L-lysine Residues Research Articles

The role of the acetyl-glutamate cycle in fumarate biosynthesis through L-ornithine supply via the ornithine-urea cycle in Aureobasidium pullulans var. aubasidani

In Aureobasidium pullulans var. Aubasidani, the ornithine-urea cycle (OUC) plays a role in fumarate biosynthesis with L-ornithine serving as a significant intermediate. The acetyl-glutamate cycle (AGC) is also responsible for synthesizing L-ornithine. But it remains unclear whether AGC can assist OUC in fumarate biosynthesis. N-acetyl-glutamate kinase and N-acetyl-gamma-glutamyl-phosphate reductase present in AGC were both encoded by the ARGBC gene in A. pullulans var. Aubasidani and N-acetyl-glutamate kinase was observed to be insensitive to feedback inhibition by L-arginine from OUC. This observation could be attributed to the presence of conserved residues, specifically L-threonine (T340) and L-arginine (R336), in the N-acetyl-glutamate kinase as opposed to the conserved L-threonine (T340) and L-lysine (K336) residues that could interact with L-arginine. Hence, AGC remained active and consistently produced L-ornithine for OUC. The deletion of the ARGBC gene in the parent glucose oxidase-deficient mutant (Δgox) led to decreased L-ornithine accumulation and reduced fumarate production in the disruptant Δgox Δargbc while complementation of the ARGBC gene enhanced accumulation of L-ornithine and restored fumarate production by the complementing strain ARGBC-H. The introduction of exogenous L-arginine clearly impacted fumarate production and cell growth in the disruptant strain Δgox Δargbc. It was concluded that achieving elevated fumarate levels through OUC, maintaining the activity of AGC for high L-ornithine accumulation was crucial. The produced fumarate had many applications.

Self-Assembly and Antimicrobial Activity of Lipopeptides Containing Lysine-Rich Tripeptides.

The conformation and self-assembly of two pairs of model lipidated tripeptides in aqueous solution are probed using a combination of spectroscopic methods along with cryogenic-transmission electron microscopy (cryo-TEM) and small-angle X-ray scattering (SAXS). The palmitoylated lipopeptides comprise C16-YKK or C16-WKK (with two l-lysine residues) or their respective derivatives containing d-lysine (k), i.e., C16-Ykk and C16-Wkk. All four molecules self-assemble into spherical micelles which show structure factor effects in SAXS profiles due to intermicellar packing in aqueous solution. Consistent with micellar structures, the tripeptides in the coronas have a largely unordered conformation, as probed using spectroscopic methods. The molecules are found to have good cytocompatibility with fibroblasts at sufficiently low concentrations, although some loss of cell viability is noted at the highest concentrations examined (above the critical aggregation concentration of the lipopeptides, determined from fluorescence dye probe measurements). Preliminary tests also showed antimicrobial activity against both Gram-negative and Gram-positive bacteria.

Staining of stratum corneum with fluorescent ε-poly-L-lysine and its application to evaluation of skin conditions.

ε-Poly-L-lysine (PLL) is a cationic polymer consisting of 25 to 35 L-lysine residues that adheres to the surface of skin as well as hair. However, the properties of PLL regarding its adhesion to the skin remain to be elucidated. In this study, we examined the staining of stratum corneum (SC) with fluorescence-labeled PLL and explored its relationship with skin condition. Alexa Fluor 488-labeled PLL (AF-PLL) was reacted with tape-stripped stratum corneum (SC), and the staining properties were monitored by fluorescence microscopy. Clinical study was performed by measuring the water content of the cheek SC and transepidermal water loss (TEWL), and the tape-stripped SC was subjected to staining with AF-PLL. AF-PLL staining of the SC was inhibited at acidic pH or by the addition of high concentration of salt solution, suggesting the involvement of ionic interaction between PLL and the SC, at least in part. The AF-PLL staining was inhibited by unlabeled PLL or various alkyl amines, but not by L-lysine monomer. AF-PLL staining was observed inside the corneocytes as well as surrounding cornified envelope. Clinical study revealed that AF-PLL staining intensity of the SC was negatively correlated with its water content and positively correlated with its TEWL. PLL can efficiently adhere to SC and AF-PLL staining of SC can be applied to evaluate skin conditions.

