Proteinogenic Research Articles

Molecular characterization of toll-like receptor 2 gene in Bikaneri camels (Camelus dromedarius)

Toll-like receptors (TLRs) are an important family of receptors constituting the first line of defense system. Tolllike receptors play crucial roles in the innate immune system by recognizing pathogen-associated molecular patterns derived from various microbes. Present investigation was carried out to characterize the coding sequence of TLR2 gene in Indian dromedary camels. Blood was collected from five Bikaneri camels and genomic DNA was isolated. Eight pairs of overlapping primers were designed for amplification of TLR2 gene coding region. All the fragments were successfully amplified using Polymerase chain reaction (PCR). The PCR products were purified and subjected to Sanger’s sequencing. TLR2 gene fragment of 3213 bp consisting of 2287 bp of coding sequence of exon 2 and 926 bp of 3’untranslated region (UTR) was successfully amplified. The sequence generated was submitted to the NCBI with accession number MW557315.1. The encoded amino acids codes for 12 leucine rich repeats (LRR) (Accession no. UBN09134.1). At the nucleotide level, the Bikaneri camels’ TLR2 gene showed highest similarity with camelids (99.30 to 99.59%) followed by vicugna pacos (97.54%). The nucleotide sequence similarity was lowest with Homo sapiens (82.37%). The amino acid sequence based phylogenetic tree revealed close relationship between camelid species followed by pig (86.2%), whereas human, gorilla, horse and donkey were distantly placed on the tree.

A transient increase of HIF-1α during the G1 phase (G1-HIF) ensures cell survival under nutritional stress

The family of hypoxia-inducible transcription factors (HIF) is activated to adapt cells to low oxygen conditions, but is also known to regulate some biological processes under normoxic conditions. Here we show that HIF-1α protein levels transiently increase during the G1 phase of the cell cycle (designated as G1-HIF) in an AMP-activated protein kinase (AMPK)-dependent manner. The transient elimination of G1-HIF by a degron system revealed its contribution to cell survival under unfavorable metabolic conditions. Indeed, G1-HIF plays a key role in the cell cycle-dependent expression of genes encoding metabolic regulators and the maintenance of mTOR activity under conditions of nutrient deprivation. Accordingly, transient elimination of G1-HIF led to a significant reduction in the concentration of key proteinogenic amino acids and carbohydrates. These data indicate that G1-HIF acts as a cell cycle-dependent surveillance factor that prevents the onset of starvation-induced apoptosis.

Hyperglucagonaemia in diabetes: altered amino acid metabolism triggers mTORC1 activation, which drives glucagon production.

Hyperglycaemia is associated with alpha cell dysfunction, leading to dysregulated glucagon secretion in type 1 and type 2 diabetes; however, the mechanisms involved are still elusive. The nutrient sensor mammalian target of rapamycin complex 1 (mTORC1) plays a major role in the maintenance of alpha cell mass and function. We studied the regulation of alpha cell mTORC1 by nutrients and its role in the development of hyperglucagonaemia in diabetes. Alpha cell mTORC1 activity was assessed by immunostaining for phosphorylation of its downstream target, the ribosomal protein S6, and glucagon, followed by confocal microscopy on pancreatic sections and flow cytometry on dispersed human and mouse islets and the alpha cell line, αTC1-6. Metabolomics and metabolic flux were studied by 13C glucose labelling in 2.8 or 16.7 mmol/l glucose followed by LC-MS analysis. To study the role of mTORC1 in mediating hyperglucagonaemia in diabetes, we generated an inducible alpha cell-specific Rptor knockout in the Akita mouse model of diabetes and tested the effects on glucose tolerance by IPGTT and on glucagon secretion. mTORC1 activity was increased in alpha cells from diabetic Akita mice in parallel to the development of hyperglycaemia and hyperglucagonaemia (two- to eightfold increase). Acute exposure of mouse and human islets to amino acids stimulated alpha cell mTORC1 (3.5-fold increase), whereas high glucose concentrations inhibited mTORC1 (1.4-fold decrease). The mTORC1 response to glucose was abolished in human and mouse diabetic alpha cells following prolonged islet exposure to high glucose levels, resulting in sustained activation of mTORC1, along with increased glucagon secretion. Metabolomics and metabolic flux analysis showed that exposure to high glucose levels enhanced glycolysis, glucose oxidation and the synthesis of glucose-derived amino acids. In addition, chronic exposure to high glucose levels increased the expression of Slc7a2 and Slc38a4, which encode amino acid transporters, as well as the levels of branched-chain amino acids and methionine cycle metabolites (~1.3-fold increase for both). Finally, conditional Rptor knockout in alpha cells from adult diabetic mice inhibited mTORC1, thereby inhibiting glucagon secretion (~sixfold decrease) and improving diabetes, despite persistent insulin deficiency. Alpha cell exposure to hyperglycaemia enhances amino acid synthesis and transport, resulting in sustained activation of mTORC1, thereby increasing glucagon secretion. mTORC1 therefore plays a major role in mediating alpha cell dysfunction in diabetes. All sequencing data are available from the Gene Expression Omnibus (GEO) repository (accession no. GSE154126; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE154126 ).

