Natural Amino Acids Research Articles

L-Proline Enhanced Whole Ovary Cryopreservation by Inhibiting Ice Crystal Growth and Reducing Oxidative Stress.

Cryopreservation and transplantation of ovaries are considered to be effective methods for preserving the fertility of female cancer patients. However, ice crystal and oxidative damage occur during the freeze-thaw cycle, significantly reducing the effectiveness of cryopreservation and limiting its clinical application. Thus, new technologies or agents must be explored to enhance ovarian cryopreservation. Recently, l-proline, a natural amino acid, has been proven to have good biocompatibility and can clear reactive oxygen species produced during cryopreservation. Whether l-proline can play a positive role in ovarian cryopreservation has not yet been explored. Here, the effect of l-proline on ovarian cryopreservation was investigated. The oxidative antioxidant system, mitochondrial function, and cell apoptosis and proliferation after thawing were systematically evaluated. Moreover, the ice crystal inhibition of l-proline was examined. Furthermore, the morphology and function of oocytes in ovaries, as well as the state of the ovaries after heterotopic renal capsule transplantation, were evaluated to validate the feasibility and reliability of this study. The above results confirm that l-proline can effectively inhibit ice crystal growth, reduce reactive oxygen species production, and enhance cryopreservation effects at the optimal concentration of 20 mM. Altogether, l-proline can significantly improve the cryopreservation effect of ovaries, which is expected to provide a new perspective for the cryopreservation of female fertility.

Strong enhancement on tetrabromobisphenol A removal in natural water matrix by adding cysteine into Cu(II)/peroxymonosulfate system over the wide pH range of 4–12: Efficiency and mechanism

Although Cu(II) can trigger peroxymonosulfate (PMS) oxidation of pollutants, it is only effective under alkaline environments and is susceptible to interference by aqueous matrix components. In this study, cysteine, a natural thiol and amino acid, was found to greatly improve the performance of tetrabromobisphenol A (TBBPA) elimination in the Cu(II)/PMS system across a broad pH range of 4.0–12.0. The cysteine/Cu(II)/PMS system achieved a 94.34 % elimination of TBBPA within 10 min at pH 7.0, which had a 7.55-fold increase in efficiency relative to the Cu(II)/PMS system, and superior to the previously-reported hydroxylamine and gallic acid-enhanced Cu(II)/PMS systems. Cu(III)-(cysteine)2, as opposed to radicals, was the main reactive species leading to the removal of TBBPA, contributing at least 91.53 %. Cysteine first coordinated with Cu(II) through sulfhydryl groups (−SH) and rapidly converted Cu(II)-(cysteine)2 to Cu(I)-(cysteine)2, then Cu(III)-(cysteine)2 was formed finally by the further oxidation of PMS on Cu(I)-(cysteine)2. This enhancement was further extended to other thiols including cysteamine, β-mercaptoethanol and mercaptosuccinic acid. Four potential degradation pathways of TBBPA were inferred from the determined nine degradation intermediates. A marked decline in the toxicity of the reaction solutions was observed after the elimination of TBBPA. Furthermore, the cysteine/Cu(II)/PMS system shown high anti-interference performance with common cations and anions (Ca(II), Mg(II), NO3–, SO42–, HCO3– and Cl–), humic acid and actual water samples. Overall, this study provided a promising approach to extend the working pH range of Cu(II)/PMS system and its anti-interference capability with actual water matrix, which was favorable for its potential utilization in water treatment.

Re-engineering of a carotenoid-binding protein based on NMR structure.

Recently, a number of message passing neural network (MPNN)-based methods have been introduced that, based on backbone atom coordinates, efficiently recover native amino acid sequences of proteins and predict modifications that result in better expressing, more soluble, and stable variants. However, usually, X-ray structures, or artificial structures generated by algorithms trained on X-ray structures, were employed to define target backbone conformations. Here, we show that commonly used algorithms ProteinMPNN and SolubleMPNN display low sequence recovery on structures determined using NMR. We subsequently propose a computational approach that we successfully apply to re-engineer AstaP, a protein that natively binds a large hydrophobic ligand astaxanthin (C40H52O4), and for which only a structure determined using NMR is currently available. The engineered variants, designated NeuroAstaP, are 51 amino acid shorter than the 22 kDa parent protein, have 38%-42% sequence identity to it, exhibit good yields, are expressed in a soluble, mostly monomeric form, and demonstrate efficient binding of carotenoids in vitro and in cells. Altogether, our work further tests the limits of using machine learning for protein engineering and paves the way for MPNN-based modification of proteins based on NMR-derived structures.

