Incorporation Of Lysine Research Articles

Proteome Dynamics: Tissue Variation in the Kinetics of Proteostasis in Intact Animals

Understanding the role of protein turnover in the maintenance of proteostasis requires accurate measurements of the rates of replacement of proteins in complex systems, such as intact animals. Moreover, any investigation of allometric scaling of protein turnover is likely to include species for which fully annotated proteomes are not available. We have used dietary administration of stable isotope labeled lysine to assess protein turnover rates for proteins from four tissues in the bank vole, Myodes glareolus. The annotated genome for this species is not available, so protein identification was attained through cross-species matching to the mouse. For proteins for which confident identifications were derived, the pattern of lysine incorporation over 40 days was used to define the rate of synthesis of individual proteins in the four tissues. The data were heavily filtered to retain a very high quality dataset of turnover rates for 1088 proteins. Comparative analysis of the four tissues revealed different median rates of degradation (kidney: 0.099 days−1; liver 0.136 days−1; heart, 0.054 days−1, and skeletal muscle, 0.035 days−1). These data were compared with protein degradation rates from other studies on intact animals or from cells in culture and indicate that both cell type and analytical methodology may contribute to variance in turnover data between different studies. These differences were not only due to tissue-specific proteins but were reflected in gene products common to all tissues. All data are available via ProteomeXchange with identifier PXD002054.

Pathogen-nematode interaction: Nitrogen supply of Listeria monocytogenes during growth in Caenorhabditis elegans.

Listeria monocytogenes is a Gram-positive facultatively intracellular human pathogen. Due to its saprophytic lifestyle, L. monocytogenes is assumed to infect and proliferate within soil organisms such as Caenorhabditis elegans. However, little is known about the nutrient usages and metabolite fluxes in this bacterium-nematode interaction. Here, we established a nematode colonization model for L. monocytogenes and a method for the efficient separation of the pathogen from the nematodal gut. Following (15)N labelling of C. elegans and gas chromatography-mass spectrometry-based (15)N isotopologue analysis, we detected a high basal metabolic rate of the nematode, and observed a significant metabolic flux from nitrogenous compounds of the nematode to listerial proteins during proliferation of the pathogen in the worm's intestine. For comparison, we also measured the N fluxes from the gut content into listerial proteins using completely (15)N-labelled Escherichia coli OP50 as food for C. elegans. In both settings, L. monocytogenes prefers the direct incorporation of histidine, arginine and lysine over their de novo biosynthesis. Our data suggest that colonization of nematodes is a strategy of L. monocytogenes to increase its access to N-rich nutrients.

Abstract 9: Temperature-responsive Cell Delivery Biopolymers for Cardiac Tissue Engineering

Background: Advances in cell therapy and material science have made tissue engineering a promising strategy for heart regeneration. We developed an injectable biomimetic reverse thermal gel (RTG) that is liquid at room temperature but gel-like at body temperature, with the ultimate goal of being able to serve as a vehicle for cell-based delivery (liquid) to targeted tissue areas (gel-phase at 37°C). In this study we tested the suitability of this biomimetic RTG on cell viability. Methods and results: We tested different biomimetic RTG systems with and without the chemical incorporation of lysine. In vitro 3D culture experiments were performed with neonatal rat ventricular myocytes (NRVM) by mixing 3x104 cells with 50 μl of polymeric solution and allowing gel formation at 37°C. The cultured cells were incubated for 21 days. For controls we used NRVMs plated on 2D traditional gelatin coated dishes. We found that the 3D polymeric matrix induces rapid coordinated contraction with improved functionality when compared with standard 2D-cultured NRVM. By immunostaining for the morphology of the sarcomere (alpha-actinin) and DAPI, we also observed that the 3D polymeric matrix stimulates cells to spread and form 3D syncytia. Conclusion: These proof-of-concept results demonstrate long-term cell viability in this unique biomimetic system and therefore provide feasibility of a polymeric cell delivery system that permits reversible liquid-to-gel transition at body temperature. These results offer potential for a tissue engineering approach to cardiac regeneration.

PRC1 is taking the lead in PcG repression.