Adsorption of proteinogenic amino acid on Al‐ and C‐doped boron nitride nanotube (BNNT)

AbstractEfficient determination of proteinogenic amino acid is of great relevance, and evaluating the intrinsic interaction of this amino acid of interest with nanotubes play a crucial role in adsorption and sensing mechanism, and in determining proteinogenic amino acid measurement index. As such, the study apparently sought to examine the adsorption of the proteinogenic amino acid (proline) on the bare surfaces of carbon boron nitride, boron nitride, and boron nitride doped with the aluminum element nanotubes. In accordant with the geometric optimization, different dispersion models: PBE0, ωB97XD, M06‐2X, and B3LYP‐D3(GD3BJ) were used; note that the latter is used only in wave function analysis studies. Studies show that the strongest adsorption belongs to boron nitride nanotubes doped with aluminum (BNAldopNT), with similar and more negative adsorption energy of ‐0.719, ‐1.393, ‐1.001, and ‐1.098eV respectively for the various benchmarked functionals utilized in the study. Then the highest energy and adsorption strength was obtained for the boron nitride nanotube (BNNT), and at the end is the carbon boron nitride nanotube (i.e. BNAldopNT > BNNT > BC2NNT). As such, in tandem with the energetic analysis carried out suggests that BNAldopNT might be a potent and preferred candidate for the adsorption of the proteinogenic amino acid (proline molecule)among other studied nanotubes, with respect to their respective adsorption energy values

Biostimulants-Based Amino Acids Augment Physio-Biochemical Responses and Promote Salinity Tolerance of Lettuce Plants (Lactuca sativa L.)

Studying the biostimulation effect of amino acids indicated their possible role in salt stress mitigation. In this investigation, six exogenous amino acids (alanine (Ala), arginine (Arg), glutamine (Glu), glycine (Gly), methionine (Met), and proline (Pro)) at 0.5 g/L were sprayed to evaluate their impact on lettuce plants cultivated under simulated salt stress conditions. Photosynthetic pigments, ion absorption, endogenous amino acids contents, catalase (CAT), and peroxidase (POD) enzyme activities were determined. A significant alleviation of salt stress was noticed when EAAs were used in the stress-induced plants, and applying Gly, Met, and Pro improved the plant status under salt stress conditions. The highest electric conductivity (568 μS/g) was testified from the control treatment (50 mM NaCl), while applying exogenous amino acids reduced electrical conductivity (EC), and the result was located between 469 and 558 μS/g. AAs alleviated Cl- anions in the lettuce leaves by 25% in comparison to control plants. Na+ cations were alleviated when the stress-induced plants were sprayed with amino acids. In contrast, applying amino acids promoted K+ uptake, and Arg presented the highest contents (3226 μg/g). AAs promoted chlorophyll (chl a and chl b) concentrations compared to the control treatment, and Met produced the maximum chl a content, while the carotene (car) contents significantly augmented when Gly, Met, and Pro were applied. AAs were highly generated in non-stressed treatment (Std) compared to the control. Under simulated salinity stress, Met and Pro application enhanced proteinogenic amino acids expression. Compared to Ctl treatment, peroxidase enzyme activities significantly diminished in the other treatments, which fell by over 40% when Gly, Met, and Pro were sprayed.