Site-specifically Phosphorylated Hsp90C-terminal Domain Variants Provide Access to Deciphering the Chaperone Code.

The ubiquitous molecular chaperone Heat shock protein 90 (Hsp90) is pivotal in many cellular processes through folding of client proteins under stressed and normal conditions. Despite intensive research on its function as a chaperone, the influence of posttranslational modifications on Hsp90 (the 'chaperone code'), and its interactions with co-chaperones and client proteins, still remains to be elucidated. The C-terminal domain (CTD) of Hsp90 is essential for formation of the active homodimer state of Hsp90 and contains recognition sites for co-chaperones and client proteins. Here we used expressed protein selenoester ligation to introduce site-selective phosphorylations in the Hsp90 CTD, while preserving the native amino acid sequence. The two phosphorylations do not affect the overall secondary structure, but in combination, slightly decrease the thermal stability of the CTD. The Hsp90 CTD functions as a chaperone in decreasing aggregation of model client proteins, but the C-terminal phosphorylations do not significantly alter the anti-aggregation activity for these clients. The optimization of expressed protein selenoester ligation (EPSL) to carry out several steps in one pot provides an efficient route to access site-specifically modified Hsp90 CTD variants, allowing the generation of Hsp90 variants with site-specific PTMs to decipher the chaperone code.

Natural Chiral Ligand Strategy: Metal-Catalyzed Reactions with Ligands Prepared from Amino Acids and Peptides

Amino acids and peptides are readily available biomolecules and can function as chiral ligands for transition metal catalysis. An example is the copper complex catalyzed 1,4-addition of dialkylzinc to acyclic enones, which employs peptide ligands. This review provides a dataset of amino acids and peptides reported in the literature proving to be effective ligands for metal-centered catalysts. Several parameters were highlighted, including amino acid combination, metal atoms, carboxyl and amino protecting groups, modification of natural amino acids, and the mechanism of catalysis. Along with analyzing physical-chemical properties, the SMILES representation for each amino acid and/or peptide was generated and made available online, providing an easy-to-use means of training machine learning models. This review offers an opportunity for the development of more efficient peptide ligands for enantioselective metal-centered catalysts. The available online dataset is a reliable manually curated table, it enables the benchmark for comparison of new terminal functional groups. Moreover, the review provides insight into the structures of the more successful peptide ligands and can be used as the foundation for the development of the next generation of peptide-based chiral ligands.

Cationic and ionizable amphiphiles based on di-hexadecyl ester of <I>L</I>-glutamic acid for liposomal transport of RNA

Various RNAs are among the most promising and actively developed therapeutic agents for the treatment of tumors, infectious diseases and a number of other pathologies associated with the dysfunction of specific genes. Some nanocarriers are used for the effective delivery of RNAs to target cells, including liposomes based on cationic and/or ionizable amphiphiles. Cationic amphiphiles contain a protonated amino group and exist as salts in an aqueous environment. Ionizable amphiphiles are a new generation of cationic lipids that exhibit reduced toxicity and immunogenicity and undergo ionization only in the acidic environment of the cell. In this work we developed a scheme for the preparation and carried out the synthesis of new cationic and ionizable amphiphiles based on natural amino acids (L-glutamic acid, glycine, beta-alanine, and gamma-aminobutyric acid). Cationic and ionizable liposomes were formed based on the obtained compounds, mixed with natural lipids (phosphatidylcholine and cholesterol), and their physicochemical characteristics (particle size, zeta potential, and storage stability) were determined. Average diameter of particles stable for 5–7 days did not exceed 100 nm. Zeta potential of cationic and ionizable liposomes was about 30 and 1 mV, respectively. The liposomal particles were used to form complexes with RNA molecules. Such RNA complexes were characterized by atomic force microscopy and their applicability for nucleic acid transport was determined.