Polycomb group (PcG) proteins constitute a major epigenetic mechanism for gene repression throughout the plant life. For a long time, the PcG mechanism has been proposed to follow a hierarchical recruitment of PcG repressive complexes (PRCs) to target genes in which the binding of PRC2 and the incorporation of H3 lysine 27 trimethyl marks led to recruitment of PRC1, which in turn mediated H2A monoubiquitination. However, recent studies have turned this model upside-down by showing that PRC1 activity can be required for PRC2 recruitment and H3K27me3 marking. Here, we review the current knowledge on plant PRC1 composition and mechanisms of repression, as well as its role during plant development.

Discovery of ATP-Competitive Inhibitors of tRNAIle Lysidine Synthetase (TilS) by High-Throughput Screening

A novel, ultrahigh-throughput, fluorescence anisotropy–based assay was developed and used to screen a 1.4-million-sample library for compounds that compete with adenosine triphosphate (ATP) for binding to Escherichia coli tRNAIle lysidine synthetase (TilS), an essential, conserved, ATP-dependent, tRNA-modifying enzyme of bacterial pathogens. TilS modifies a cytidine base in the anticodon loop of Ile2 tRNA by attaching lysine, thereby altering codon recognition of the CAU anticodon from AUG (methionine) to AUA (isoleucine). A scintillation proximity assay for the incorporation of lysine into Ile2 tRNA was used to eliminate false positives in the initial screen resulting from detection artifacts as well as compounds competitive with the fluorescent label instead of ATP, and to measure inhibitor potencies against E. coli and Pseudomonas aeruginosa TilS isozymes. The tRNAIle substrate for P. aeruginosa TilS was identified for the first time to enable these measurements. ATP-competitive binding of inhibitors was confirmed by one-dimensional ligand-observe nuclear magnetic resonance. A preliminary structure–activity relationship is shown for two inhibitor series.

Absolute quantitation of host cell proteins in recombinant human monoclonal antibodies with an automated CZE‐ESI‐MS/MS system

We report the first use of CZE for absolute characterization of host cell proteins (HCPs) in recombinant human monoclonal antibodies. An electrokinetically pumped nanoelectrospray interface was used to couple CZE with a tandem mass spectrometer. Three isotopic-labeled peptides (LSFDKDAMVAR, VDIVENQAMDTR, and LVSDEMVVELIEK) were synthesized by direct incorporation of an isotope-labeled lysine or arginine. The heavy-labeled peptides were spiked in the HCP digests at known concentrations. After CZE-ESI-MS/MS analysis, the peaks of native and isotopic-labeled peptides were extracted with mass tolerance ≤ 5 ppm from the electropherograms, and the ratios of peak area between native and isotopic-labeled peptides pairs were calculated. Calibration curves (the ratios of peak area versus spiked peptide amount) with R(2) values of 0.999, 0.997, and 0.999 were obtained for the three HCP peptides, and the absolute amounts of the three proteins present were determined to be at the picomole level in a 20 μg sample of digested HCPs. The target proteins were present at the 7-30 ppt level in the purified HCP samples.

Histone H3 Lysine 14 (H3K14) Acetylation Facilitates DNA Repair in a Positioned Nucleosome by Stabilizing the Binding of the Chromatin Remodeler RSC (Remodels Structure of Chromatin)

Histone H3 acetylation is induced by UV damage in yeast and may play an important role in regulating the repair of UV photolesions in nucleosome-loaded genomic loci. However, it remains elusive how H3 acetylation facilitates repair. We generated a strongly positioned nucleosome containing homogeneously acetylated H3 at Lys-14 (H3K14ac) and investigated possible mechanisms by which H3K14 acetylation modulates repair. We show that H3K14ac does not alter nucleosome unfolding dynamics or enhance the repair of UV-induced cyclobutane pyrimidine dimers by UV photolyase. Importantly, however, nucleosomes with H3K14ac have a higher affinity for purified chromatin remodeling complex RSC (Remodels the Structure of Chromatin) and show greater cyclobutane pyrimidine dimer repair compared with unacetylated nucleosomes. Our study indicates that, by anchoring RSC, H3K14 acetylation plays an important role in the unfolding of strongly positioned nucleosomes during repair of UV damage.

Identification of novel protein functions and signaling mechanisms by genetics and quantitative phosphoproteomics in Caenorhabditis elegans.

