Leucine Dipeptides Research Articles

Gold and Silver Nanoparticles as Biosensors: Characterization of Surface and Changes in the Adsorption of Leucine Dipeptide under the Influence of Substituent Changes.

Early detection of diseases can increase the chances of successful treatment and survival. Therefore, it is necessary to develop a method for detecting or sensing biomolecules that cause trouble in living organisms. Disease sensors should possess specific properties, such as selectivity, reproducibility, stability, sensitivity, and morphology, for their routine application in medical diagnosis and treatment. This work focuses on biosensors in the form of surface-functionalized gold (AuNPs) and silver nanoparticles (AgNPs) prepared using a less-time-consuming, inexpensive, and efficient synthesis route. This allows for the production of highly pure and stable (non-aggregating without stabilizers) nanoparticles with a well-defined spherical shape, a desired diameter, and a monodisperse distribution in an aqueous environment, as confirmed by transmission electron microscopy with energy-dispersive X-ray spectroscopy (TEM-EDS), X-ray diffraction (XRD), photoelectron spectroscopy (XPS), ultraviolet-visible (UV-VIS) spectroscopy, and dynamic light scattering (DLS). Thus, these nanoparticles can be used routinely as biomarker sensors and drug-delivery platforms for precision medicine treatment. The NPs' surface was coated with phosphonate dipeptides of L-leucine (Leu; l-Leu-C(R1)(R2)PO3H2), and their adsorption was monitored using SERS. Reproducible spectra were analyzed to determine the orientation of the dipeptides (coating layers) on the nanoparticles' surface. The appropriate R2 side chain of the dipeptide can be selected to control the arrangement of these dipeptides. This allows for the proper formation of a layer covering the nanoparticles while also simultaneously interacting with the surrounding biological environment, such as cells, tissues, and biological fluids.

Mammalian telomeric RNA (TERRA) can be translated to produce valine–arginine and glycine–leucine dipeptide repeat proteins

Mammalian telomeres consist of (TTAGGG)n repeats. Transcription of the C-rich strand generates a G-rich RNA, termed TERRA, containing G-quadruplex structures. Recent discoveries in several human nucleotide expansion diseases revealed that RNA transcripts containing long runs of 3 or 6 nt repeats which can form strong secondary structures can be translated in multiple frames to generate homopeptide or dipeptide repeat proteins, and multiple studies have shown them to be toxic in cells. We noted that the translation of TERRA would generate two dipeptide repeat proteins: highly charged repeating valine-arginine (VR)n and hydrophobic repeating glycine-leucine (GL)n. Here, we synthesized these two dipeptide proteins and raised polyclonal antibodies to VR. The VR dipeptide repeat protein binds nucleic acids and localizes strongly to replication forks in DNA. Both VR and GL form long 8-nm filaments with amyloid properties. Using labeled antibodies to VR and laser scanning confocal microscopy, threefold to fourfold more VR was observed in the nuclei of cell lines containing elevated TERRA as contrasted to a primary fibroblast line. Induction of telomere dysfunction via knockdown of TRF2 led to higher amounts of VR, and alteration of TERRA levels using a locked nucleic acid (LNA) GapmeR led to large nuclear VR aggregates. These observations suggest that telomeres, in particular in cells undergoing telomere dysfunction, may express two dipeptide repeat proteins with potentially strong biological properties.

Low-Dose Doxycycline Treatment Normalizes Levels of Some Salivary Metabolites Associated with Oral Microbiota in Patients with Primary Sjögren's Syndrome.