Prolinyl Nucleotides Drive Enzyme‐Free Genetic Copying of RNA

AbstractProline is one of the proteinogenic amino acids. It is found in all kingdoms of life. It also has remarkable activity as an organocatalyst and is of structural importance in many folded polypeptides. Here, we show that prolinyl nucleotides with a phosphoramidate linkage are active building blocks in enzyme‐ and ribozyme‐free copying of RNA in the presence of monosubstituted imidazoles as organocatalysts. Both dinucleotides and mononucleotides are incorporated at the terminus of RNA primers in aqueous buffer, as instructed by the template sequence, in up to eight consecutive extension steps. Our results show that condensation products of amino acids and ribonucleotides can act like nucleoside triphosphates in media devoid of enzymes or ribozymes. Prolinyl nucleotides are metastable building blocks, readily activated by catalysts, helping to explain why the combination of α‐amino acids and nucleic acids was selected in molecular evolution.

Prolinyl Nucleotides Drive Enzyme-Free Genetic Copying of RNA.

Proline is one of the proteinogenic amino acids. It is found in all kingdoms of life. It also has remarkable activity as an organocatalyst and is of structural importance in many folded polypeptides. Here, we show that prolinyl nucleotides with a phosphoramidate linkage are active building blocks in enzyme- and ribozyme-free copying of RNA in the presence of monosubstituted imidazoles as organocatalysts. Both dinucleotides and mononucleotides are incorporated at the terminus of RNA primers in aqueous buffer, as instructed by the template sequence, in up to eight consecutive extension steps. Our results show that condensation products of amino acids and ribonucleotides can act like nucleoside triphosphates in media devoid of enzymes or ribozymes. Prolinyl nucleotides are metastable building blocks, readily activated by catalysts, helping to explain why the combination of α-amino acids and nucleic acids was selected in molecular evolution.

Catalytic N‐H Insertion Reactions with α‐Diazoacetates: An Efficient Method for Enantioselective Amino Acid Synthesis

AbstractAs the monomers of proteins, amino acids are critical in chemistry and biology, playing important roles in multiple fields such as synthetic chemistry, medicinal chemistry, and materials chemistry. Unlike proteinogenic amino acids, unnatural amino acids can be difficult to obtain from living organisms and generally are synthesized chemically. Recently, a method involving the asymmetric N−H bond carbene insertion reaction with α‐diazoacetates as the carbene precursor has shown high enantioselectivity and good functional group tolerance, allowing for the facile synthesis of natural and unnatural amino acids and the introduction of new concepts in catalysis.

An R2R3-MYB Transcription Factor RoMYB10 Regulates Anthocyanin Biosynthesis in Black Raspberry