Stable isotope labeling by amino acids combined with mass spectrometry is a widely used methodology for measuring relative changes in protein and phosphorylation levels at a global level. We have applied this method to the model organism Caenorhabditis elegans in combination with RNAi-mediated gene knockdown by feeding the nematode on pre-labeled lysine auxotroph Escherichia coli. In this chapter, we describe in details the generation of the E. coli strain, incorporation of heavy isotope-labeled lysine in C. elegans, and the procedure for a comprehensive global phosphoproteomic experiment.

Specificity Determinants for Lysine Incorporation in Staphylococcus aureus Peptidoglycan as Revealed by the Structure of a MurE Enzyme Ternary Complex

Formation of the peptidoglycan stem pentapeptide requires the insertion of both L and D amino acids by the ATP-dependent ligase enzymes MurC, -D, -E, and -F. The stereochemical control of the third position amino acid in the pentapeptide is crucial to maintain the fidelity of later biosynthetic steps contributing to cell morphology, antibiotic resistance, and pathogenesis. Here we determined the x-ray crystal structure of Staphylococcus aureus MurE UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:meso-2,6-diaminopimelate ligase (MurE) (E.C. 6.3.2.7) at 1.8 Å resolution in the presence of ADP and the reaction product, UDP-MurNAc-L-Ala-γ-D-Glu-L-Lys. This structure provides for the first time a molecular understanding of how this Gram-positive enzyme discriminates between L-lysine and D,L-diaminopimelic acid, the predominant amino acid that replaces L-lysine in Gram-negative peptidoglycan. Despite the presence of a consensus sequence previously implicated in the selection of the third position residue in the stem pentapeptide in S. aureus MurE, the structure shows that only part of this sequence is involved in the selection of L-lysine. Instead, other parts of the protein contribute substrate-selecting residues, resulting in a lysine-binding pocket based on charge characteristics. Despite the absolute specificity for L-lysine, S. aureus MurE binds this substrate relatively poorly. In vivo analysis and metabolomic data reveal that this is compensated for by high cytoplasmic L-lysine concentrations. Therefore, both metabolic and structural constraints maintain the structural integrity of the staphylococcal peptidoglycan. This study provides a novel focus for S. aureus-directed antimicrobials based on dual targeting of essential amino acid biogenesis and its linkage to cell wall assembly.

Native SILAC: Metabolic Labeling of Proteins in Prototroph Microorganisms Based on Lysine Synthesis Regulation

Mass spectrometry (MS)-based quantitative proteomics has matured into a methodology able to detect and quantitate essentially all proteins of model microorganisms, allowing for unprecedented depth in systematic protein analyses. The most accurate quantitation approaches currently require lysine auxotrophic strains, which precludes analysis of most existing mutants, strain collections, or commercially important strains (e.g. those used for brewing or for the biotechnological production of metabolites). Here, we used MS-based proteomics to determine the global response of prototrophic yeast and bacteria to exogenous lysine. Unexpectedly, down-regulation of lysine synthesis in the presence of exogenous lysine is achieved via different mechanisms in different yeast strains. In each case, however, lysine in the medium down-regulates its biosynthesis, allowing for metabolic proteome labeling with heavy-isotope-containing lysine. This strategy of native stable isotope labeling by amino acids in cell culture (nSILAC) overcomes the limitations of previous approaches and can be used for the efficient production of protein standards for absolute SILAC quantitation in model microorganisms. As proof of principle, we have used nSILAC to globally analyze yeast proteome changes during salt stress.

EED and KDM6B Coordinate the First Mammalian Cell Lineage Commitment To Ensure Embryo Implantation