Saliva is a complex oral fluid, and plays a major role in oral health. Primary Sjögren’s syndrome (pSS), as an autoimmune disease that typically causes hyposalivation. In the present study, salivary metabolites were studied from stimulated saliva samples (n = 15) of female patients with pSS in a group treated with low-dose doxycycline (LDD), saliva samples (n = 10) of non-treated female patients with pSS, and saliva samples (n = 14) of healthy age-matched females as controls. Saliva samples were analyzed with liquid chromatography mass spectrometry (LC-MS) based on the non-targeted metabolomics method. The saliva metabolite profile differed between pSS patients and the healthy control (HC). In the pSS patients, the LDD treatment normalized saliva levels of several metabolites, including tyrosine glutamine dipeptide, phenylalanine isoleucine dipeptide, valine leucine dipeptide, phenylalanine, pantothenic acid (vitamin B5), urocanic acid, and salivary lipid cholesteryl palmitic acid (CE 16:0), to levels seen in the saliva samples of the HC. In conclusion, the data showed that pSS is associated with an altered saliva metabolite profile compared to the HC and that the LLD treatment normalized levels of several metabolites associated with dysbiosis of oral microbiota in pSS patients. The role of the saliva metabolome in pSS pathology needs to be further studied to clarify if saliva metabolite levels can be used to predict or monitor the progress and treatment of pSS.

Synthesis of phenylalanine and leucine dipeptide functionalized silica-based nanoporous material as a safe UV filter for sunscreen

To allay concerns that sunscreen ingredients are harmful to skin health, we have developed phenylalanine (Phe) and leucine (Leu) dipeptide functionalized silica-based nanoporous material (Phe-Leu-OMe-Meso) as a safe ultraviolet filter for skin protection from UVA/UVB sun irradiation. The selected peptide-based functional molecules are composed of natural amino acids, which have obvious advantages compared with other biological molecules and organic molecules, such as low toxicity and high stability. Octyl salicylate (OS) is adopted as the drug molecule to observe the adsorption behavior of the nanoporous materials. The results confirm that Phe groups effectively improve the OS loaded capacity of Phe-Leu-OMe-Meso by aromatic π–π stacking interaction, and the experimental data obey the pseudo-second-order kinetic model and the Langmuir, Freundlich, Temkin isotherm model. Moreover, in vitro evaluation of the spectrophotometric sun protection factor indicates that Leu groups make Phe-Leu-OMe-Meso possess an excellent sunscreen efficacy close to that of mesoporous TiO2 and exhibit a synergistic effect with OS on sun protection. In summary, the dipeptide functionalization of silica-based nanoporous material has an excellent prospect in the application of sunscreen.

Novel modified leucine and phenylalanine dipeptides modulate viability and attachment of cancer cells

Here, we describe the synthesis and biological characterization of 32 novel phenylalanine and leucine dipeptides modified on both the N and C termini by salicylic acid and aromatic or alicyclic amines, respectively. All compounds displayed antiproliferative activity in the tested cancer cell lines and eight of the compounds exhibited single digit micromolar GI50 values. Treated cells rapidly detached from surface of tissue culture dishes and we found that focal adhesion kinase (FAK), p130CAS and paxillin, which are important regulators of cell adhesion, were dephosphorylated at Y397, Y410 and Y118, respectively. The most potent compound reduced proliferation in the HCT-116 cell line in a dose-dependent manner, as shown by a decrease in 5-bromo-2′-deoxyuridine incorporation into DNA. Furthermore, this compound increased the levels of several apoptotic markers, including activated caspases, and increased site-specific poly-(ADP-ribose)polymerase (PARP) cleavage.

Antineoplastic activity of linear leucine homodipeptides and their potential mechanisms of action.