Natural alleles of LEAFY and WAPO1 interact to regulate spikelet number per spike in wheat

Key messageSpecific combinations ofLFY andWAPO1 natural alleles maximize spikelet number per spike in wheat. Spikelet number per spike (SNS) is an important yield component in wheat that determines the maximum number of grains that can be formed in a wheat spike. In wheat, loss-of-function mutations in LEAFY (LFY) or its interacting protein WHEAT ORTHOLOG OF APO1 (WAPO1) significantly reduce SNS by reducing the rate of formation of spikelet meristems. In previous studies, we identified a natural amino acid change in WAPO1 (C47F) that significantly increases SNS in hexaploid wheat. In this study, we searched for natural variants in LFY that were associated with differences in SNS and detected significant effects in the LFY-B region in a nested association mapping population. We generated a large mapping population and confirmed that the LFY-B polymorphism R80S is linked with the differences in SNS, suggesting that LFY-B is the likely causal gene. A haplotype analysis revealed two amino acid changes P34L and R80S, which were both enriched during wheat domestication and breeding suggesting positive selection. We also explored the interactions between the LFY and WAPO1 natural variants for SNS using biparental populations and identified significant interaction, in which the positive effect of the 80S and 34L alleles from LFY-B was only detected in the WAPO-A1 47F background but not in the 47C background. Based on these results, we propose that the allele combination WAPO-A1-47F/LFY-B 34L 80S can be used in wheat breeding programs to maximize SNS and increase grain yield potential in wheat.

Surface immobilization and properties optimization of phage hydrolase against Gram-negative bacteria

Immobilized hydrolase not only reduces the production of antibiotic-resistant bacteria, but also effectively improves the stability of hydrolase in external use. In this study, phage hydrolase LysSSE1 against Gram-negative bacteria were surface immobilized and optimized for their bactericidal activity. Different anti-pathogen surface materials were prepared, where LysSSE1 was immobilized on the glass surface with a silica-affinity peptide and into which different peptide linkers were introduced. Immobilized enzymes inserted into the natural amino acid peptide linker exhibited higher bactericidal activity, greater stability, and more consistent bactericidal performance compared to those without the peptide linker. Among these immobilized enzymes, LysSSE1-NL-SiAP1 exhibited the strongest bactericidal activity and the best repeatable bactericidal performance, which only reduced the original performance by about 5% after three bactericidal cycles. Modeling analysis suggested that the presence of peptide linker might increase the molecular flexibility of the proximal hydrolase domain to better interact with the bacterial substrate. Our surface immobilization strategy could be extended to other lytic proteins, providing support for the development of surface sterilization methods and materials.

Palladium‐Catalyzed Directing‐Group‐Mediated γ‐C(sp3)−H Glycosylation for Synthesis of C‐Alkyl Glycosides

AbstractC‐alkyl glycosides play a crucial role in various bioactive compounds. However, the synthesis of C‐alkyl glycosides poses significant challenges, particularly through C(sp3)−H glycosylation. Here, we report a set of reactions for constructing C‐alkyl glycosides through directing‐group‐mediated functionalization of unactivated γ‐C(sp3)−H bonds under mild conditions. These reactions not only achieve high regioselectivity and stereoselectivity in glycosylation, but also exhibit a wide substrate scope. They are compatible with both arene and alkane substrates, as well as natural and unnatural amino acid substrates. Mechanistic studies have shown that the directing‐group 8‐aminoquinoline (AQ) and picolinamide (PA) may affect the chirality of the β‐carbon of L‐valine through a sterically favorable trans‐palladacycle intermediate, resulting in (R) or (S) configuration of glycosylated amino acid, respectively. These reactions are promising to provide a convenient and powerful tool for constructing C‐alkyl glycosides and carbohydrate‐based drugs in the future.

Long Stability of Atomically Precise Red Emissive Copper Nanoclusters within the Gel and Their Use As a Potential Catalyst and Fluorescent Ink.