At present, there are many excellent cultivars of raspberry worldwide with different fruit colors, such as yellow, red, purple and black. Anthocyanin accumulation is responsible for flower and fruit coloration in plants. Anthocyanins belong to flavonoids which are natural pigments widely distributed in plants. The MYB transcription factor (TF) plays a vital regulatory role in the metabolic pathway of anthocyanins. Here, RoMYB10, an R2R3-MYB in black raspberry (Rubus occidentalis L.), was isolated. The basic physical and chemical properties, conserved motif distribution, evolutionary relationship and subcellular localization were analyzed for the RoMYB10 encoded amino acid sequence. The contents of anthocyanin were determined in four different colored raspberry fruits by high-performance liquid chromatography–tandem mass spectrometry (HPLC-MS). The expression levels of MYB10 genes in various tissues and fruits at different developmental stages of the four different colored raspberries were detected by real-time fluorescence quantitative PCR (qRT-PCR). The results showed that the contents of anthocyanin and the expression levels of the MYB10 gene were the highest in black raspberry. Moreover, the expression of MYB10 in the fruit was significantly higher than in other tissues, especially in ripe and mottled fruits. When ectopic overexpression of RoMYB10 occurred in tobacco, the expression levels of the RoMYB10 gene and total anthocyanin contents in transgenic tobacco plants were significantly higher than those in wild-type (WT) plants. In addition, overexpression of RoMYB10 up-regulated the key genes in the anthocyanin biosynthesis, such as NtCHS, NtCHI, NtF3H, NtANS and NtUFGT. Our study suggests that RoMYB10 is involved in the regulating of anthocyanin biosynthesis in raspberry and has potential as a molecular tool for manipulating biosynthesis of anthocyanins in fruits using metabolic engineering.

Research Note: Evaluation of standardized ileal amino acid digestibility in feed ingredients for Pekin ducks

The digestibility of crude protein (CP) and amino acids (AA) in feedstuffs including corn, soybean meal (SBM), and corn distillers dried grains with solubles (DDGS) was investigated in White Pekin ducks. The test ingredients were the sole source of AA and CP. A nitrogen-free diet was also formulated for determining endogenous losses of AA and nitrogen. Birds were fed a standard starter diet for the first 15 d posthatch. On d 16, 96 ducklings (860 ± 50 g BW) were selected and allocated into 1 of 4 dietary treatments containing the test ingredients with 6 replicates per treatment in a randomized complete block design. All the crumbled assay diets were offered ad-libitum for 5 d and contained chromic oxide as an indigestible marker at 0.05%. On d 21, ducks were euthanized to collect the ileal digesta for digestibility analysis. Basal endogenous losses were abundant in glutamine, aspartic acid, leucine, proline, and serine, consecutively. For apparent ileal digestibility (AID), lysine and methionine were the highest (P < 0.05) in SBM, followed by corn and DDGS. For threonine, the highest values (P < 0.05) were similarly noted for SBM, followed by DDGS and corn; the values were 80.44, 69.88, and 64.89%, consecutively. Considering standardized ileal digestibility (SID), higher values (P < 0.05) for SBM were similarly noted for all the amino acids including lysine, methionine, and threonine; the values were 89.40, 93.58, and 86.50%, respectively. Conclusively, improved AA and CP digestibility was noted with SBM. Dietary protein source affected the extent of digestibility at the distal ileum; and the utilization of digestible amino acid coefficients during ducks' feed formulation is emphasized.

Evaluation of Linkers' Influence on Peptide-Based Piezoelectric Biosensors' Sensitivity to Aldehydes in the Gas Phase.

Recent findings qualified aldehydes as potential biomarkers for disease diagnosis. One of the possibilities is to use electrochemical biosensors in point-of-care (PoC), but these need further development to overcome some limitations. Currently, the primary goal is to enhance their metrological parameters in terms of sensitivity and selectivity. Previous findings indicate that peptide OBPP4 (KLLFDSLTDLKKKMSEC-NH2) is a promising candidate for further development of aldehyde-sensitive biosensors. To increase the affinity of a receptor layer to long-chain aldehydes, a structure stabilization of the peptide active site via the incorporation of different linkers was studied. Indeed, the incorporation of linkers improved sensitivity to and binding of aldehydes in comparison to that of the original peptide-based biosensor. The tendency to adopt disordered structures was diminished owing to the implementation of suitable linkers. Therefore, to improve the metrological characteristics of peptide-based piezoelectric biosensors, linkers were added at the C-terminus of OBPP4 peptide (KLLFDSLTDLKKKMSE-linker-C-NH2). Those linkers consist of proteinogenic amino acids from group one: glycine, L-proline, L-serine, and non proteinogenic amino acids from group two: β-alanine, 4-aminobutyric acid, and 6-aminohexanoic acid. Linkers were evaluated with in silico studies, followed by experimental verification. All studied linkers enhanced the detection of aldehydes in the gas phase. The highest difference in frequency (60 Hz, nonanal) was observed between original peptide-based biosensors and ones based on peptides modified with the GSGSGS linker. It allowed evaluation of the limit of detection for nonanal at the level of 2 ppm, which is nine times lower than that of the original peptide. The highest sensitivity values were also obtained for the GSGSGS linker: 0.3312, 0.4281, and 0.4676 Hz/ppm for pentanal, octanal, and nonanal, respectively. An order of magnitude increase in sensitivity was observed for the six linkers used. Generally, the linker's rigidity and the number of amino acid residues are much more essential for biosensors' metrological characteristics than the amino acid sequence itself. It was found that the longer the linkers, the better the effect on docking efficiency.

