Amino Lysine Research Articles

Non-enzymatic reaction of glycosyl oxazoline with peptides

Recently, a number of chemoenzymatic strategies have been explored for achieving preparation of homogeneous glycopeptides and glycoproteins, especially by using endoglycanases and glycosyl oxazolines. However, concomitant occurrence of non-enzymatic reactions has been reported, but no further characterization of the byproducts was conducted. In this work, we made an attempt to identify the side product by using model substrates. Analysis of the product allowed us to propose that the oxazoline ring was attacked by the amino group of lysine, leading to the formation of disubstituted acetamidine.

The Remarkable Character of Porphobilinogen Synthase.

Porphobilinogen synthase (PBGS), also known as 5-aminolevulinate dehydratase, is an essential enzyme in the biosynthesis of all tetrapyrroles, which function in respiration, photosynthesis, and methanogenesis. Throughout evolution, PBGS adapted to a diversity of cellular niches and evolved to use an unusual variety of metal ions both for catalytic function and to control protein multimerization. With regard to the active site, some PBGSs require Zn2+; a subset of those, including human PBGS, contain a constellation of cysteine residues that acts as a sink for the environmental toxin Pb2+. PBGSs that do not require the soft metal ion Zn2+ at the active site instead are suspected of using the hard metal Mg2+. The most unexpected property of the PBGS family of enzymes is a dissociative allosteric mechanism that utilizes an equilibrium of architecturally and functionally distinct protein assemblies. The high-activity assembly is an octamer in which intersubunit interactions modulate active-site lid motion. This octamer can dissociate to dimer, the dimer can undergo a hinge twist, and the twisted dimer can assemble to a low-activity hexamer. The hexamer does not have the intersubunit interactions required to stabilize a closed conformation of the active site lid. PBGS active site chemistry benefits from a closed lid because porphobilinogen biosynthesis includes Schiff base formation, which requires deprotonated lysine amino groups. N-terminal and C-terminal sequence extensions dictate whether a specific species of PBGS can sample the hexameric assembly. The bulk of species (nearly all except animals and yeasts) use Mg2+ as an allosteric activator. Mg2+ functions allosterically by binding to an intersubunit interface that is present in the octamer but absent in the hexamer. This conformational selection allosteric mechanism is purported to be essential to avoid the untimely accumulation of phototoxic chlorophyll precursors in plants. For those PBGSs that do not use the allosteric Mg2+, there is a spatially equivalent arginine-derived guanidium group. Deprotonation of this residue promotes formation of the hexamer and accounts for the basic arm of the bell-shaped pH vs activity profile of human PBGS. A human inborn error of metabolism known as ALAD porphyria is attributed to PBGS variants that favor the hexameric assembly. The existence of one such variant, F12L, which dramatically stabilizes the human PBGS hexamer, allowed crystal structure determination for the hexamer. Without this crystal structure and octameric PBGS structures containing the allosteric Mg2+, it would have been difficult to decipher the structural basis for PBGS allostery. The requirement for multimer dissociation as an intermediate step in PBGS allostery was established by monitoring subunit disproportionation during the turnover-dependent transition of heteromeric PBGS (comprised of human wild type and F12L) from hexamer to octamer. One outcome of these studies was the definition of the dissociative morpheein model of protein allostery. The phylogenetically variable time scales for PBGS multimer interconversion result in atypical kinetic and biophysical behaviors. These behaviors can serve to identify other proteins that use the morpheein model of protein allostery.

Novel fluorescent substrates for detection of trypsin activity and inhibitor screening by self-quenching

A group of self-quenching-based substrates with two fluorescent peptides for detection of trypsin activity was designed and synthesized. The substrates could be easily synthesized using simple solid-phase peptide synthesis techniques. Two fluorescent peptide substrates for trypsin were conjugated to the amino groups of lysine as a branched unit. The fluorescence of these substrates was self-quenched owing to the highly assembled fluorophores on the substrates. The release of these concentrated fluorophores by proteases allows for fluorescence recovery. Self-quenching reduced the fluorescence of the substrates by 64.1%, and the fluorescence intensity was recovered by the release of the fluorophores from the substrate peptides via tryptic cleavage. The kinetic assay revealed that the kcat/Km values of the substrates were almost comparable to those of the standard fluorescent probe, peptide-MCA. The detection limit for trypsin was 111pM, and the calculation of IC50 and Ki values for the Bowman–Birk inhibitor was achieved using these substrates. These easily synthesizable self-quenching-based substrates have the potential to be useful for the detection of other disease-related protease activities.