Herein, an amphiphile-based hydrogel (with 5% DMF) containing natural amino acid residue has been used to prepare and stabilize red-emitting CuNCs for several months. Though different methods have been attempted, amphiphile and 4-mercaptobenzoic acid (4-MBA)-containing hydrogels are pinpointed to be the base medium to stabilize this new Cu-cluster. From a MALDI-TOF MS analysis it was found that it is a Cu8-atom cluster stabilized by three 4-MBA ligands. Copper acetate monohydrate (Cu(CH3COO)2·H2O) has been used as a copper precursor, and l-ascorbic acid has been used as a reducing agent. FEG-TEM analysis shows that the Cu cluster has an average size of 2.83 nm. Interestingly, these clusters can be used as a fluorescent ink with a visibility of the solid state under a UV-lamp with an excitation of 365 nm. This envisaged applying these CuNCs for anticounterfeiting. These Cu-clusters show an excitation of 420 nm with an emission of 620 nm, as is evident from the fluorescence spectroscopic analysis. Based on our knowledge, this is the first example of making and consequently stabilizing Cu-clusters using hydrogel as a template for a few months. Moreover, these CuNCs can also be used as a catalyst for the reduction of nitro derivatives to their amine derivatives in aqueous medium.

Modular Design and Self-assembly of pH-Responsive Multidomain Peptides Incorporating Non-natural Ionic Amino Acids.

Stimuli-responsive peptides, particularly pH-responsive variants, hold significant promise in biomedical and technological applications by leveraging the broad pH spectrum inherent to biological environments. However, the limited number of natural pH-responsive amino acids within biologically relevant pH ranges presents challenges for designing rational pH-responsive peptide assemblies. In our study, we introduce a novel approach by incorporating a library of non-natural amino acids featuring chemically diverse tertiary amine side chains. Hydrophobic and ionic properties of these non-natural amino acids facilitate their incorporation into the assembly domain when uncharged, and electrostatic repulsion promotes disassembly under lower pH conditions. Furthermore, we observed a direct relationship between the number of substitutions and the hydrophobicity of these amino acids, influencing their pH-responsive properties and enabling rational design based on desired transitional pH ranges. The structure-activity relationship of these pH-responsive peptides was evaluated by assessing their antimicrobial properties, as their antimicrobial activity is triggered by the disassembly of peptides to release active monomers. This approach not only enhances the specificity and controllability of pH responsiveness but also broadens the scope of peptide materials in biomedical and technological applications.

Exploiting the chemical diversity space of phosphopeptide binding to nasopharyngeal carcinoma PLK1 PBD domain with unnatural amino acid building blocks by using QSAR-based genetic optimization

ABSTRACT Human polo-like kinase 1 (PLK1) has been recognized as an attractive therapeutic target against nasopharyngeal carcinoma (NPC). The kinase contains a conserved polo-box domain (PBD) that exhibits a wide specificity across various substrates. Previously, we explored natural amino acid preference in PLK1 PBD-binding phosphopeptides. However, limited to the short sequence only natural amino acids cannot guarantee the sufficient exploitation of chemical and structural diversity of the phosphopeptides. Here, we described a genetic optimization (GO) strategy to systematically optimize a 104-sized 6-mer phosphopeptide array towards increasing affinity to PLK1 PBD domain by using 20 natural plus 34 unnatural amino acids as basic building blocks. A QSAR predictor was created to guide the GO optimization and then evaluated rigorously at molecular and cellular levels. Three unnatural phosphopeptides uPP8, uPP15 and uPP20 were designed as potent binders with K d = 0.18, 0.42 and 0.08 μM, respectively, in which the uPP20 also possessed a good anti-tumor activity against human NPC cells when fused with cell permeation sequence. In addition, we defined a relaxed 6-mer motif for the preferential PLK1 PBD-binding phosphosites, namely [Φ/П]-3-[ζ]-2-[ζ]-1-[pT/pS]0-[Φ/П]+1-[Φ]+2, where the symbols Φ, ζ and П represent hydrophobic, polar and aromatic amino acid types, respectively.

De Novo Biosynthesis of L-Azetidine-2-Carboxylic Acid in Escherichia coli Strains for Powdery Mildew Treatment.