A Study of Type II ɛ-PL Degrading Enzyme (pldII) in Streptomyces albulus through the CRISPRi System.

ε-Poly-L-lysine (ε-PL) is a widely used antibacterial peptide polymerized of 25–35 L-lysine residues. The antibacterial effect of ε-PL is closely related to the polymerization degree. However, the mechanism of ε-PL degradation in S. albulus remains unclear. This study utilized the integrative plasmid pSET152-based CRISPRi system to transcriptionally repress the ε-PL degrading enzyme (pldII). The expression of pldII is regulated by changing the recognition site of dCas9. Through the ε-PL bacteriostatic experiments of repression strains, it was found that the repression of pldII improves the antibacterial effect of the ε-PL product. The consecutive MALDI-TOF-MS results confirmed that the molecular weight distribution of the ε-PL was changed after repression. The repression strain S1 showed a particular peak with a polymerization degree of 44, and other repression strains also generated ε-PL with a polymerization degree of over 40. Furthermore, the homology modeling and substrate docking of pldII, a typical endo-type metallopeptidase, were performed to resolve the degradation mechanism of ε-PL in S. albulus. The hydrolysis of ε-PL within pldII, initiated from the N-terminus by two amino acid-binding residues, Thr194 and Glu281, led to varying levels of polymerization of ε-PL.

Tandem Repeat of a Short Human Chemerin-Derived Peptide and Its Nontoxic d-Lysine-Containing Enantiomer Display Broad-Spectrum Antimicrobial and Antitubercular Activities.

To design novel antimicrobial peptides by utilizing the sequence of the human host defense protein, chemerin, a seven-residue amphipathic stretch located in the amino acid region, 109-115, was identified, which possesses the highest density of hydrophobic and positively charged residues. Although this 7-mer peptide was inactive toward microorganisms, its 14-mer tandem repeat (Chem-KVL) was highly active against different bacteria including methicillin-resistant Staphylococcus aureus, a multidrug-resistant Staphylococcus aureus strain, and slow- and fast-growing mycobacterial species. The selective enantiomeric substitutions of its two l-lysine residues were attempted to confer cell selectivity and proteolytic stability to Chem-KVL. Chem-8dK with a d-lysine replacement in its middle (eighth position) showed the lowest hemolytic activity against human red blood cells among Chem-KVL analogues and maintained high antimicrobial properties. Chem-8dK showed in vivo efficacy against Pseudomonas aeruginosa infection in BALB/c mice and inhibited the development of resistance in this microorganism up to 30 serial passages and growth of intracellular mycobacteria in THP-1 cells.

D- and Unnatural Amino Acid Substituted Antimicrobial Peptides With Improved Proteolytic Resistance and Their Proteolytic Degradation Characteristics.

The transition of antimicrobial peptides (AMPs) from the laboratory to market has been severely hindered by their instability toward proteases in biological systems. In the present study, we synthesized derivatives of the cationic AMP Pep05 (KRLFKKLLKYLRKF) by substituting L-amino acid residues with D- and unnatural amino acids, such as D-lysine, D-arginine, L-2,4-diaminobutanoic acid (Dab), L-2,3-diaminopropionic acid (Dap), L-homoarginine, 4-aminobutanoic acid (Aib), and L-thienylalanine, and evaluated their antimicrobial activities, toxicities, and stabilities toward trypsin, plasma proteases, and secreted bacterial proteases. In addition to measuring changes in the concentration of the intact peptides, LC-MS was used to identify the degradation products of the modified AMPs in the presence of trypsin and plasma proteases to determine degradation pathways and examine whether the amino acid substitutions afforded improved proteolytic resistance. The results revealed that both D- and unnatural amino acids enhanced the stabilities of the peptides toward proteases. The derivative DP06, in which all of the L-lysine and L-arginine residues were replaced by D-amino acids, displayed remarkable stability and mild toxicity in vitro but only slight activity and severe toxicity in vivo, indicating a significant difference between the in vivo and in vitro results. Unexpectedly, we found that the incorporation of a single Aib residue at the N-terminus of compound UP09 afforded remarkably enhanced plasma stability and improved activity in vivo. Hence, this derivative may represent a candidate AMP for further optimization, providing a new strategy for the design of novel AMPs with improved bioavailability.