Galaxamide is a rare cyclic homopentapeptide composed of three leucines and two N-methyl leucines isolated from marine algae Galaxaura filamentosa. The strong antitumor activity of this compound makes it a promising candidate for tumor therapy. The synthesis of galaxamide, however, is a complex process, and it has poor water solubility. On the basis of its special chemical composition, we designed a series of linear leucine homopeptides. Among seven dipeptide derivatives, five compounds with terminal protection groups and methyl substitution of the hydrogen in the amido group showed remarkable inhibitory effects against various cancer cells. N-tertbutyl-D-leucine-N-methyl-D-leucinebenzyl (A7), the only stereomer condensed by two D-leucines, showed the highest antineoplastic activity. A7-treated cells showed cell cycle arrest and morphological changes typical of cells undergoing apoptosis. The population of Annexin-V positive/propidium iodide-negative cells also increased, indicating the induction of early apoptosis. A7 promoted the cleavage of caspase-9 and caspase-3, as well as increased intracellular Ca levels and decreased the mitochondrial membrane potential. Collectively, certain linear leucine dipeptides derived from cyclic pentapeptide are able to inhibit tumor cell proliferation through cell cycle arrest and apoptosis induction. The N-methyl group in the side chain and the D/L conformation of the amino-acid residue are critical for their activity.

Oral Administration of Glycine and Leucine Dipeptides Improves Skin Hydration and Elasticity in UVB-Irradiated Hairless Mice.

Placenta is a special organ that contains many nutrients such as growth factors, minerals, and bioactive peptides. Dipeptides of glycine and leucine are major components of porcine placenta extracts (PPE) that has been used as an alternative of human placenta extracts. In this study, we investigated whether major peptides of PPE, Glycyl-L-Leucine (Gly-Leu), L-Leucyl-Glycine (Leu-Gly), and L-Leucyl-L-Leucine (Leu-Leu), affect skin hydration and elasticity in vitro and in vivo. We found that Gly-Leu and Leu-Gly dipeptides induced the expression of transglutaminase 1 in normal human epidermal keratinocytes (NHEKs) whereas Leu-Leu dipeptides did not. Treatment with Gly-Leu or Leu-Gly significantly increased hyaluronan (HA) synthesis in NHEKs and the upregulation of hyaluronan synthase 2 (HAS2) mRNA level was confirmed. In addition, elastase activity was inhibited in NHEKs treated with Gly-Leu or Leu-Gly dipeptides. Oral administration of Gly-Leu or Leu-Gly dipeptides increased skin hydration and elasticity in UVB-irradiated hairless mice. The significant upregulation of HA in UVB-irradiated hairless mice was observed in response to oral administration of Gly-Leu or Leu-Gly. These results suggest that the major dipeptides of porcine placenta, Gly-Leu and Leu-Gly, are potentially active ingredients for skin moisturization formulations.

A self-assembling fluorescent dipeptide conjugate for cell labelling

Derivatives of fluorophore FITC (fluorescein isothiocyanate) are widely used in bioassays to label proteins and cells. An N-terminal leucine dipeptide is attached to FITC, and we show that this simple conjugate molecule is cytocompatible and is uptaken by cells (human dermal and corneal fibroblasts) in contrast to FITC itself. Co-localisation shows that FITC-LL segregates in peri-nuclear and intracellular vesicle regions. Above a critical aggregation concentration, the conjugate is shown to self-assemble into beta-sheet nanostructures comprising molecular bilayers.

Correction to Balanced Protein-Water Interactions Improve Properties of Disordered Proteins and Non-Specific Protein Association.

In our paper1 we did not properly acknowledge the contribution by Head-Gordon and co-workers2 (ref 41 of our manuscript). These authors used a similar modification of the mixing rules for protein–water interactions, although the correction was applied on a per-atom basis to both the characteristic distance (sigma) and energy (epsilon) parameters rather than using a single scaling factor for epsilon as we did. Head-Gordon and co-workers used extensive solvation free energy data on model organic compounds to fit their parameters, whereas we used FRET efficiencies for a single unfolded protein (cold shock protein) to estimate our scaling parameter. Overall, we reached qualitatively similar conclusions regarding the solvation free energies of force fields with Amber 943 Lennard-Jones parameters being too unfavorable, as reported previously by these authors.2 The more systematic approach of Nerenberg et al. showed a mean signed error of solvation free energies close to zero for their optimized force field.2 We would also like to make a specific correction to the sentence on page 5115, which is inaccurate: “Recently, a full parametrization of protein Lennard-Jones parameters to reproduce solvation free energies showed some promising results, but unfortunately resulted in very unstable folded proteins”. Instead, it should read as follows: “Recently, a rescaling of protein–water Lennard-Jones parameters to reproduce solvation free energies showed some promising results for correcting protein–protein interactions using concentrated solutions of glycine and leucine dipeptides. In conjunction with a 12-10 van der Waals (vdW) term to replace the standard 12-6 vdW potential to model the nonelectrostatic part of the hydrogen bonding between the backbone carbonyl oxygens and amide protons, this modification resulted in stable folded proteins.”