Dietary Manipulation of Amino Acids for Cancer Therapy.

Cancer cells cannot proliferate and survive unless they obtain sufficient levels of the 20 proteinogenic amino acids (AAs). Unlike normal cells, cancer cells have genetic and metabolic alterations that may limit their capacity to obtain adequate levels of the 20 AAs in challenging metabolic environments. However, since normal diets provide all AAs at relatively constant levels and ratios, these potentially lethal genetic and metabolic defects are eventually harmless to cancer cells. If we temporarily replace the normal diet of cancer patients with artificial diets in which the levels of specific AAs are manipulated, cancer cells may be unable to proliferate and survive. This article reviews in vivo studies that have evaluated the antitumor activity of diets restricted in or supplemented with the 20 proteinogenic AAs, individually and in combination. It also reviews our recent studies that show that manipulating the levels of several AAs simultaneously can lead to marked survival improvements in mice with metastatic cancers.

Design of Novel Mixed-Mode Columns for Analysis of Amino Acids Derivatized with 4-Fluoro-7-nitro-2,1,3-benzoxadiazole and the Evaluation of Retention Properties

Three-dimensional (3D) HPLC methods are useful tools for the selective analysis of biomolecules in complicated real-world matrices, however, the columns applicable for the second dimension are still limited. In the present study, two types of mixed-mode columns (Singularity MX-102 and 103) were newly designed/developed focusing on the separation of N-protected chiral amino acids. The retention profiles of the two mixed-mode columns were evaluated using 10 proteinogenic amino acids with various chemical properties, namely, alanine, asparagine (Asn), glutamine, glycine, lysine (Lys), phenylalanine, proline, serine (Ser), threonine and valine (Val), and compared to that of a mixed-mode column (Singularity MX-001) previously adopted for the 3D-HPLC analysis of chiral amino acids. The amino acids were derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) and detected by their fluorescence (ex. 470 nm, em. 530 nm). For the mobile phases, mixed solutions of acetonitrile/methanol containing formic acid were used. As a result, NBD-Val eluted the fastest for all the tested mixed-mode columns. However, the NBD-amino acids most strongly retained on the columns were different (Asn for MX-001, Ser for MX-102 and Lys for MX-103). Also, the elution orders of the 10 NBD-amino acids on the three mixed-mode columns were not the same as that on the reversed-phase C18 column, and characteristic elution orders and retention properties were observed for the individual mixed-mode columns. These results indicated that mixed-mode columns reported in the present study have different retention mechanisms, and the development of highly selective 3D-HPLC systems using these columns is expected.

Synthesis and Anti-Inflammatory Evaluation of a Library of Chiral Derivatives of Xanthones Conjugated with Proteinogenic Amino Acids.