Non-isocyanate urethane linkage formation using l-lysine residues as amine sources.

Bio-based polyurethane materials are broadly applied in medicine as drug delivery systems. Nevertheless, their synthesis comprises the use of petroleum-based toxic amines, isocyanates and polyols, and their biocompatibility or functionalization is limited. Therefore, the use of lysine residues as amine sources to create non-isocyanate urethane (NIU) linkages was investigated. Therefore, a five-membered biscyclic carbonate (BCC) was firstly synthetized and reacted with a protected lysine, a tripeptide and a heptapeptide to confirm the urethane linkage formation with lysine moiety and to optimize reaction conditions. Afterwards, the reactions between BCC and a model protein, elastin-like protein (ELP), and β-Lactoglobulin (BLG) obtained from whey protein, respectively, were performed. The synthesized protein materials were structural, thermally and morphologically characterized to confirm the urethane linkage formation. The results demonstrate that using both simple and more complex source of amines (lysine), urethane linkages were effectively achieved. This pioneering approach opens the possibility of using proteins to develop non-isocyanate polyurethanes (NIPUs) with tailored properties.

Assessment of Proteus mirabilis Antigen Immunological Complexes by Atomic Force Microscopy.

Atomic force microscopy (AFM) is being increasingly used to directly measure protein interactions in nearly physiological environments. Here, protocols for atomic force microscopy (AFM) for visualization of antigen-antibody complexes are presented. The technique is used to demonstrate complexes formed by rheumatoid arthritis patient antibodies with lipopolysaccharide (LPS) isolated from P. mirabilis (O3) strain S1959 and a synthetic antigen (LPS epitope of 6 N-alpha-(D-galacturonoyl)-L-lysine residues).

Effect of £-Poly-l-lysine on a Glucose Sensor Based on Glucose Oxidase and Ferricyanide Ion

ε-Poly-L-lysine (εPL) is a homopolymer of L-lysine residues with linkages between the α-carboxyl and ε-amino groups. εPL exists as a polycationic species under acidic and neutral conditions, and can form a polyion complex with negatively charged glucose oxidase (GOx). We previously reported that εPL significantly promoted a GOx enzymatic reaction using ferricyanide ion ([Fe(CN)6]3-) as the oxidant. Here, we construct a GOx-immobilized electrode using εPL and glutaraldehyde, and show that [Fe(CN)6]3- can efficiently act as the mediator in this electrode. Ferricyanide ion failed to function adequately as the mediator when bovine serum albumin or other polyamines (e.g., polyallylamine, α-poly-L-lysine) were used instead of εPL. The GOx-immobilized electrode using εPL successfully responded to glucose even when the [Fe(CN)6]3- concentration was as low as 1 mM, and exhibited a wide dynamic range of up to several tens of mM. Thus, εPL is considered to be a useful additive for glucose sensors based on the [Fe(CN)6]4-/3--mediated GOx catalytic current.

Macroporous Hydrogels Composed Entirely of Synthetic Polypeptides: Biocompatible and Enzyme Biodegradable 3D Cellular Scaffolds.

Synthetic polypeptides are a class of bioinspired polymers with well demonstrated biocompatibility, enzyme biodegradability, and cell adhesive properties, making them promising materials for the preparation of macroporous hydrogels as 3D cellular scaffolds. Three-dimensional macroporous hydrogels composed entirely of biocompatible and enzyme biodegradable synthetic polypeptides have thus been prepared. Under cryoconditions, macroporous hydrogels in the form of macroporous cryogels were prepared using a single copolymer component through direct EDC/sulfo-NHS zero-length cross-linking between poly(l-glutamic acid) (PLG) and poly(l-lysine) (PLL) residues on a PLG-r-PLL random copolypeptide chain. The resulting macroporous cryogels were found to contain large interconnected pores (≥100 μm) highly suitable for tissue engineering applications. Tuning the relative ratios of the amino acid components could result in cryogels with very different pore structures, swelling, and mechanical properties, suitable for developing gels for a range of possible soft tissue engineering applications. These cryogels were shown to be enzymatically biodegradable and demonstrated excellent biocompatibility, cell attachment and cell proliferation profiles with mammalian fibroblast (NIH-3T3) cells, demonstrating the appeal of these novel cryogels as highly suitable cellular scaffolds.

