D-amino Acids Research Articles

Rational Engineering of an Antimalarial Peptide with Enhanced Proteolytic Stability and Preserved Parasite Invasion Inhibitory Activity

We describe the rational chemical engineering to enhance proteolytic stability of a chimeric peptide using a combination of unique strategies that involve the incorporation of a series of D-amino acids...

D-Peptide cell culture scaffolds with enhanced antibacterial and controllable release properties.

The development of peptide-based hydrogels characterized by both high biostability and potent antimicrobial activity, aimed at combating multidrug-resistant bacterial infections and providing scaffolds for cell cultures, continues to pose a significant challenge. The susceptibility of antimicrobial peptides (AMPs) to degradation by cations, serum, and proteases restricted their applications in clinical environments. In this study, we designed a peptide sequence (termed D-IK1) entirely consisting of D-amino acids, an enantiomer of a previously reported AMP IK1. Our results demonstrated remarkably improved antibacterial and anticancer activities of D-IK1 as compared to IK1. D-IK1 self-assembled into hydrogels that effectively inhibited bacterial and cancer cell growth by the controlled and sustained release of D-IK1. Importantly, D-IK1 was extremely stable in salt solutions and resisted serum and protease degradation. In addition, the D-IK1 hydrogel fostered cell adhesion and proliferation, proving its viability as a 3D scaffold for cell culture applications. Our research presents a versatile, highly stable antibacterial hydrogel scaffold with potential widespread applications in cell culture, wound healing, and the eradication of multidrug-resistant bacterial infections.

Synergistic D-Amino Acids Based Antimicrobial Cocktails Formulated via High-Throughput Screening and Machine Learning.

Antimicrobial resistance (AMR) from pathogenic bacterial biofilms has become a global health issue while developing novel antimicrobials is inefficient and costly. Combining existing multiple drugs with enhanced efficacy and/or reduced toxicity may be a promising approach to treat AMR. D-amino acids mixtures coupled with antibiotics can provide new therapies for drug-resistance infection with reduced toxicity by lower drug dosage requirements. However, iterative trial-and-error experiments are not tenable to prioritize credible drug formulations, owing to the extremely large number of possible combinations. Herein, a new avenue is provide to accelerate the exploration of desirable antimicrobial formulations via high-throughput screening and machine learning optimization. Such an intelligent method can navigate the large search space and rapidly identify the D-amino acid mixtures with the highest anti-biofilm efficiency and also the synergisms between D-amino acid mixtures and antibiotics. The optimized drug cocktails exhibit high antimicrobial efficacy while remaining non-toxic, which is demonstrated not only from in vitro assessments but also the first in vivo study using a lung infection mouse model.

Development of LAG-3/FGL1 blocking peptide and combination with radiotherapy for cancer immunotherapy

Aside from antibodies, peptides show great potential as immune checkpoint inhibitors (ICIs) due to several advantages, such as better tumor penetration and lower cost. Lymphocyte-activation gene 3 (LAG-3) is an immune checkpoint which can induce T cell dysfunction through interaction with its soluble ligand fibrinogen like protein-1 (FGL1). Here, we found that LAG-3 expression was higher than programmed cell death protein 1 (PD-1) in multiple human cancers by TCGA databases, and successfully identified a LAG-3 binding peptide LFP-6 by phage display bio-panning, which specifically blocks the interaction of LAG-3/FGL1 but not LAG-3/MHC-II. Subsequently, d-amino acids were introduced to substitute the N- and C-terminus of LFP-6 to obtain the proteolysis-resistant peptide LFP-D1, which restores T cell function in vitro and inhibits tumor growth in vivo. Further, a bispecific peptide LFOP targeting both PD-1/PD-L1 and LAG-3/FGL1 was designed by conjugating LFP-D1 with PD-1/PD-L1 blocking peptide OPBP-1(8–12), which activates T cell with enhanced proliferation and IFN-γ production. More importantly, LFOP combined with radiotherapy significantly improve the T cell infiltration in tumor and elevate systemic antitumor immune response. In conclusion, we developed a novel peptide blocking LAG-3/FGL1 which can restore T cell function, and the bispecific peptide synergizes with radiotherapy to further enhance the antitumor immune response.