The first mammalian cell lineage commitment is the formation of the trophectoderm (TE) and the inner cell mass (ICM) lineages during preimplantation development. Proper development of the TE and ICM lineages is dependent upon establishment of specific transcriptional programs. However, the epigenetic mechanisms that functionally contribute to establish TE- and ICM-specific transcriptional programs are poorly understood. Here, we show that proper development of the TE and ICM lineages is coordinated via combinatorial regulation of embryonic ectoderm development (EED) and lysine-specific demethylase 6B (KDM6B). During blastocyst formation, the relative levels of EED and KDM6B expression determine altered polycomb repressor 2 (PRC2) complex recruitment and incorporation of the repressive histone H3 lysine 27 trimethylation (H3K27Me3) mark at the chromatin domains of TE-specific master regulators CDX2 and GATA3, leading to their activation in the TE lineage and repression in the ICM lineage. Furthermore, ectopic gain of EED along with depletion of KDM6B in preimplantation mouse embryos abrogates CDX2 and GATA3 expression in the nascent TE lineage. The loss of CDX2 and GATA3 in the nascent TE lineage results in improper TE development, leading to failure in embryo implantation to the uterus. Our study delineates a novel epigenetic mechanism that orchestrates proper development of the first mammalian cell lineages.

The Incorporation of Lysine into the Peroxidase Peptide Reactivity Assay for Skin Sensitization Assessments

To establish further a practical quantitative in chemico reactivity assay for screening contact allergens, lysine peptide was incorporated into a liquid chromatography and tandem mass spectrometry-based assay for reactivity assessments of hapten and pre-/pro-hapten chemical sensitizers. Loss of peptide was determined following 24 h coincubation with test chemical using a concentration-response study design. A total of 70 chemicals were tested in discrete reactions with cysteine or lysine peptide, in the presence and absence of horseradish peroxidase-hydrogen peroxide oxidation system. An empirically derived prediction model for discriminating sensitizers from nonsensitizers resulted in an accuracy of 83% for 26 haptens, 19 pre-/pro-haptens, and 25 nonsensitizers. Four sensitizers were shown to selectively react with lysine including two strong/extreme and two weak sensitizers. In addition, seven sensitizers were identified as having higher reactivity toward lysine compared with cysteine. The majority of sensitizing chemicals (27/45) were reactive toward both cysteine and lysine peptides. An estimate of the relative reactivity potency was determined based on the concentration of test chemical that depletes peptide at or above a threshold positive value. Here, we report the use of EC15 as one example to illustrate the use of the model for screening the skin sensitization potential of novel chemicals. Results from this initial assessment highlight the utility of lysine for assessing a chemical's potential to elicit sensitization reactions or induce hypersensitivity. This approach has the potential to qualitatively and quantitatively evaluate an important mechanism in contact allergy for hazard and quantitative risk assessments without animal testing.

Characterization and immobilization of purified Aspergillus flavipesl-methioninase: continuous production of methanethiol

To immobilize the purified Aspergillus flavipesl-methioninase on solid carriers for continuous production of methanethiol with high purity, by the enzymatic methods. The purified l-methioninase was immobilized using different methods, and physicochemical and kinetic studies for the potent immobilized enzyme were conducted parallel to the soluble one. The activity of the purified extracellular enzyme was 1·8-fold higher than intracellular one from submerged cultures of A. flavipes. Among the tested methods, polyacrylamide (42·2%), Ca-alginate (40·9%) and chitin (40·8%) displayed the highest immobilization efficiency. The thermal inactivation rate was strongly decreased for chitin-immobilized enzyme (0·222 s⁻¹) comparing to soluble enzyme (0·51 s⁻¹). Enzyme immobilization efficiency was greatly improved using 4·0% glutaraldehyde and 41·6/6·3 (T/C) as spacers for chitin and polyacrylamide-enzyme conjugates, comparing to their controls. Also the incorporation of lysine, glutathione, cysteine and dithiothreitol as active site protectants significantly enhance the catalytic efficiency of immobilized enzyme. The activity of enzyme was increased by 4·5- and 3·5-fold using glutathione plus DDT and glutathione plus methionine, for chitin and polyacrylamide enzyme, respectively. Chitin enzyme gave a plausible stability till fourth cycle for production of methanethiol under controlled system. Applying GC and HNMR analysis, methanethiol has identical chemical structure to the standard compound. Technically, a new method for continuous production of pure methanethiol, with broad applications, was developed using a simple low expenses method.