Stretch-Induced Helical Conformations in Poly(l-lysine)/Hyaluronic Acid Multilayers.

We investigate the effect of stretching on the secondary structure of cross-linked poly(l-lysine)/hyaluronic acid (PLL/HA) multilayers. We show that stretching these films induces changes in the secondary structure of PLL chains. Our results suggest that not only α- but also 310-helices might form in the film under stretching. Such 310-helices have never been observed for PLL so far. These changes of the secondary structure of PLL are reversible, i.e., when returning to the nonstretched state one recovers the initial film structure. Using molecular dynamics simulations of chains composed of 20 l-lysine residues (PLL20), we find that these chains never adopt a helical conformation in water. In contrast, when the end-to-end distance of the chains is restrained to values smaller than the mean end-to-end distance of free chains, a distance domain rarely explored by the free chains, helical conformations become accessible. Moreover, the formation of not only α- but also 310-helices is predicted by the simulations. These results suggest that the change of the end-to-end distance of PLL chains in the stretched film is at the origin of the helix formation.

Enhancement of ε-poly-L-lysine (ε-PL) production by a novel producer Bacillus cereus using metabolic precursors and glucose feeding.

Epsilon poly-l-lysine (ε-PL) is a homo-biopolymer with approximately 25–30 l-lysine residues. It is a promising natural biopolymer widely used in food and pharmaceutical industry. The present work reports enhanced production of ε-PL with a novel producer Bacillus cereus using amino acids and TCA cycle intermediates in the fermentation medium. Among the various amino acids and TCA cycle intermediates tested 2 mM l-aspartic acid and 5 mM citric acid gave ε-PL yield of 145.5 and 230 mg/L, respectively. A combination of citric acid after 24 h and l-aspartic acid after 36 h improved ε-PL yield from 85 mg/L (control) to 335 mg/L. Glucose feeding strategy along with metabolic precursors was employed which further enhanced ε-PL yield to 565 mg/L. Thus, more than sixfold increase in ε-PL yield was achieved suggesting the potential of Bacillus cereus as a novel ε-PL producer.

ε-Poly-L-lysine peptide chain length regulated by the linkers connecting the transmembrane domains of ε-Poly-L-lysine synthetase.

ε-Poly-l-lysine (ε-PL), consisting of 25 to 35 l-lysine residues with linkages between the α-carboxyl groups and ε-amino groups, is produced by Streptomyces albulus NBRC14147. ε-PL synthetase (Pls) is a membrane protein with six transmembrane domains (TM1 to TM6) as well as both an adenylation domain and a thiolation domain, characteristic of the nonribosomal peptide synthetases. Pls directly generates ε-PL chain length diversity (25- to 35-mer), but the processes that control the chain length of ε-PL during the polymerization reaction are still not fully understood. Here, we report on the identification of Pls amino acid residues involved in the regulation of the ε-PL chain length. From approximately 12,000 variants generated by random mutagenesis, we found 8 Pls variants that produced shorter chains of ε-PL. These variants have one or more mutations in two linker regions connecting the TM1 and TM2 domains and the TM3 and TM4 domains. In the Pls catalytic mechanism, the growing chain of ε-PL is not tethered to the enzyme, implying that the enzyme must hold the growing chain until the polymerization reaction is complete. Our findings reveal that the linker regions are important contributors to grasp the growing chain of ε-PL.

Binding ability of a thymine-functionalized oligolysine towards nucleic acids

In this Letter, we investigated the binding properties towards nucleic acids of a thymine-functionalized oligolysine, composed of nucleobase-bearing amino acid moieties and underivatized l-lysine residues alternate in the backbone. The basic nucleopeptide proved to be well soluble in water and able to interact with both DNA and RNA, as suggested by circular dichroism, UV and surface plasmon resonance studies performed on the thymine-containing oligomer with both adenine-containing DNA (dA12) and RNA (rA12 and poly rA) molecules. In both cases the thymine-functionalized oligolysine was proven to form complexes characterized by a 1:1 T/A stoichiometric ratio, as evidenced by CD titration. UV melting experiments revealed that the complex formed between the homothymine oligolysine and rA12 RNA was more stable than the complex with dA12 DNA probably due to the additional H-bonding of the 2′-OH groups in RNA, that reinforces the overall interaction with the nucleopeptide. Finally, human serum stability assays were conducted on the thymine-bearing nucleopeptide which showed a half-life of 45min.