Reactions of Gluten with Anhydride Derivatives

Gluten was extracted from wheat flour followed by chemical modification of groups such as the free e-amino group of lysine in proteins with acid anhydrides such as, maleic anhydride (2), Diacetyl-d-tartaric anhydride (3), 7-oxa bicyclo [2.2.1] hept-5-enecis -2, 3dicarboxylic anhydride (4), 9, 10-dihydroanthracene9,10-α, β-succinic anhydride (5) and Triphenylphosphoranylidenesuccinic Anhydride (6). Attachment of such side groups will alter repulsive or attractive forces among polypeptides to cause certain structural rearrangement, which in turn may cause changes in functional properties of modified gluten. Also different properties were determined comparing with original gluten

Formation and spectral properties of metal ion complexes of tripeptides

The aim of this study was to develop complexes that could be used to eleviate the inflammation associated with arthritis by facilitating the dermal absorption of copper. To this end, copper(II) complexes of two tripeptides sarcosyl-l-histidyl-l-lysine (L1) and sarcosyl-l-lysyl-l-histidine (L2) were studied using potentiometric and spectroscopic (UV–vis, ESR, 1H NMR) techniques as well as molecular mechanics (MM) calculations. Since Zn(II) and Ni(II) are in vivo competitors of Cu(II), equilibrium constants of H+, Cu(II), Ni(II) and Zn(II) with the peptides were measured in aqueous solution at 25±0.01°C and an ionic strength of 0.15M (NaCl) using glass electrode potentiometry. In the pH range 2.00–11.00, four species; MLH, ML, MLH−1, and MLH−2 were observed. 1H NMR identified the binding sites to be the imidazole nitrogen, the amide nitrogen and the terminal amino group. The ε-amino group of lysine did not coordinate to the Cu(II). The ML2H−2 species involved coordination of two deprotonated amide groups to give a neutral complex. UV/vis and EPR spectroscopy confirmed the mode of coordination, which was different in L1 and L2. Molecular mechanics calculations were used to support the structures postulated from potentiometric and spectroscopic data. Speciation calculations of Cu(II) using a computer model of blood plasma indicated that Sar-Lys-His (L2) was the best at mobilising copper in silico.

Synchrotron Small-Angle X-ray Scattering Study of Cross-Linked Polymeric Micelles.

Polymeric micelles of methoxypoly(ethylene glycol)-b-poly(lactide) containing lysine units (mPEG-PLA-Lys4) were cross-linked by reacting of lysine moieties with a bifunctional bis(N-hydroxy-succinimide ester). The micelles were characterized in aqueous solution using dynamic light scattering, transmission electron microscopy, and synchrotron small-angle X-ray scattering. The mPEG-PLA-Lys4 was synthesized through the ring-opening polymerization of N6-carbobenzyloxy-L-lysine N-carboxyanhydride with amine-terminated mPEG-PLA and subsequent deprotection. The polymeric micelles showed enhanced micelle stability after cross-linking, which was confirmed by adding sodium dodecyl sulfate as a destabilizing agent. The average diameters measured via dynamic light scattering were 19.1 nm and 29.2 nm for non-cross-linked polymeric micelles (NCPMs) and cross-linked polymeric micelles (CPMs), respectively. The transmission electron microscopy images showed that the size of the polymeric micelles increased slightly due to cross-linking, which was in good agreement with the DLS measurements. The overall structures and internal structural changes of NCPMs and CPMs in aqueous solution were studied in detail using synchrotron X-ray scattering method. According to the structural parameters of X-ray scattering analysis, CPMs with a more densely packed core structure were formed by reacting bifunctional cross-linking agents with lysine amino groups located in the innermost core of the polymeric micelles.

Effects of peptide acetylation and dimethylation on electrospray ionization efficiency.