Large-scaled metabolic profiling of human dermal fibroblasts derived from pseudoxanthoma elasticum patients and healthy controls.

Mutations in the ABC transporter ABCC6 were recently identified as cause of Pseudoxanthoma elasticum (PXE), a rare genetic disorder characterized by progressive mineralization of elastic fibers. We used an untargeted metabolic approach to identify biochemical differences between human dermal fibroblasts from healthy controls and PXE patients in an attempt to find a link between ABCC6 deficiency, cellular metabolic alterations and disease pathogenesis. 358 compounds were identified by mass spectrometry covering lipids, amino acids, peptides, carbohydrates, nucleotides, vitamins and cofactors, xenobiotics and energy metabolites. We found substantial differences in glycerophospholipid composition, leucine dipeptides, and polypeptides as well as alterations in pantothenate and guanine metabolism to be significantly associated with PXE pathogenesis. These findings can be linked to extracellular matrix remodeling and increased oxidative stress, which reflect characteristic hallmarks of PXE. Our study could facilitate a better understanding of biochemical pathways involved in soft tissue mineralization.

Efficient parameterization of torsional terms for force fields.

A novel method is presented for fitting force-field dihedral angles using an ensemble of structures generated from an ab initio Monte Carlo simulation. Importance sampling is used to achieve an efficient algorithm using a low level of theory to minimize the system at each step with the dihedral angles constrained, followed by dihedral fitting using the single point energies at a higher level of theory. The resulting method is an order of magnitude more efficient than the traditional method of doing a constrained scan over each dihedral independently. Also as the sampling is more uniformly distributed, the full surface is approximated to a greater accuracy. The dihedral fitting is done with a nonlinear optimization method to vary the phase as well as the force constant. The utility of the method is demonstrated by fitting dihedrals of methyl L-lactate, diisopropyl fluorophosphate, isopentenyl phosphate, a leucine dipeptide, and two inhibitors of Signal Transducer and Activator of Transcription 5. The results show that the Monte Carlo scheme is more efficient than constrained scans and is particularly effective at approximating the underlying potential energy surface when the dihedral degrees are coupled.

Using Phenotype MicroArrays to Determine Culture Conditions That Induce or Repress Toxin Production by Clostridium difficile and Other Microorganisms

Toxin production is a central issue in the pathogenesis of Clostridium difficile and many other pathogenic microorganisms. Toxin synthesis is influenced by a variety of known and unknown factors of genetics, physiology, and environment. To facilitate the study of toxin production by C. difficile, we have developed a new, reliable, quantitative, and robust cell-based cytotoxicity assay. Then we combined this new assay with Phenotype MicroArrays (PM) technology which provides high throughput testing of culture conditions. This allowed us to quantitatively measure toxin production by C. difficile type strain ATCC 9689 under 768 culture conditions. The culture conditions include different carbon, nitrogen, phosphorus, and sulfur sources. Among these, 89 conditions produced strong toxin induction and 31 produced strong toxin repression. Strong toxin inducers included adenine, guanosine, arginine dipeptides, γ-D-Glu-Gly, methylamine, and others. Some leucine dipeptides and the triple-leucine tripeptide were among the strongest toxin repressors. While some results are consistent with previous observations, others are new observations that provide insights into toxin regulation and pathogenesis of C. difficile. Additionally, we have demonstrated that this combined assay technology can be applied broadly to a wide range of toxin producing microorganisms. This study is the first demonstration of simultaneous assessment of a large number of culture conditions influencing bacterial toxin production. The new functional cytotoxin quantitation method developed provides a valuable tool for studying toxigenic microorganisms and may also find applications in clinical and epidemiological research.