Anti-tumor activities of the host-defense peptide hymenochirin-1B

The hymenochirins are a family of cationic, amphipathic, α-helical host-defense peptides, first isolated from skin secretions of the Congo clawed frog Hymenochirus boettgeri (Pipidae). Of the four hymenochirins tested, hymenochirin-1B (IKLSPETKDNLKKVLKGAIKGAIVAKMV.NH2) shows the greatest cytotoxic potency against non-small cell lung adenocarcinoma A549 cells (LC50=2.5±0.2μM), breast adenocarcinoma MDA-MB-231 cells (LC50=9.0±0.3μM), colorectal adenocarcinoma HT-29 cells (LC50=9.7±0.2μM), and hepatocarcinoma HepG2 cells (LC50=22.5±1.4μM) with appreciably less hemolytic activity against human erythrocytes (LC50=213±18μM). Structure–activity relationships were investigated by synthesizing analogs of hymenochirin-1B in which Pro5, Glu6 and Asp9on the hydrophilic face of the helix were replaced by one or more L-lysine or D-lysine residues. The [D9K] analog displays the greatest increase in potency against all four cell lines (up to 6fold) but hemolytic activity also increases (LC50=174±12μM). The [D9k] and [E6k,D9k] analogs retain relatively high cytotoxic potency against the tumor cells (LC50 in the range 2.1–21μM) but show reduced hemolytic activity (LC50>300μM). The data suggest that hymenochirin-1B has therapeutic potential as a template to generate potent, non-toxic anti-cancer agents.

An analog of the host-defense peptide hymenochirin-1B with potent broad-spectrum activity against multidrug-resistant bacteria and immunomodulatory properties

Hymenochirin-1B (IKLSPETKDN10LKKVLKGAIK20GAIAVAKMV.NH2) is a cationic, amphipathic, α-helical, host-defense peptide, first isolated from skin secretions of the Congo clawed frog Hymenochirus boettgeri (Pipidae). Structure-activity relationships were investigated by synthesizing analogs in which the Pro5, Glu6 and Asp9 on the hydrophilic face of the α-helix are substituted by one or more l-lysine or d-lysine residues. Although replacement with l-lysine generates analogs with increased antimicrobial potency against a range of Gram-positive and Gram-negative bacteria (up to 8-fold), the peptides are more hemolytic. Increasing the cationicity of hymenochirin-1B while reducing the helicity by substitutions with d-lysine generates analogs that are between 2 and 8 fold more potent than the native peptide and are equally or less hemolytic. [E6k,D9k]hymenochirin-1B represents a candidate for drug development as it shows high potency against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and a range of Gram-negative bacteria, including multidrug-resistant strains of Acinetobacter baumannii and Stenotrophomonas maltophilia (MIC in the range 0.8–3.1μM) and NDM-1 carbapenemase-producing clinical isolates of Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae and Citrobacter freundii (MIC in the range 3.1–6.25μM), and low hemolytic activity (LC50=302μM). [E6k,D9k]hymenochirin-1B, at a concentration of 2.5μM, significantly (P<0.05) stimulates the production of the anti-inflammatory cytokines IL-4 and IL-10 by human peripheral blood mononuclear cells but is without significant effect on production of the pro-inflammatory cytokines TNF-α and IL-17.