Peptide acetylation and dimethylation have been widely used to derivatize primary amino groups (peptide N-termini and the ε-amino group of lysines) for chemical isotope labeling of quantitative proteomics or for affinity tag labeling for selection and enrichment of labeled peptides. However, peptide acetylation results in signal suppression during electrospray ionization (ESI) due to charge neutralization. In contrast, dimethylated peptides show increased ionization efficiency after derivatization, since dimethylation increases hydrophobicity and maintains a positive charge on the peptide under common LC conditions. In this study, we quantitatively compared the ESI efficiencies of acetylated and dimethylated model peptides and tryptic peptides of BSA. Dimethylated peptides showed higher ionization efficiency than acetylated peptides for both model peptides and tryptic BSA peptides. At the proteome level, peptide dimethylation led to better protein identification than peptide acetylation when tryptic peptides of mouse brain lysate were analyzed with LC-ESI-MS/MS. These results demonstrate that dimethylation of tryptic peptides enhanced ESI efficiency and provided up to two-fold improved protein identification sensitivity in comparison with acetylation.

Caffeine-loaded whey protein hydrogels reinforced with gellan and enriched with calcium chloride

Gellan gum and CaCl2 were exploited to modulate the textural and techno-functional properties of heat-set whey protein hydrogels. Enrichment with CaCl2 increased the amount of released caffeine from the protein hydrogel in conjunction with decreasing cohesiveness index and microstructural features modification. Gellan bundles as visualised by microscopic images conferred a remarkable reinforcing influence to gel samples; enrichment with gellan at 0.5 mg mL−1 increased the gel hardness value approximately 6.4 fold. Based on Fourier transform infrared (FTIR) spectroscopy it was suggested that gellan and CaCl2 enrichments decreased the β-sheet content of the protein gel matrix in favour of random coil structures. FTIR spectroscopy also proposed cation-π interactions between Ca2+ ions and electron-rich amide bonds of whey proteins, as well as interactions between carboxyl groups of gellan and the ε-amino group of lysine in β-lactoglobulin.

Aminothiazoles as Potent and Selective Sirt2 Inhibitors: A Structure-Activity Relationship Study.

Sirtuins are NAD(+)-dependent protein deacylases that cleave off acetyl but also other acyl groups from the ε-amino group of lysines in histones and other substrate proteins. Dysregulation of human Sirt2 (hSirt2) activity has been associated with the pathogenesis of cancer, inflammation, and neurodegeneration, which makes the modulation of hSirt2 activity a promising strategy for pharmaceutical intervention. The sirtuin rearranging ligands (SirReals) have recently been discovered by us as highly potent and isotype-selective hSirt2 inhibitors. Here, we present a well-defined structure-activity relationship study, which rationalizes the unique features of the SirReals and probes the limits of modifications on this scaffold regarding inhibitor potency. Moreover, we present a crystal structure of hSirt2 in complex with an optimized SirReal derivative that exhibits an improved in vitro activity. Lastly, we show cellular hyperacetylation of the hSirt2 targeted tubulin caused by our improved lead structure.

Self-delivery of a peptide-based prodrug for tumor-targeting therapy

A novel self-delivered prodrug system was fabricated for tumor-targeting therapy. In this nanosystem, the Arg-Gly-Asp-Ser (RGDS) tetrapeptide was used to improve the therapeutic index to integrin-overexpressing tumor cells. The antitumorous drug camptothecin was further appended to the e-amino group of lysine by 20-O-succinyl linkage and controllably released via hydrolytic cleavage. Prodrug molecules self-assembled into fibrillar nano-architectures and achieved the capability of self-delivery after being injected subcutaneously into mice. Introduction of hydrophobic myristic acid favored the self-assembly and enhanced the cellular internalization of the prodrugs. In vitro and in vivo studies demonstrated that the self-assembled nanofibers could effectively target integrinoverexpressing tumorous cells and inhibit tumor growth via RGD-mediated specific targeting. Therefore, the traditional idea that fibrillar structures hold low therapeutic efficacy due to poor cell uptake can be challenged.