In recent decades, the relationship between drug chirality and biological activity has been assuming enormous importance in medicinal chemistry. Particularly, chiral derivatives of xanthones (CDXs) have interesting biological activities, including enantioselective anti-inflammatory activity. Herein, the synthesis of a library of CDXs is described, by coupling a carboxyxanthone (1) with both enantiomers of proteinogenic amino esters as chiral building blocks (2-31), following the chiral pool strategy. The coupling reactions were performed at room temperature with good yields (from 44 to 99.9%) and very high enantiomeric purity, with most of them presenting an enantiomeric ratio close to 100%. To afford the respective amino acid derivatives (32-61), the ester group of the CDXs was hydrolyzed in mild alkaline conditions. Consequently, in this work, sixty new derivatives of CDXs were synthetized. The cytocompatibility and anti-inflammatory activity in the presence of M1 macrophages were studied for forty-four of the new synthesized CDXs. A significant decrease in the levels of a proinflammatory cytokine targeted in the treatment of several inflammatory diseases, namely interleukin 6 (IL-6), was achieved in the presence of many CDXs. The amino ester of L-tyrosine (X1AELT) was the most effective in reducing IL-6 production (52.2 ± 13.2%) by LPS-stimulated macrophages. Moreover, it was ≈1.2 times better than the D-enantiomer. Indeed, enantioselectivity was observed for the majority of the tested compounds. Thus, their evaluation as promising anti-inflammatory drugs should be considered.

Ornithine is the central intermediate in the arginine degradative pathway and its regulation in Bacillus subtilis

The Gram-positive bacterium Bacillus subtilis can utilize several proteinogenic and non-proteinogenic amino acids as sources of carbon, nitrogen, and energy. The utilization of the amino acids arginine, citrulline, and ornithine is catalyzed by enzymes encoded in the rocABC and rocDEF operons and by the rocG gene. The expression of these genes is controlled by the alternative sigma factor SigL. RNA polymerase associated with this sigma factor depends on ATP-hydrolyzing transcription activators to initiate transcription. The RocR protein acts as a transcription activator for the roc genes. However, the details of amino acid metabolism via this pathway are unknown. Here, we investigated the contributions of all enzymes of the Roc pathway to the degradation of arginine, citrulline, and ornithine. We identified the previously uncharacterized RocB protein as responsible for the conversion of citrulline to ornithine. Invitro assays with the purified enzyme suggest that RocB acts as a manganese-dependent N-carbamoyl-L-ornithine hydrolase that cleaves citrulline to form ornithine and carbamate. Moreover, the molecular effector that triggers transcription activation by RocR has not been unequivocally identified. Using a combination of transcription reporter assays and biochemical experiments, we demonstrate that ornithine is the molecular inducer of RocR activity. Taken together, our work suggests that binding of ATP to RocR triggers its hexamerization, and binding of ornithine then allows ATP hydrolysis and activation of roc gene transcription. Thus, ornithine is the central molecule of the roc degradative pathway as it is the common intermediate of arginine and citrulline degradation and the molecular effector of RocR.

TRPM4 drives cerebral edema by switching to alternative splicing isoform after experimental traumatic brain injury.

Traumatic brain injury (TBI) affects people of all ages and is recognized as a major cause of death and disability worldwide, also brings heavy life burden to patients and their families. However, the treatment of secondary injury after TBI is still scarce. Alternative splicing (AS) is a crucial post-transcriptional regulatory mechanism associated with various physiological processes, while the contribution of AS in treatment after TBI is poorly illuminated. In this study, we performed and analyzed the transcriptome and proteome datasets of brain tissue at multiple time points in a controlled cortical impact (CCI) mouse model. We found that AS, as an independent change against the transcriptional level, is a novel mechanism linked to cerebral edema after TBI. Bioinformatics analysis further indicated that the transformation of splicing isoforms after TBI was related to cerebral edema. Accordingly, we found that the fourth exon of transient receptor potential channel melastatin 4 (Trpm4) abrogated skipping at 72 h after TBI, resulting in a frameshift of the encoded amino acid and an increase in the proportion of spliced isoforms. Using magnetic resonance imaging (MRI), we have shown in the numbers of 3nEx isoforms of Trpm4 may be positively correlated with volume of cerebral edema. Thus alternative splicing of Trpm4 becomes a noteworthy mechanism of potential influence on edema. In summary, alternative splicing of Trpm4 may drive cerebral edema after TBI. Trpm4 is a potential therapeutic targeting cerebral edema in patients with TBI.