Chemogenetic emulation of intraneuronal oxidative stress affects synaptic plasticity

Overproduction of reactive oxygen species (ROS) and oxidative cell damage are commonly associated with most brain pathologies [1, 2]. Dysregulation of redox homeostasis in the aging brain is thought to be responsible for impaired synaptic transmission and plasticity, leading to reduced neuronal computational capacity and learning and memory deficits. Studying the contribution of oxidative stress to the development of diseases, such as age-related dementia and Alzheimer’s disease, is complex due to the lack of methods for modeling isolated oxidative damage in individual cell types [3]. We introduce a chemogenetic approach utilizing D-amino acid oxidase (DAAO) from yeast to produce hydrogen peroxide intraneuronally, which is one of the most stable ROS [4]. H2O2 generation was evaluated in primary cultured neurons and acute mouse brain slices through the utilization of a genetically encoded fluorescent biosensor, HyPer7, to validate the methodology [5]. The changes in the fluorescence signal of HyPer7 after treating neurons that expressed DAAO with D-Norvaline (D-Nva), a substrate for DAAO, confirmed the targeted production of H2O2 through chemogenetics. Using electrophysiological recordings in acute brain slices, we demonstrated that intraneuronal oxidative stress induced by chemogenetics did not affect basal synaptic transmission and the probability of neurotransmitter release from presynaptic terminals. However, it diminished long-term potentiation (LTP) at the single-cell level. Astrocytes have the ability to metabolize d-amino acids, rendering the proposed approach ineffective in vivo experiments. Consequently, in vivo testing of the tool was necessary for validation. To achieve this, an optical setup for exciting and detecting the HyPer7 signal was developed and implanted into the mouse brain via optical fibers. By using this approach, we were able to demonstrate the generation of H2O2 in DAAO-expressing neurons in vivo, upon intraperitoneal administration of D-amino acids. The results demonstrate that using a DAAO-based chemogenetic tool, along with electrophysiological recordings, clarifies numerous unanswered queries regarding the part of ROS-dependent signaling in typical brain activities and the impact of oxidative stress on the development of cognitive aging and preliminary neurodegenerative stages. The suggested method is valuable for detecting initial indicators of neuronal oxidative stress. Additionally, it can be used for evaluating probable antioxidants that can effectively combat neuronal oxidative harm.

Chemical Nose Strategy with Metabolic Labeling and "Antibiotic-Responsive Spectrum" Enables Accurate and Rapid Pathogen Identification.

The worldwide antimicrobial resistance (AMR) dilemma urgently requires rapid and accurate pathogen phenotype discrimination and antibiotic resistance identification. The conventional protocols are either time-consuming or depend on expensive instrumentations. Herein, we demonstrate a metabolic-labeling-assisted chemical nose strategy for phenotyping classification and antibiotic resistance identification of pathogens based on the "antibiotic-responsive spectrum" of different pathogens. d-Amino acids with click handles were metabolically incorporated into the cell wall of pathogens for further clicking with dibenzocyclooctyne-functionalized upconversion nanoparticles (DBCO-UCNPs) in the presence/absence of six types of antibiotics, which generates seven-channel sensing responses. With the assistance of machine learning algorithms, eight types of pathogens, including three types of antibiotic-resistant bacteria, can be well classified and discriminated in terms of microbial taxonomies, Gram phenotypes, and antibiotic resistance. The present metabolic-labeling-assisted strategy exhibits good anti-interference capability and improved discrimination ability rooted in the unique sensing mechanism. Sensitive identification of pathogens with 100% accuracy from artificial urinary tract infection samples at a concentration as low as 105 CFU/mL was achieved. Pathogens outside of the training set can also be discriminated well. This clearly demonstrated the potential of the present strategy in the identification of unknown pathogens in clinical samples.