Site-Specific Labeling of Protein Lysine Residues and N-Terminal Amino Groups with Indoles and Indole-Derivatives.

Indoles and indole-derivatives can be used to site-specifically label proteins on lysine and N-terminal amino groups under mild, nondenaturing reaction conditions. Hen egg white lysozyme (HEWL) and α-lactalbumin were labeled with indole, fluoroindole, or fluoroindole-2-carboxylate via electrophilic aromatic substitutions to lysine side chain Nε- and N-terminal amino imines, formed in situ in the presence of formaldehyde. The reaction is highly site-selective, easily controlled by temperature, and does not eliminate the native charge of the protein, unlike many other common lysine-specific labeling strategies. (19)F NMR was used to monitor reaction progression, and in the case of HEWL, unique resonances for each labeled side chain could be resolved. We demonstrate that the indole tags are highly selective for primary amino groups. (19)F NMR demonstrates that each lysine exhibits a different rate of conjugation to indoles making it possible to employ these tags as a means of probing surface topology by NMR or mass spectrometry. Given the site-specificity of this tagging method, the mildness of the reaction conditions (aqueous, buffered, or unbuffered) and the low stoichiometry required for the reaction, indole-derivatives should serve as a valuable addition to the bioconjugation toolkit. We propose that labeling lysine side chains and N-terminal amino groups with indoles is a versatile and general strategy for bioconjugations with substituted indoles having broad implications for protein functionalization.

Lysine acetyltransfer supports platelet function

The reversible acetylation of protein lysine ε-amino groups, catalyzed by lysine acetyltransferases and deacetylases, serves as a molecular switch in the orchestration of diverse cellular activities. Here, we aimed to investigate the role of lysine acetyltransfer in platelet function. Proteomics methods identified 552 acetyllysine (acK) modifications on 273 platelet proteins that serve as candidate substrates for lysine acetyltransferases. Bioinformatics analyses of the identified acK-modified platelet proteins supported roles for the lysine acetyltransferase p300 in the regulation of actin-mediated platelet processes. Biochemical experiments showed that platelets express p300, which is activated in an Src kinase-dependent manner upon platelet stimulation with the platelet glycoproteinVI agonist collagen-related peptide (CRP). Inhibition of platelet p300 abrogated CRP-stimulated lysine acetylation of actin, filamin, and cortactin, as well as F-actin polymerization, integrin activation, and platelet aggregation. Super-resolution visualization of platelet actin-rich adhesion structures revealed abundant acK protein colocalized with platelet actin cytoskeletal proteins. Inhibition of p300 blocked platelet filopodium formation and the spreading of platelets on fibrinogen and collagen surfaces. In whole blood, p300 inhibition prevented the formation of platelet aggregates under shear, suggesting a physiologic role for lysine acetyltransferase activity in platelet function. Together, our findings reveal lysine acetyltransfer to be a potential regulator of platelet actin dynamics, and potential roles for lysine acetylation in the molecular coordination of platelet activation and function.

A facile label-free colorimetric sensor for Hg2+ based on Hg-triangular silver nanoplates with amalgam-like structure

In this contribution, we have developed a rapid method for the highly selective detection of mercury ions (Hg2+) using citrate-stabilized triangular silver nanoplates (AgNPs). The colorimetric sensor strategy is based on the aggregation of Hg2+-coated AgNPs with the assistance of two amino groups of lysine, resulting in a color change of AgNP solution from blue to mauve or gray. The sensor enables the analysis of Hg2+ with a minimum detectable concentration that corresponds to 50nM. Meanwhile, the strategy offers excellent selectivity toward Hg2+ against other cations. The validation of this method is carried out by analysis of Hg2+ in tap water with satisfying recoveries over 91.35–106%.

Homocitrulline: a new marker for differentiating acute from chronic renal failure.