Simple estimation of absolute free energies for biomolecules

One reason that free energy difference calculations are notoriously difficult in molecular systems is due to insufficient conformational overlap, or similarity, between the two states or systems of interest. The degree of overlap is irrelevant, however, if the absolute free energy of each state can be computed. We present a method for calculating the absolute free energy that employs a simple construction of an exactly computable reference system which possesses high overlap with the state of interest. The approach requires only a physical ensemble of conformations generated via simulation and an auxiliary calculation of approximately equal central-processing-unit cost. Moreover, the calculations can converge to the correct free energy value even when the physical ensemble is incomplete or improperly distributed. As a "proof of principle," we use the approach to correctly predict free energies for test systems where the absolute values can be calculated exactly and also to predict the conformational equilibrium for leucine dipeptide in implicit solvent.

Solubility of proline–leucine dipeptide in water and in aqueous sodium chloride solutions from T = (288.15 to 313.15) K

Solubility of proline–leucine dipeptide, in water and in aqueous sodium chloride solutions, was measured at T = (288.15, 298.15, 308.15 and 313.15) K as a function of electrolyte concentration m = (0.1, 0.25, 0.5, 0.75 and 1) mol · kg −1 of water. Solubility data has been evaluated from density measurements using a vibrating tube densimeter. It has been observed that sodium chloride renders the dipeptide proline–leucine more soluble in water. Salting-in coefficients and standard free energies of transfer of proline–leucine, from water to aqueous sodium chloride solutions, have been calculated from the solubility data. Standard enthalpies and entropies of transfer have also been estimated and interpreted in terms of electrostatic and hydrophobic perturbed domains in the hydration shells of the dipeptide and of the cation and anion of the salt, as a function of temperature and salt concentration.

Peptide Conformational Equilibria Computed via a Single-Stage Shifting Protocol

We study the conformational equilibria of two peptides using a novel statistical mechanics approach designed for calculating free energy differences between highly dissimilar conformational states. Our results elucidate the contrasting roles of entropy in implicitly solvated leucine dipeptide and decaglycine. The method extends earlier work by Voter and overcomes the notorious "overlap" problem in free energy computations by constructing a mathematically equivalent calculation with high conformational similarity. The approach requires only equilibrium simulations of the two states of interest, without the need for sampling transition states. We discuss possible extensions and optimizations of the approach.

Synthesis of the Phenylserine−Leucine Dipeptide Fragment Present in the Antibiotic Lysobactin from an Aziridine-2-imide Precursor

The ring expansion of (2R′,3S′) or (2S′,3R′)-N-(α-amino acyl)-aziridine-2-imides and the mild hydrolysis of the resulting oxazoline-4-imides gives (2R,3S)-phenylserine−leucine or (2S,3R)-phenylserine−leucine dipeptides. In particular, the latter fragment is present in the depsipeptide antibiotic Lysobactin.

Synthesis of the Phenylserine−Leucine Dipeptide Fragment Present in the Antibiotic Lysobactin from an Aziridine-2-imide Precursor

Synthesis of the Phenylserine−Leucine Dipeptide Fragment Present in the Antibiotic Lysobactin from an Aziridine-2-imide Precursor

The neuroinvasiveness of a murine retrovirus is influenced by a dileucine-containing sequence in the cytoplasmic tail of glycosylated Gag.

