fatty Acid.

The interaction of drugs with human serum albumin (HSA) is an important element of therapy. Albumin affects the distribution of the drug substance in the body, as well as its pharmacokinetic and pharmacodynamic properties. On the one hand, inflammation and protein glycation, directly associated with many pathological conditions and old age, can cause structural and functional modification of HSA, causing binding disorders. On the other hand, the widespread availability of various dietary supplements that affect the content of fatty acids in the body means that knowledge of the binding activity of transporting proteins, especially in people with chronic diseases, e.g., diabetes, will achieve satisfactory results of the selected therapy. Therefore, the aim of the present study was to evaluate the effect of a mixture of fatty acids (FA) with different saturated and unsaturated acids on the affinity of acetohexamide (AH), a drug with hypoglycaemic activity for glycated albumin, simulating the state of diabetes in the body. Based on fluorescence studies, we can conclude that the presence of both saturated and unsaturated FA disturbs the binding of AH to glycated albumin. Acetohexamide binds more strongly to defatted albumin than to albumin in the presence of fatty acids. The competitive binding of AH and FA to albumin may influence the concentration of free drug fraction and thus its therapeutic effect.

Membrane Binding of Neuronal Calcium Sensor-1: Highly Specific Interaction with Phosphatidylinositol-3-Phosphate.

Neuronal calcium sensors are a family of N-terminally myristoylated membrane-binding proteins possessing a different intracellular localization and thereby targeting unique signaling partner(s). Apart from the myristoyl group, the membrane attachment of these proteins may be modulated by their N-terminal positively charged residues responsible for specific recognition of the membrane components. Here, we examined the interaction of neuronal calcium sensor-1 (NCS-1) with natural membranes of different lipid composition as well as individual phospholipids in form of multilamellar liposomes or immobilized monolayers and characterized the role of myristoyl group and N-terminal lysine residues in membrane binding and phospholipid preference of the protein. NCS-1 binds to photoreceptor and hippocampal membranes in a Ca2+-independent manner and the binding is attenuated in the absence of myristoyl group. Meanwhile, the interaction with photoreceptor membranes is less dependent on myristoylation and more sensitive to replacement of K3, K7, and/or K9 of NCS-1 by glutamic acid, reflecting affinity of the protein to negatively charged phospholipids. Consistently, among the major phospholipids, NCS-1 preferentially interacts with phosphatidylserine and phosphatidylinositol with micromolar affinity and the interaction with the former is inhibited upon mutating of N-terminal lysines of the protein. Remarkably, NCS-1 demonstrates pronounced specific binding to phosphoinositides with high preference for phosphatidylinositol-3-phosphate. The binding does not depend on myristoylation and, unexpectedly, is not sensitive to the charge inversion mutations. Instead, phosphatidylinositol-3-phosphate can be recognized by a specific site located in the N-terminal region of the protein. These data provide important novel insights into the general mechanism of membrane binding of NCS-1 and its targeting to specific phospholipids ensuring involvement of the protein in phosphoinositide-regulated signaling pathways.

Computational Design of Myristoylated Cell-Penetrating Peptides Targeting Oncogenic K-Ras.G12D at the Effector-Binding Membrane Interface.

A number of small inhibitors have been developed in recent years to target the cancer-driving protein, K-Ras. In this study, we propose and design a novel way of targeting oncogenic K-Ras4B.G12D with myristoylated cell-penetrating peptides which become membrane-anchored and lock the protein into an inactive state. In all atom molecular dynamics simulations, such peptides associate with K-Ras4B exclusively at the effector-binding region, which, in turn, is expected to hinder the binding of downstream effector proteins (e.g., C-Raf). The myristoylated R9 (Arg9) peptide locks K-Ras4B.G12D into orientations that are unfavorable for effector binding. After breaking the cyclic structure and myristoylation, a cell-penetrating peptide cyclorasin 9A5, which was designed for targeting the Ras/Raf interface, is also found to be effective in targeting the Ras/membrane interface. The myristoylated peptides likely have high cell permeability because of their mixed cationic/hydrophobic character at the N-terminus, while simultaneously the subsequent multiple charges help to maintain a close association of the peptide with the K-Ras4B.G12D effector-binding lobe. Targeting protein-membrane interfaces is starting to attract attention very recently, thanks to our understanding of the signaling mechanism of an increased number of peripheral membrane proteins. The strategy used in this study has potential applications in the design of drugs against K-Ras4B-driven cancers. It also provides insights into the general principles of targeting protein-membrane interfaces.

Binding to medium and long chain fatty acyls is a common property of HEAT and ARM repeat modules.

Covalent post-translational modification (PTM) of proteins with acyl groups of various carbon chain-lengths regulates diverse biological processes ranging from chromatin dynamics to subcellular localization. While the YEATS domain has been found to be a prominent reader of acetylation and other short acyl modifications, whether additional acyl-lysine reader domains exist, particularly for longer carbon chains, is unclear. Here, we employed a quantitative proteomic approach using various modified peptide baits to identify reader proteins of various acyl modifications. We discovered that proteins harboring HEAT and ARM repeats bind to lysine myristoylated peptides. Recombinant HEAT and ARM repeats bind to myristoylated peptides independent of the peptide sequence or the position of the myristoyl group. Indeed, HEAT and ARM repeats bind directly to medium- and long-chain free fatty acids (MCFA and LCFA). Lipidomic experiments suggest that MCFAs and LCFAs interact with HEAT and ARM repeat proteins in mammalian cells. Finally, treatment of cells with exogenous MCFAs and inhibitors of MCFA-CoA synthases increase the transactivation activity of the ARM repeat protein ß-catenin. Taken together, our results suggest an unappreciated role for fatty acids in the regulation of proteins harboring HEAT or ARM repeats.