Transformation of the naturally occurring frog skin peptide, alyteserin-2a into a potent, non-toxic anti-cancer agent

Alyteserin-2a (ILGKLLSTAAGLLSNL.NH(2)) is a cationic, amphipathic α-helical cell-penetrating peptide, first isolated from skin secretions of the midwife toad Alytes obstetricans. Structure-activity relationships were investigated by synthesizing analogs of alyteserin-2a in which amino acids on the hydrophobic face of the helix were replaced by L-tryptophan and amino acids on the hydrophilic face were replaced by one or more L-lysine or D-lysine residues. The Trp-containing peptides display increased cytotoxic activity against non-small cell lung adenocarcinoma A549 cells (up to 11-fold), but hemolytic activity against human erythrocytes increases in parallel. The potency of the N15K analog against A549 cells (LC(50) = 13 μM) increases sixfold relative to alyteserin-2a and the therapeutic index (ratio of LC(50) for erythrocytes and tumor cells) increases twofold. Incorporation of a D-Lys(11) residue into the N15K analog generates a peptide that retains potency against A549 cells (LC(50) = 15 μM) but whose therapeutic index is 13-fold elevated relative to the native peptide. [G11k, N15K] alyteserin-2a is also active against human hepatocarcinoma HepG2 cells (LC(50) = 26 μM), breast adenocarcinoma MDA-MB-231 cells (LC(50) = 20 μM), and colorectal adenocarcinoma HT-29 cells (LC(50) = 28 μM). [G11k, N15K] alyteserin-2a, in concentrations as low as 1 μg/mL, significantly (P < 0.05) inhibits the release of the immune-suppressive cytokines IL-10 and TGF-β from unstimulated and concanavalin A-stimulated peripheral blood mononuclear cells. The data suggest a strategy of increasing the cationicity while reducing the helicity of naturally occurring amphipathic α-helical peptides to generate analogs with improved cytotoxicity against tumor cells but decreased activity against non-neoplastic cells.

Synthesis of a diaminopropanoic acid-based nucleoamino acid and assembly of cationic nucleopeptides for biomedical applications

In this work, we report a synthetic approach to a Fmoc-protected nucleoamino acid, based on L-diaminopropanoic acid, carrying the DNA nucleobase on the alpha-amino group by means of an amide bond, suitable for the solid-phase synthesis of novel nucleopeptides of potential interest in biomedicine. After ESI-MS and NMR characterization this building block was used for the assembly of a thymine-functionalized nucleopeptide, composed of nucleobase-containing L-diaminopropanoic acid moieties and underivatized L-lysine residues alternated in the backbone. Circular dichroism studies performed on the cationic nucleopeptide and adenine-containing DNA and RNA molecules suggested that the thymine-containing peptide is able to interact with both DNA and RNA. In particular, a significant conformational variation in the RNA structure was suggested by CD studies. Human serum stability assays were also conducted on the cationic nucleopeptide, which was found to be highly resistant to enzymatic degradation.

Effect of the oligopeptide length on the DNA condensation process in aqueous-salt solutions

The methods of circular dichroism, ultraviolet spectrophotometry, dynamic light scattering, viscometry, dynamic birefringence, and atomic force microscopy are employed to study the conformational properties of the DNA molecule during its complexation with oligopeptides. Samples with c 2, 3, and 5 monomeric L-lysine residues are used. Experimental data evidence that in the diluted solutions (with a low ionic strength) of DNA with oligopeptides (with 3 and 5 monomeric units) the condensation of the macromolecule is observed at specific peptide concentrations. For the bipeptide the DNA condensation in the solution is not observed. In the DNA solutions with a high ionic strength (1 M NaCl), oligopeptides do not affect the macromolecule size.

Occurrence, Biosynthesis, Biodegradation, and Industrial and Medical Applications of a Naturally Occurring ε-Poly-L-lysine

ε-Poly-L-lysine (ε-PL) consists of 25-35 L-lysine residues with linkages between the α-carboxyl groups and the ε-amino groups. It exhibits antimicrobial activity against a spectrum of microorganisms, including bacteria and fungi. Because of its high levels of safety and biodegradability, it is used as a food preservative in several countries. We recently identified an ε-PL synthetase (Pls) as a membrane protein, and investigated the catalytic mechanism. Pls was found to be an unusual non-ribosomal peptide synthetase (NRPS)-like peptide synthetase producing ε-PL with chain-length diversity. In addition, transcriptional analysis of pls and a kinetic study of Pls further suggested that the Pls catalytic function is regulated by intracellular ATP, high levels of which are required for full enzymatic activity. Furthermore, it was found that acidic pH conditions during ε-PL fermentation are necessary for the accumulation of intracellular ATP, rather than inhibition of the ε-PL-degrading enzyme.