D -Alanine Affects the Circadian Clock to Regulate Glucose Metabolism in the Kidney.

Key Points d-Alanine affects the circadian clock to regulate gluconeogenesis in the kidney. d-Alanine itself has a clear intrinsic circadian rhythm, which is regulated by urinary excretion, and acts on the circadian rhythm. d-Alanine is a signal activator for circadian rhythm and gluconeogenesis through circadian transcriptional network. Background The aberrant glucose circadian rhythm is associated with the pathogenesis of diabetes. Similar to glucose metabolism in the kidney and liver, d-alanine, a rare enantiomer of alanine, shows circadian alteration, although the effect of d-alanine on glucose metabolism has not been explored. Here, we show that d-alanine acts on the circadian clock and affects glucose metabolism in the kidney. Methods The blood and urinary levels of d-alanine in mice were measured using two-dimensional high-performance liquid chromatography system. Metabolic effects of d-alanine were analyzed in mice and in primary culture of kidney proximal tubular cells from mice. Behavioral and gene expression analyses of circadian rhythm were performed using mice bred under constant darkness. Results d-Alanine levels in blood exhibited a clear intrinsic circadian rhythm. Since this rhythm was regulated by the kidney through urinary excretion, we examined the effect of d-alanine on the kidney. In the kidney, d-alanine induced the expressions of genes involved in gluconeogenesis and circadian rhythm. Treatment of d-alanine mediated glucose production in mice. Ex vivo glucose production assay demonstrated that the treatment of d-alanine induced glucose production in primary culture of kidney proximal tubular cells, where d-amino acids are known to be reabsorbed, but not in that of liver cells. Gluconeogenetic effect of d-alanine has an intraday variation, and this effect was in part mediated through circadian transcriptional network. Under constant darkness, treatment of d-alanine normalized the circadian cycle of behavior and kidney gene expressions. Conclusions d-Alanine induces gluconeogenesis in the kidney and adjusts the period of the circadian clock. Normalization of circadian cycle by d-alanine may provide the therapeutic options for life style–related diseases and shift workers.

Relations between Glucose and d-Amino Acids in the Modulation of Biochemical and Functional Properties of Rodent Islets of Langerhans.

The cell-to-cell signaling role of d-amino acids (d-AAs) in the mammalian endocrine system, particularly in the islets of Langerhans, has drawn growing interest for their potential involvement in modulating glucose metabolism. Previous studies found colocalization of serine racemase [produces d-serine (d-Ser)] and d-alanine (d-Ala) within insulin-secreting beta cells and d-aspartate (d-Asp) within glucagon-secreting alpha cells. Expressed in the islets, functional N-methyl-d-aspartate receptors are involved in the modulation of glucose-stimulated insulin secretion and have binding sites for several d-AAs. However, knowledge of the regulation of d-AA levels in the islets during glucose stimulation as well as the response of islets to different levels of extracellular d-AAs is limited. In this study, we determined the intracellular and extracellular levels of d-Ser, d-Ala, and d-Asp in cultures of isolated rodent islets exposed to different levels of extracellular glucose. We found that the intracellular levels of the enantiomers demonstrated large variability and, in general, were not affected by extracellular glucose levels. However, significantly lower levels of extracellular d-Ser and d-Ala were observed in the islet media supplemented with 20 mM concentration of glucose compared to the control condition utilizing 3 mM glucose. Glucose-induced oscillations of intracellular free calcium concentration ([Ca2+]i), a proxy for insulin secretion, were modulated by the exogenous application of d-Ser and d-Ala but not by their l-stereoisomers. Our results provide new insights into the roles of d-AAs in the biochemistry and function of pancreatic islets.

Design of Chiral β-Double Helices from γ-Peptide Foldamers.