The tempo and intensity of retroviral neuropathogenesis are dependent on the capacity of the virus to invade the central nervous system. For murine leukemia viruses, an important determinant of neuroinvasiveness is the virus-encoded protein glycosylated Gag, the function of which in the virus life cycle is not known. While this protein is dispensable for virus replication, mutations which prevent its expression slow the spread of virus in vivo and restrict virus dissemination to the brain. To further explore the function of this protein, we compared two viruses, CasFrKP (KP) and CasFrKP41 (KP41), which differ dramatically in neurovirulence. KP expresses high early viremia titers, is neuroinvasive, and induces clinical neurologic disease in 100% of neonatally inoculated mice, with an incubation period of 18 to 23 days. In contrast, KP41 expresses early viremia titers 100- fold lower than those of KP, exhibits attenuated neuroinvasiveness, and induces clinical neurologic disease infrequently, with a relatively long incubation period. The genomes of these two viruses differ by only 10 nucleotides, resulting in differences at five residues, all located within the N-terminal cytoplasmic tail of glycosylated Gag. In this study, using KP as the parental virus, we systematically mutated each of the five amino acid residues to those of KP41 and found that substitution mutation of two membrane-proximal residues, E53 and L56, to K and P, respectively produced the greatest effect on early viremia kinetics and neurovirulence. These mutations disrupted the KP sequence E53FLL56, the leucine dipeptide of which suggests the possibility that it may represent a sorting signal for glycosylated Gag. Supporting this idea was the finding that alteration of this sequence motif increased the level of cell surface expression of the protein, which suggests that analysis of the intracellular trafficking of glycosylated Gag may provide further clues to its function.

Synthesis of salicyl-peptides and their effect on human platelet aggregationin vitro

A series of leucine dipeptide amides containing at their N-terminal amino group the salicyl-residue [(o)-RO-C6H4-CO-, where R=H or CH3CO] have been synthesized by conventional solution techniques and tested for their inhibitory activity on human platelet aggregationin vitro induced by collagen, ADP or adrenaline. The salicyl-peptide (o)-HO-C6H4-CO-Leu-Asp(OBzl)-NH2 was found to exert strong inhibitory activity on platelet aggregation induced by collagen with an IC50 value 4.5 mM. The corresponding dipetide H2N-Leu-Asp(OVzl)-NH2 was also examined and was found to be less active, indicating that the presence of the lipophilicβ-benzyl ester group in combination with the salicyl group enhance the inhibitory activity. All the other salicyl-peptides examined either didn't show any inhibitory or aggregatory activity or a slight inhibition at the concentration of 9–10 mm.

Tyrosyl Fluorescence Decays and Rotational Dynamics in Tyrosine Monomers and in Dipeptides

Picosecond time-correlated single-photon counting was used to measure fluorescence lifetimes and fluorescence anisotropy decays of tyrosine and the tyrosine–alanine and tyrosine–leucine dipeptides. After excitation of tyrosine at 287 nm two emitting species were observed, one at 303 nm with a lifetime of 3.3 ns and another at 340 nm with a lifetime of 360 ps. The rotational correlation time of tyrosine at 303 nm is 38 ps in water at pH 7 and depends linearly on viscosity with a slope of 44 ps/cP, consistent with Stokes–Einstein–Debye theory. We calculated a value of 45 ns for the radiative lifetime of tyrosine, yielding a fluorescence quantum yield of 0.07. The dipeptides Tyr–Ala and Tyr–Leu exhibit two- or three-exponential decays. The amplitudes of the decay components for three-exponential fits correlate closely with the populations of rotamers in these peptides as determined by NMR. The quenching of dipeptide fluorescence is shown to depend on the solvent polarity, strongly supporting the hypothesis that tyrosyl fluorescence in peptides is quenched by charge transfer. The rotational correlation times of tyrosine, Tyr–Ala, and Tyr–Leu increase linearly with the van der Waals volumes. However, rotational relaxation is somewhat faster than expected from Stokes–Einstein–Debye theory with “stick” boundary conditions.