L-Azetidine-2-carboxylic acid (L-Aze), a natural nonproteinogenic amino acid found widely in plants, has recently been identified as an environmentally friendly agent for controlling powdery mildew with low toxicity. In this study, a biological route for L-Aze production via the methionine salvage pathway (Yang Cycle) was first in silico designed for Escherichia coli. Subsequently, systematic engineering strategies were employed to enhance the production efficiency, including the enhancement of the 5-phosphoribosyl 1-pyrophosphate (PRPP) supply, construction of the ATP-adenine cycle, and engineering of the strain's resistance to L-Aze. The final strain produced L-Aze from glucose with a titer of 568.5 mg/L. The antifungal activity of the produced L-Aze in the fermentation broth was also confirmed for treating powdery mildew in cucurbits. This approach not only provides a sustainable and green route for pesticide production to control powdery mildew but also expands our understanding of the exogenous construction of the Yang Cycle in E. coli.

β-Selective 2-Deoxy- and 2,6-Dideoxyglucosylations Catalyzed by Bis-Thioureas.

We present methods for β-selective 2-deoxy- and 2,6-dideoxyglucosylations of natural products, carbohydrates, and amino acids using bis-thiourea hydrogen-bond-donor catalysts. Disarming ester protecting groups were necessary to counter the high reactivity of 2-deoxyglycosyl electrophiles toward non-stereospecific SN1 pathways. Alcohol and phenol nucleophiles with both base- and acid-sensitive functionalities were compatible with the catalytic protocol, enabling access to a wide array of 2-deoxy-β-O-glucosides.

Identification of a novel intronic variant in COL4A2 gene associated with fetal severe cerebral encephalomalacia and subdural hemorrhage

BackgroundGenetic variants in COL4A2 are less common than those of COL4A1 and their fetal clinical phenotype has not been well described to date. We present a fetus from China with an intronic variant in COL4A2 associated with a prenatal diagnosis of severe cerebral encephalomalacia and subdural hemorrhage.MethodsWhole exome sequencing (WES) was applied to screen potential genetic causes. Bioinformatic analysis was performed to predict the pathogenicity of the variant. In in vitro experiment, the minigene assays were performed to assess the variant’s effect.ResultsIn this proband, we observed ventriculomegaly, subdural hemorrhage, and extensive encephalomalacia that initially suggested cerebral hypoxic-ischemic and/or hemorrhagic lesions. WES identified a de novo heterozygous variant c.549 + 5G > A in COL4A2 gene. This novel variant leads to the skipping of exon 8, which induces the loss of 24 native amino acids, resulting in a shortened COL4A2 protein (p.Pro161_Gly184del).ConclusionOur study demonstrated that c.549 + 5G > A in COL4A2 gene is a disease-causing variant by aberrant splicing. This finding enriches the variant spectrum of COL4A2 gene, which not only improves the understanding of the fetal neurological disorders associated with hypoxic-ischemic and hemorrhagic lesions from a clinical perspective but also provides guidance on genetic diagnosis and counseling.

Directed evolution of aminoacyl-tRNA synthetases through in vivo hypermutation.

Genetic code expansion (GCE) has become a critical tool in biology by enabling the site-specific incorporation of non-canonical amino acids (ncAAs) into proteins. Central to GCE is the development of orthogonal aminoacyl-tRNA synthetase (aaRS)/tRNA pairs wherein engineered aaRSs recognize chosen ncAAs and charge them onto tRNAs that decode blank codons ( e.g ., the amber stop codon). Many orthogonal aaRS/tRNA pairs covering a wide range of ncAAs have been generated by directed evolution, yet the evolution of new aaRS/tRNA pairs by standard strategies remains a labor-intensive process that often produces aaRS/tRNA pairs with suboptimal ncAA incorporation efficiencies. In this study, we present a strategy for evolving aaRSs that leverages OrthoRep to drive their continuous hypermutation in yeast. We demonstrate our strategy in 8 independent aaRS evolution campaigns starting from 4 different aaRS/tRNA parents targeting 7 distinct ncAAs. We observed the rapid evolution of multiple novel aaRSs capable of incorporating an overall range of 13 ncAAs tested into proteins in response to the amber codon. Some evolved systems reached efficiencies for amber codon-specified ncAA-dependent translation comparable to translation with natural amino acids specified by sense codons in yeast. Additionally, we discovered a surprising aaRS that evolved to self-regulate its own expression for greater dependency on ncAAs for translation. These findings demonstrate the potential of OrthoRep-driven aaRS evolution platforms in supporting the continued growth of GCE technologies.