Chirality is ubiquitous in nature, and homochirality is manifested in many biomolecules. Although β-double helices are rare in peptides and proteins, they consist of alternating L- and D-amino acids. No peptide double helices with homochiral amino acids have been observed. Here, we report chiral β-double helices constructed from γ-peptides consisting of alternating achiral (E)-α,β-unsaturated 4,4-dimethyl γ-amino acids and chiral (E)-α,β-unsaturated γ-amino acids in both single crystals and in solution. The two independent strands of the same peptide intertwine to form a β-double helix structure, and it is stabilized by inter-strand hydrogen bonds. The peptides with chiral (E)-α,β-unsaturated γ-amino acids derived from α-L-amino acids adopt a (P)-β-double helix, whereas peptides consisting of (E)-α,β-unsaturated γ-amino acids derived from α-D-amino acids adopt an (M)-β-double helix conformation. The circular dichroism (CD) signature of the (P) and (M)-β-double helices and the stability of these peptides at higher temperatures were examined. Furthermore, ion transport studies suggested that these peptides transport ions across membranes. Even though the structural analogy suggests that these new β-double helices are structurally different from those of the α-peptide β-double helices, they retain ion transport activity. The results reported here may open new avenues in the design of functional foldamers.

D-Amino acids: new clinical pathways for brain diseases.

Free d-amino acids (d-AAs) are emerging as a novel and important class of signaling molecules in many organs, including the brain and endocrine systems. There has been considerable progress in our understanding of the fundamental roles of these atypical messengers, with increasingly recognized implications in a wide range of neuropathologies, including schizophrenia (SCZ), epilepsy, Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), substance abuse, and chronic pain, among others. Research has enabled the discovery that d-serine, d-aspartate and more recently d-cysteine are essential for the healthy development and function of the central nervous system (CNS). We discuss recent progress that has profoundly transformed our vision of numerous physiological processes but has also shown how d-AAs are now offering therapeutic promise in clinical settings for several human diseases.

Analysis of D-amino acids secreted from murine islets of Langerhans using Marfey’s reagent and reversed phase LC-MS/MS

D-amino acids (D-AAs) are important signaling molecules due to their ability to bind ionotropic N-methyl-D-aspartate receptors. D-serine (D-Ser), D-alanine (D-Ala), and D-aspartate (D-Asp) have been found individually in the endocrine portion of the pancreas, the islets of Langerhans, and/or their secretions. However, there has been no report of a comprehensive assessment of D-AAs in islet secretions. To evaluate the release of these compounds, the effectiveness of both 1-(9-fluorenyl)-ethyl chloroformate (FLEC reagent) and 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (Marfey’s reagent, MR) in separation of D/L-AA enantiomeric pairs in islet-specific buffers were evaluated. MR-derivatized D/L AAs showed greater than baseline resolution (Rs ≥ 1.5) of 13 enantiomeric pairs when using a non-linear gradient and an acidic mobile phase system, while FLEC-derivatized AAs exhibited limited resolution on both biphenyl and C18 columns. The optimized MR method yielded highly reproducible separations with retention times less than 1% RSD. Excellent linearity between the analyte concentrations and response (R2 > 0.98) were obtained, with less than 15% RSD for all analyte responses. Most analytes had an LOD at or below 100 nM, except for L-Ala (200 nM). The optimized MR method was used to quantify D-AAs in secretions of 150 murine islets after incubation in 3- and 20-mM glucose. In response to both solutions, D-Ser and D-glutamine were tentatively identified via comparison of retention time and quantifier-to-qualifer ion ratios with standards, and from spiking experiments. Both were secreted in low quantities which did not differ significantly in either low (D-Ser: 44 ± 2 fmol islet-1h−1; D-Gln: 300 ± 100 fmol islet-1h−1) or high (D-Ser: 23 ± 1 fmol islet-1h−1; D-Gln: 120 ± 50 fmol islet-1h−1) glucose across 3 biological replicates. The method developed is robust and can be applied to further examine the release of D-AAs and their potential roles in islet physiology.