Carbamylation is a non-enzymatic post-translational modification of proteins characterized by the addition of isocyanic acid to amino groups. As isocyanic acid mainly originates from the spontaneous dissociation of urea, carbamylation rate is increased during renal failure. The aim of the study was to evaluate serum homocitrulline (HCit), which results from the carbamylation of ε-amino groups of lysine (Lys) residues, in acute renal failure (ARF) and to determine if it could be useful for differentiating acute from chronic renal failure (CRF). In total, 213 patients with renal failure referred to the nephrology department of the university hospital of Reims were included. Patients were classified into three groups: patients with ARF (ARF group, n=39), patients with CRF complicated with ARF (A/CRF group, n=29) and patients with CRF (CRF group, n=145). Serum HCit concentrations were measured by LC-MS/MS. Concentration kinetics of HCit and urea were studied in patients suffering from ARF. The HCit thresholds distinguishing ARF and CRF were investigated. HCit concentrations increased in ARF patients reaching a peak delayed compared to urea concentration peak. HCit concentrations were positively correlated with urea concentrations (r=0.51) and with the time elapsed since the estimated onset of ARF (r=0.57). Serum HCit concentrations were higher (p<0.05) in CRF group compared to ARF group. The receiver operating characteristic curve analysis showed that HCit concentrations <289 μmol/mol Lys were predictive of ARF (Sensitivity: 83%, Specificity: 72%, AUC: 0.856). Our results demonstrate that HCit is a promising biomarker for distinguishing between ARF and CRF patients.

Mass spectrometric identification of isocyanate-induced modifications of keratins in human skin

In the current paper we show that exposure of human callus to isocyanates leads to covalent modifications within keratin proteins. Mass spectrometric analyses of pronase digests of keratin isolated from exposed callus show that both mono- and di-adducts (for di-isocyanates) are predominantly formed on the ε-amino group of lysine. In addition, numerous modified tryptic keratin fragments were identified, demonstrating rather random lysine modification. Interestingly, preliminary experiments demonstrate that in case of MDI a similar lysine di-adduct was formed with lung elastin. Our data support the hypothesis that skin sensitization through antigenic modifications of skin proteins by isocyanates could play a role in occupational isocyanate-induced asthma. It is further envisaged that the elucidated adducts will also have great potential for use as biomarkers to assess skin exposure to isocyanates. Advantageously, the various lysine adducts display the presence of a characteristic daughter fragment at m/z 173.1 [lysine-NCO]+, enabling generic and rapid screening for exposure to isocyanates.

A colorimetric silver nanoparticle-based assay for Hg(II) using lysine as a particle-linking reagent

We describe a colorimetric method for the determination of Hg(II) that is based on the aggregation of silver nanoparticles (AgNPs) in the presence of Hg(II) and lysine. If Hg(II) ions are added to unmodified AgNPs, they will deposit on the surface where they will be reduced to Hg(0) by NaBH4 to form a shell around the AgNPs. Upon addition of lysine to the Hg(II)-capped AgNPs, the aggregation of the AgNPs will be induced because the two amino groups of lysine have a high affinity for Hg(II). Aggregation results in a color change from orange to yellowish. The effect was exploited to design a colorimetric assay for Hg(II) that has a 1 nM detection limit.

Use of the guanidination reaction for determining reactive lysine, bioavailable lysine and gut endogenous lysine.

Determining the bioavailability of lysine in foods and feedstuffs is important since lysine is often the first limiting indispensable amino acid in diets for intensively farmed livestock (pigs and poultry) and also in many cereal-based diets consumed by humans. When foods or feedstuffs are heat processed, lysine can undergo Maillard reactions to produce nutritionally unavailable products. The guanidination reaction, the reaction of O-methylisourea with the side chain amino group of lysine that produces homoarginine, has been used to determine the unmodified lysine (reactive lysine) in processed foods and feedstuffs and also true ileal digestible reactive lysine (bioavailable lysine). The advantages of the guanidination method in comparison with other reactive lysine methods such as the fluorodinitrobenzene, trinitrobenzenesulphonic acid and dye-binding methods are that it is very specific for reactive lysine and also that the method is relatively straightforward to conduct. The specificity of the guanidination reaction for the lysine side chain amino group is particularly important, since ileal digesta will contain N-terminal groups in the form of free amino acids and peptides. The main disadvantage is that complete conversion of lysine to homoarginine is required, yet it is not straightforward to test for complete guanidination in processed foods and feedstuffs. Another disadvantage is that the guanidination reaction conditions may vary for different food types and sometimes within the same food type. Consequently, food-specific guanidination reaction conditions may be required and more work is needed to optimise the reaction conditions across different foods and feedstuffs.