Mechanism of Lysine on the Formation of Glycyrrhetic Acid Elastic Vesicles

Elastic vesicles of glycyrrhetic acid (GA) with lysine in the aqueous phase were prepared by the film-high pressure homogenization method. The influence of lysine on the formation of GA elastic vesicles was evaluated in this work. The lysine salt of GA was synthesized and entrapped in elastic vesicles as a control formulation. The particle size, zeta-potential, entrapment efficiency, phase transformation temperature, deformability of the elastic vesicles, and the permeation of GA through rat skin were investigated. By the incorporation of lysine into elastic vesicles the particle size decreased slightly and the phase transformation temperature decreased while the entrapment efficiency and deformability of the elastic vesicles increased obviously. The drug loading reached 1.5 mg·mL - 1 , which was 30 times higher than that of GA elastic vesicles without lysine. The drug loading and deformability of the elastic vesicles was remarkably higher than that obtained using the lysine salt. In addition, the cumulative amount of GA permeation through rat skin within 8 h and the residual amount in the skin were found to be 4.3 times and 9.2 times higher than that of the vesicles without lysine, which was higher than that of the elastic vesicles with the lysine salt of GA. These results indicate that lysine forms an ion associate with GA, which takes part in the formation of vesicle membranes and subsequently increases the membraneʹs fluidity. Such a high drug loading capability is a result of the synergistic effect between lysine and the elastic vesicles. (Article)

Regulation of Angiogenesis by Histone Chaperone HIRA-mediated Incorporation of Lysine 56-acetylated Histone H3.3 at Chromatin Domains of Endothelial Genes

Angiogenesis is critically dependent on endothelial cell-specific transcriptional mechanisms. However, the molecular processes that regulate chromatin domains and thereby dictate transcription of key endothelial genes are poorly understood. Here, we report that, in endothelial cells, angiogenic signal-mediated transcriptional induction of Vegfr1 (vascular endothelial growth factor receptor 1) is dependent on the histone chaperone, HIRA (histone cell cycle regulation-defective homolog A). Our molecular analyses revealed that, in response to angiogenic signals, HIRA is induced in endothelial cells and mediates incorporation of lysine 56 acetylated histone H3.3 (H3acK56) at the chromatin domain of Vegfr1. HIRA-mediated incorporation of H3acK56 is a general mechanism associated with transcriptional induction of several angiogenic genes in endothelial cells. Depletion of HIRA inhibits H3acK56 incorporation and transcriptional induction of Vegfr1 and other angiogenic genes. Our functional analyses revealed that depletion of HIRA abrogates endothelial network formation on Matrigel and inhibits angiogenesis in an in vivo Matrigel plug assay. Furthermore, analysis in a laser-induced choroidal neovascularization model showed that depletion of HIRA significantly inhibits neovascularization. Our results for the first time decipher a histone chaperone (HIRA)-dependent molecular mechanism in endothelial gene regulation and indicate that histone chaperones could be new targets for angiogenesis therapy.

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Incorporation of lysine from microbial origin into tissue protein of Iberian and Landrace × Large White piglets

The distinct incorporation of lysine from microbial origin in tissues has been investigated in Iberian and Landrace × Large White piglets of 47 ± 3 d of age ( n = 8/genotype) given 15N-labelled ammonium sulphate added to the diet. Four piglets of each breed were allocated in metabolic cages to prevent coprophagy. All piglets consumed the labelled diet for 10 days and then were slaughtered. Viscera and carcass weights were recorded and gastrointestinal tissues carefully handled. Samples of carcass, liver, and bacterial pellets isolated from the last third of the small intestine, were hydrolyzed, desalted by ion-exchange, and the amino acids eluted analyzed for 15N-lysine enrichment in a GC-combustion/isotope ratio mass-spectrometer. Background 15N-lysine in tissues was also determined in 3 piglets per breed. Liver enrichment was considerably higher in Landrace × Large White than in Iberian piglets (0.0273 ± 0.0013 vs. 0.0167 ± 0.0013 APE, P < 0.05) that, opposite to our expectations, suggests higher incorporation of microbial lysine in tissues of Landrace × Large White piglets. Protein shortage in the experimental diet relative to requirements could have enhanced microbial lysine absorption in the conventional piglets.