Expanded Substrate Specificity in D-Amino Acid Transaminases: A Case Study of Transaminase from Blastococcus saxobsidens.

Enzymes with expanded substrate specificity are good starting points for the design of biocatalysts for target reactions. However, the structural basis of the expanded substrate specificity is still elusive, especially in the superfamily of pyridoxal-5'-phosphate-dependent transaminases, which are characterized by a conserved organization of both the active site and functional dimer. Here, we analyze the structure-function relationships in a non-canonical D-amino acid transaminase from Blastococcus saxobsidens, which is active towards D-amino acids and primary (R)-amines. A detailed study of the enzyme includes a kinetic analysis of its substrate scope and a structural analysis of the holoenzyme and its complex with phenylhydrazine-a reversible inhibitor and analogue of (R)-1-phenylethylamine-a benchmark substrate of (R)-selective amine transaminases. We suggest that the features of the active site of transaminase from B. saxobsidens, such as the flexibility of the R34 and R96 residues, the lack of bulky residues in the β-turn at the entrance to the active site, and the short O-pocket loop, facilitate the binding of substrates with and without α-carboxylate groups. The proposed structural determinants of the expanded substrate specificity can be used for the design of transaminases for the stereoselective amination of keto compounds.

Sculpting Secondary Structure of a Cyclic Peptide: Conformational Analysis of a Cyclic Hexapeptide Containing a Combination of l-Leu, d-Leu, and Aib Residues.

We have previously reported that cyclo(l-Leu-d-Leu-Aib-l-Leu-d-Leu-Aib) (2), a cyclic hexapeptide consisting of heterochiral l-Leu and d-Leu (l-Leu-d-Leu) residues with achiral 2-aminoisobutyric acid (Aib) residues, forms a figure-8 conformation. In this study, we newly designed cyclo(l-Leu-d-Leu-Aib-d-Leu-l-Leu-Aib)+ (4), an epimer of 2, and examined the conformational differences between 2 and 4 by X-ray crystallographic analysis. Peptide 4 formed a planar cyclic conformation with an antiparallel β-sheet hydrogen-bonding pattern. This investigation demonstrates the potential to manipulate the molecular conformation of cyclic peptides by simply arranging the l- and d-amino acids and emphasizes that diverse conformations can be obtained by using cyclic peptides. Harnessing cyclic peptides as platforms for distinct molecular structures is a promising approach to expanding the chemical space for various applications.

Artificial Peptide-Protein Necrosomes Promote Cell Death.

The presence of disordered region or large interacting surface within proteins significantly challenges the development of targeted drugs, commonly known as the "undruggable" issue. Here, we report a heterogeneous peptide-protein assembling strategy to selectively phosphorylate proteins, thereby activating the necroptotic signaling pathway and promoting cell necroptosis. Inspired by the structures of natural necrosomes formed by receptor interacting protein kinases (RIPK) 1 and 3, the kinase-biomimetic peptides are rationally designed by incorporating natural or D -amino acids, or connecting D -amino acids in a retro-inverso (DRI) manner, leading to one RIPK3-biomimetic peptide PR3 and three RIPK1-biomimetic peptides. Individual peptides undergo self-assembly into nanofibrils, whereas mixing RIPK1-biomimetic peptides with PR3 accelerates and enhances assembly of PR3. In particular, RIPK1-biomimetic peptide DRI-PR1 exhibits reliable binding affinity with protein RIPK3, resulting in specific cytotoxicity to colon cancer cells that overexpress RIPK3. Mechanistic studies reveal the increased phosphorylation of RIPK3 induced by RIPK1-biomimetic peptides, elucidating the activation of the necroptotic signaling pathway responsible for cell death without an obvious increase in secretion of inflammatory cytokines. Our findings highlight the potential of peptide-protein hybrid aggregation as a promising approach to address the "undruggable" issue and provide alternative strategies for overcoming cancer resistance in the future.