4-Hydroxy-2-nonenal (4-HNE) and Its Lipation Product 2-Pentylpyrrole Lysine (2-PPL) in Peanuts.

After synthesis of a deuterated 4-hydroxy-2-nonenal (4-HNE) standard, the formation of 4-HNE during heating of peanut oil and whole peanuts, respectively, was measured by GC-MS. Whereas a significant increase in 4-HNE levels was observed for peanut oil, the amount of 4-HNE decreased when whole peanuts were roasted due to lipation reactions with amino acid side chains of the proteins. The ε-amino group of lysine was identified as the favored reaction partner of 4-HNE. After heating N(α)-acetyl-l-lysine and 4-HNE, a Schiff base, a novel pyridinium derivative, a 2-pentylpyrrol derivative and, following reduction and hydrolysis, a reduced, cyclized Michael adduct were identified. 2-Amino-6-(2-pentyl-1H-pyrrol-1-yl)hexanoic acid (2-PPL) was synthesized and quantitated in peanut proteins, which had been incubated with various amounts of 4-HNE by HPLC-ESI-MS/MS after enzymatic hydrolysis. At low 4-HNE concentrations the modification of lysine could be entirely explained by the formation of 2-PPL. Additionally, 2-PPL was quantified for the first time in peanut samples, and an increase depending on the roasting time was observed. 2-PPL represents a suitable marker to evaluate the extent of food protein lipation by 4-HNE.

How Y357F, Y276F mutants affect the methylation activity of PRDM9: QM/MM MD and free energy simulations.

Histone methyltransferase PRDM9 catalyzes the methylation of H3K4me2 (histone 3 dimethylated lysine 4) to H3K4me3 (histone 3 trimethylated lysine 4) by transferring the methyl group from S-adenosyl methionine (AdoMet). PRDM9 is the major determinant of the meiotic recombination hotspot and the enrichment of H3K4me3 at the hotspot defines the initiation site of meiotic recombination. In PRDM9, two conserved tyrosine residues Tyr357 and Tyr276 surrounding the amino group of the substrate lysine may influence the methylation activity through hydrogen bond interactions with AdoMet or the substrate lysine. In this study, quantum mechanical/molecular mechanical (QM/MM) molecular dynamics (MD) and free energy simulations were performed to reveal the methylation processes catalyzed by wild type PRDM9, its Y357F, and Y276F mutants, respectively. The different roles of Tyr357 and Tyr276 in the methylation activity of PRDM9 were also investigated and compared. The calculated free energy barriers of the methyl transfers suggest that the Y276F mutation decreases the catalytic activity of the methyl transfer, while the Y357F mutation does not change the catalytic activity of the methyl transfer. The reactant complex conformations generated in the QM/MM MD simulations show that the reactive configuration can be formed in the Y357F mutant but not in the Y276F mutant.

Effect of succinylation on physicochemical and functional properties of milk protein concentrate

Milk protein concentrate (MPC) is a complete dairy protein ingredient (containing both caseins and whey proteins) available with protein concentrations ranging from 40 to 90%. However, MPC powders are poorly soluble which thus, restricts their potential use for food application. Succinylation involves chemical derivatisation of ϵ-amino group of lysine in proteins and enhances solubility of less soluble proteins. In the present investigation, highest degree of succinylation was achieved at the level of 4mol of succinic anhydride/mole of lysine in MPC. Findings from intrinsic tryptophan intensities stated that succinylation of milk proteins showed structural modification and increase in hydrophobicity. Further, the effects of succinylation on the functional properties (solubility, water- and oil-binding capacities, viscosity, foaming capacity and stability, emulsion activity and stability) of MPC were evaluated. Succinylation of proteins significantly (P<0.05) increased solubility as a result of altered charge on protein and reduced particle size of native MPC, which in turn improved other functional properties of native MPC. The microstructure of succinylated MPC at different degrees of succinylation by scanning electron microscopy revealed that the size and number of white patch like structures present on protein increased with degree of succinylation.