Advanced Identification of Proteins in Uncharacterized Proteomes by Pulsed in Vivo Stable Isotope Labeling-based Mass Spectrometry

Despite progress in the characterization of their genomes, proteomes of several model organisms are often only poorly characterized. This problem is aggravated by the presence of large numbers of expressed sequence tag clones that lack homologues in other species, which makes it difficult to identify new proteins irrespective of whether such molecules are involved in species-specific biological processes. We have used a pulsed stable isotope labeling with amino acids in cell culture (SILAC)-based mass spectrometry method, which is based on the detection of paired peptides after [(13)C(6)]lysine incorporation into proteins in vivo, to greatly increase the confidence of protein identification in cross-species database searches. The method was applied to identify nearly 3000 proteins in regenerating tails of the urodele amphibian Notophthalmus viridescens, which possesses outstanding capabilities in the regeneration of complex tissues. We reason that pulsed in vivo SILAC represents a versatile tool to identify new proteins in species for which only limited sequence information exists.

Investigating Conformational Ensembles in Alanine Based Peptides Using Vibrational and Ecd Spectroscopy

Short alanine based peptides are of interest to the protein community, due, in part to their departure from the statistical coil model. These peptides are too small to assume major secondary structures, but rather, have been found to adopt an ensemble of conformations in aqueous solution, with a predominance of PPII. However, experimental evidence suggests that the presence of charged residues might induce the sampling of multiple turn conformations, thus leading to a more compact structure of the peptide. To check this further, we measured the amide I profiles of the FTIR, Raman and VCD spectrum of H-(AAKAAW)-OH, and subsequently simulated the vibrational spectra using an excitonic coupling model, with NMR coupling constant and end-to-end distance constraints. We included multiple conformations: PPII, s-strand, R helix, L helix, and turns. The alanine residues experienced a high propensity for PPII structure, ∼70%, while ∼20% for s-strand conformations and smaller percentages for other coil structures. Lysine, however showed a larger propensity for s-strand ∼30% than the alanine residues, but the PPII content for lysine is still high (∼42%). We obtained an end-to-end distance of 10A, which is in accordance with FRET measurements of the end to end distance of H-Dbo-AAKAAW)-OH, (Dbo: 2,3-diazabicyclo[2.2.2]oct-2-ene-labeled asparagine). This distance is indicative of a rather compact peptide sampling many different coil structures, including a high PPII content, as well as turn structures. The charge lysine residue results in more turn structures being sampled by the succeeding alanine residue. UV circular dichroism (UV-CD) spectra of H-(AAKAAW)-OH and H-(AAAAAW)-OH indicate a higher PPII content for the latter peptide. These data show that the incorporation of lysine yields indeed a more compact conformation.

The effects of cross‐link density and chemistry on the calcification potential of diamine‐extended glutaraldehyde‐fixed bioprosthetic heart‐valve materials

Despite indications that GA (glutaraldehyde)-crosslinked tissues remain prone to long-term degradation and calcification, it is still the reagent of choice in the fixation of bioprosthetic heart valves. We have shown previously that increased GA concentrations and diamine extension of cross-links with lysine incorporation lead to mitigated in vivo calcification, mainly of porcine aortic-wall tissue. The present study was performed to assess the correlation between the cross-link density of all three commonly used tissue types [PW (porcine aortic wall), PL (porcine aortic leaflet) and BP (bovine pericardium)] and tissue calcification in the subcutaneous rat model after GA treatment with or without lysine. The effect of lysine enhancement, and increased GA concentration in the presence of lysine, resulted in significant increases in tissue cross-linking in all three tissue types. Although increased GA concentration on its own resulted in decreased calcification without an increase in cross-link density, overall positive correlations were found between denaturation temperature and RPD (resistance towards protease degradation) [correlation coefficient (rho) values: rhoPW =0.922, rhoPL =0.783 and rhoBP =0.955], whereas negative correlations existed between RPD and calcification (rhoPW=-0.836, rhoPL=-0.929 and rhoBP=-0.579). The combination of lysine enhancement and an increase in GA concentration from 0.2 to 3% resulted in 79, 44 and 56% decreases in calcification in PW, PL and BP. In the case of BP, a decrease in calcification of 81% could be achieved merely by adding lysine extension to low-concentration (0.2 %) GA cross-linking. Thus it is concluded that the increase in cross-link density achieved by lysine incorporation, and by increased GA concentration in the presence of lysine, results in significant and marked decreases in calcification of all three types of tissues commonly used in bioprosthetic heart valves.