A newly identified enzyme from Japanese common squid Todarodes pacificus has the ability to biosynthesize d-aspartate

Amino acids exist in two chiral forms, namely L and D. Although l-amino acids are predominant in vivo, certain limited circumstances have reported the usage of d-amino acids. d-aspartate (Asp), among them, plays crucial physiological roles in living organisms and is biosynthesized from L-Asp by the enzyme named aspartate racemase (AspRase). D-Asp is known to accumulate in large amounts in the nervous system of cephalopods. To understand the function of D-Asp in nervous system in more detail, it is necessary to elucidate its metabolic pathway; however, AspRase gene has not been identified in cephalopods as in the case of mammals. In this study, we successfully identified a novel gene encoding AspRase from the optic ganglion of Japanese common squid Todarodes pacificus. Our discovery of the squid AspRase challenges the prevailing assumption that AspRases across different animals share similar structures. Surprisingly, the squid AspRase is a unique enzyme that differs significantly from known AspRases, being structurally and phylogenetically related to aspartate aminotransferase (AST) and possessing both AspRase and AST activities. The optimum pH and temperature for AspRase activity using L-Asp as a substrate are approximately 7.0 and 20 °C, respectively. Moreover, we have found that AspRase activity is enhanced in the presence of 2-oxoacids. These findings have far-reaching implications for the understanding of enzymology and suggest that yet-to-be-identified mammalian AspRases may also be phylogenetically related to AST, rather than conventional AspRases. Furthermore, our results provide valuable insights into the evolution of the D-Asp biosynthetic pathway.

Separation and Identification of Isoleucine Enantiomers and Diastereomers Using an Original Chiral Resolution Labeling Reagent.

D-Amino acids, which are present in small amounts in living organisms, are responsible for a variety of physiological functions. Some bioactive/biomolecular peptides also contain D-amino acids in their sequences; such peptides express different functions than peptides composed only of L-form amino acids. Among the 20 amino acids that make up proteins, threonine (Thr) and isoleucine (Ile) have two chiral carbons and thus have two enantiomers and diastereomers. These stereoisomers have been previously analyzed through HPLC using chiral columns or chiral resolution labeling reagents. However, the separation and identification of these stereoisomers are highly laborious and complicated. Herein, we propose an analytical method for the separation and identification of Ile stereoisomers through LC-MS using our original chiral resolution labeling reagent, 1-fluoro-2,4-dinitrophenyl-5-L-valine-N,N-dimethylethylenediamine-amide (L-FDVDA) and a PBr column packed with pentabromobenzyl-modified silica gel. Twenty DL-amino acids including Thr stereoisomers (41 amino acids including glycine) were separated and identified using C18 column. Ile stereoisomers could be separated using not a C18 column but a PBr column. Additionally, we showed that peptides containing Thr and Ile stereoisomers can be accurately detected through labeling with L-FDVDA.

D-tyrosine enhances disoctyl dimethyl ammonium chloride on alleviating SRB corrosion

Microbiologically influenced corrosion (MIC) caused by sulfate reducing bacteria (SRB) is a serious challenge in many industries, but biofilm greatly decreases the toxicity of bactericides to cell inside. d-amino acids are potential enhancers for bactericides due to their excellent performance on biofilm inhibition. However, the mechanism of d-amino acid cooperating with bactericides for MIC inhibition is still unknown. In this study, d-tyrosine（D-Tyr）and disoctyl dimethyl ammonium chloride (DDAC) were selected as the typical d-amino acid and bactericide, respectively, to evaluate their synergetic inhibition on the corrosion caused by Desulfovibrio vulgaris. D-Tyr obviously enhanced the role of DDAC in inhibiting corrosion with high corrosion inhibition efficiency at 77.23 %. The attachment of EPS and live cells on the coupon surface decreased in the presence of D-Try, leading to more cells directly exposed to DDAC. Besides, D-Try decreased the amount of live cells on the surface and thus reduced the utilization of Fe by SRB and corrosion current. Moreover, dead cells settling to the coupon surface may form a protective lay to retard the contact between live SRB and Fe, leading to slow cathode reaction and less corrosion. Therefore, D-Tyr can reduce the coverage of biofilm, thereby reducing its protective effect on SRB and achieving better corrosion inhibition effect. This work provides a new strategy for improving bactericides and inhibiting MIC.