Recent advances in pyridoxal phosphate-dependent enzymes and their applications

Pyridoxal phosphate (PLP), the active form of vitamin B6, is an important coenzyme in various enzyme-catalyzed reactions. PLP-dependent enzymes can catalyze a variety of chemical reactions, such as racemization, decarboxylation, β-addition, β-elimination, retro-aldol cleavage, transamination, and α-elimination. They are biologically synthesized a powerful tool for a variety of natural amino acids, non-natural amino acids and their related compounds. This article details the structural features and catalytic mechanisms of typical PLP-dependent enzymes such as ω-transaminase, lysine decarboxylase, threonine aldolase, and L-tyrosine phenol-lyase, and reviews the research progress in molecular modification and industrial applications of these enzymes. Finally, this article provides an outlook on the future development of PLP-dependent enzymes, including in vivo regeneration system and industrial applications of PLP cofactors, and discusses the tremendous potential of these enzymes in biocatalytic applications.

Therapeutic peptides identification via kernel risk sensitive loss-based k-nearest neighbor model and multi-Laplacian regularization.

Therapeutic peptides are therapeutic agents synthesized from natural amino acids, which can be used as carriers for precisely transporting drugs and can activate the immune system for preventing and treating various diseases. However, screening therapeutic peptides using biochemical assays is expensive, time-consuming, and limited by experimental conditions and biological samples, and there may be ethical considerations in the clinical stage. In contrast, screening therapeutic peptides using machine learning and computational methods is efficient, automated, and can accurately predict potential therapeutic peptides. In this study, a k-nearest neighbor model based on multi-Laplacian and kernel risk sensitive loss was proposed, which introduces a kernel risk loss function derived from the K-local hyperplane distance nearest neighbor model as well as combining the Laplacian regularization method to predict therapeutic peptides. The findings indicated that the suggested approach achieved satisfactory results and could effectively predict therapeutic peptide sequences.

Alkylation of Glycine Derivatives through a Synergistic Single-Electron Transfer and Halogen-Atom Transfer Process.

Here, we present a versatile method for forming C(sp3)-C(sp3) bonds, enabling the synthesis of a range of natural and non-natural amino acids. This approach utilizes readily available glycine derivatives and alkyl iodides, combining single-electron transfer and halogen-atom transfer processes. The utility of this step-economic and redox-economic C(sp3)-C(sp3) bond formation is further highlighted in the late-stage site-selective modifications of the glycine residue in short peptides.

A Versatile Method for Site-Specific Chemical Installation of Aromatic Posttranslational Modification Analogs into Proteins.

Posttranslational modifications (PTMs) of proteins play central roles in regulating the protein structure, interactome, and functions. A notable modification site is the aromatic side chain of Tyr, which undergoes modifications such as phosphorylation and nitration. Despite the biological and physiological importance of Tyr-PTMs, our current understanding of the mechanisms by which these modifications contribute to human health and disease remains incomplete. This knowledge gap arises from the absence of natural amino acids that can mimic these PTMs and the lack of synthetic tools for the site-specific introduction of aromatic PTMs into proteins. Herein, we describe a facile method for the site-specific chemical installation of aromatic PTMs into proteins through palladium-mediated S-C(sp2) bond formation under ambient conditions. We demonstrate the incorporation of novel PTMs such as Tyr-nitration and phosphorylation analogs to synthetic and recombinantly expressed Cys-containing peptides and proteins within minutes and in good yields. To demonstrate the versatility of our approach, we employed it to prepare 10 site-specifically modified proteins, including nitrated and phosphorylated analogs of Myc and Max proteins. Furthermore, we prepared a focused library of site-specifically nitrated and phosphorylated α-synuclein (α-Syn) protein, which enabled, for the first time, deciphering the role of these competing modifications in regulating α-Syn conformation aggregation in vitro. Our strategy offers advantages over synthetic or semisynthetic approaches, as it enables rapid and selective transfer of rarely explored aromatic PTMs into recombinant proteins, thus facilitating the generation of novel libraries of homogeneous posttranslationally modified proteins for biomarker discovery, mechanistic studies, and drug discovery.