Simulation of oriented NMR spectra: Combining molecular dynamics and chemical shift tensor calculations.

Solid state NMR is widely used to study the orientation and other structural features of proteins and peptides in lipid bilayers. Using data obtained by PISEMA (Polarization Inversion Spin Exchange at Magic Angle) experiments, periodic spectral patterns arise from well-aligned α-helical molecules. Significant problems in the interpretation of PISEMA spectra may arise for systems that do not form perfectly defined secondary structures, like α-helices, or the signal pattern is disturbed by molecular motion. Here, we present a new method that combines molecular dynamics simulation with tensorial orientational constraints (MDOC) and chemical shift tensor calculations for the simulation and interpretation of PISEMA-like spectra. The calculations include the spectra arising from non α-helical molecules and molecules with non-uniform intrinsic mobility. In a first step, dipolar or quadrupolar interaction tensors drive molecular rotations and reorientations to obtain the proper mean values as observed in corresponding NMR experiments. In a second step, the coordinate snapshots of the MDOC simulations are geometry optimized with the isotropic 15 N chemical shifts as constraints using Bond Polarization Theory (BPT) to provide reliable 15 N CS tensor data. The averaged dipolar 1 H-15 N couplings and the δzz tensor components can then be combined to simulate PISEMA patterns. We apply this method to the ß-helical peptide gramicidin A (gA) and demonstrate that this method enables the assignment of most PISEMA resonances. In addition, MDOC simulations provide local order parameters for the calculated sites. These local order parameters reveal large differences in backbone mobility between L- and D-amino acids of gA.

A raman spectroscopic investigation of conformation of flavin adenine dinucleotide, a coenzyme of d-amino acid oxidase

Based on silver nanoparticles application, surface-enhanced Raman scattering spectra of D-amino acid oxidase from pig kidney were recorded and analyzed. Spectral parameters characteristic of conformational changes in cofactor flavin adenine dinucleotide during activation of the enzyme by D-amino acids were revealed. It was found that enzyme substrate specificity determines the amount of time from the start of of the conformational changes of flavin adenine dinucleotide until they no longer occur: in the presence of D-alanine, registration of the said conformational changes takes up just a few seconds, while it takes 10 min in the presence of D-serine .

Topoisomeric Membrane-Active Peptides: A Review of the Last Two Decades.

In recent decades, bioactive peptides have been gaining recognition in various biomedical areas, such as intracellular drug delivery (cell-penetrating peptides, CPPs) or anti-infective action (antimicrobial peptides, AMPs), closely associated to their distinct mode of interaction with biological membranes. Exploiting the interaction of membrane-active peptides with diverse targets (healthy, tumoral, bacterial or parasitic cell membranes) is opening encouraging prospects for peptides in therapeutics. However, ordinary peptides formed by L-amino acids are easily decomposed by proteases in biological fluids. One way to sidestep this limitation is to use topoisomers, namely versions of the peptide made up of D-amino acids in either canonic (enantio) or inverted (retroenantio) sequence. Rearranging peptide sequences in this fashion provides a certain degree of native structure mimicry that, in appropriate contexts, may deliver desirable biological activity while avoiding protease degradation. In this review, we will focus on recent accounts of membrane-active topoisomeric peptides with therapeutic applications as CPP drug delivery vectors, or as antimicrobial and anticancer candidates. We will also discuss the most common modes of interaction of these peptides with their membrane targets.