Improved Acylation of Pseudoproline: Masked Threonine in Flow Peptide Chemistry

Pseudoproline derivatives are incorporated into polypeptides as aggregation disrupters during synthesis, from which the unmodified side chains can be recovered during the final step (resin cleavage). However, direct coupling to pseudoproline is challenging and thus dipeptides of pseudoproline, Fmoc-Xaa-Yaa(ΨMe,Mepro)-OH, are traditionally introduced, which requires the entire library of Fmoc-Xaa-Thr(ΨMe,Mepro)-OH in stock, making this approach expensive. Here, we show how the in situ acylation of the incorporated H-Thr(ΨPro)– can be done successful in flow peptide chemistry, with almost all proteinogenic amino acids (Xaa or X). The Xaa-Thr(ΨPro) amide bond was established with a reagent excess of three to five equivalents, with better than 75% efficiency for most Xaa and 70% in the case of Met. Only for Asp was the efficiency too low (8%) to support the direct use of pseudoproline derivatives.

Intestinal Amino Acid Transport and Metabolic Health.

Amino acids derived from protein digestion are important nutrients for the growth and maintenance of organisms. Approximately half of the 20 proteinogenic amino acids can be synthesized by mammalian organisms, while the other half are essential and must be acquired from the nutrition. Absorption of amino acids is mediated by a set of amino acid transporters together with transport of di- and tripeptides. They provide amino acids for systemic needs and for enterocyte metabolism. Absorption is largely complete at the end of the small intestine. The large intestine mediates the uptake of amino acids derived from bacterial metabolism and endogenous sources. Lack of amino acid transporters and peptide transporter delays the absorption of amino acids and changes sensing and usage of amino acids by the intestine. This can affect metabolic health through amino acid restriction, sensing of amino acids, and production of antimicrobial peptides.

Expanding the substrate scope of pyrrolysyl-transfer RNA synthetase enzymes to include non-α-amino acids in vitro and in vivo

The absence of orthogonal aminoacyl-transfer RNA (tRNA) synthetases that accept non-l-α-amino acids is a primary bottleneck hindering the in vivo translation of sequence-defined hetero-oligomers and biomaterials. Here we report that pyrrolysyl-tRNA synthetase (PylRS) and certain PylRS variants accept α-hydroxy, α-thio and N-formyl-l-α-amino acids, as well as α-carboxy acid monomers that are precursors to polyketide natural products. These monomers are accommodated and accepted by the translation apparatus in vitro; those with reactive nucleophiles are incorporated into proteins in vivo. High-resolution structural analysis of the complex formed between one PylRS enzyme and a m-substituted 2-benzylmalonic acid derivative revealed an active site that discriminates prochiral carboxylates and accommodates the large size and distinct electrostatics of an α-carboxy substituent. This work emphasizes the potential of PylRS-derived enzymes for acylating tRNA with monomers whose α-substituent diverges substantially from the α-amine of proteinogenic amino acids. These enzymes or derivatives thereof could synergize with natural or evolved ribosomes and/or translation factors to generate diverse sequence-defined non-protein heteropolymers.

Chitosan complexes with amino acids and whey peptides: Sensory and antioxidant properties

Chitosan reacts with amino acids and hydrolyzed whey proteins to produce biologically active complexes that can be used in functional foods. The research objective was to obtain chitosan biocomposites with peptides and amino acids with improved antioxidant and sensory properties. &#x0D; The research featured biocomposites of chitosan and succinylated chitosan with whey peptides and amino acids. The methods of pH metry and spectrophotometry were employed to study the interaction parameters between polysaccharides and peptides, while colorimetry and spectrophotometry served to describe the amino acids content. The antiradical effect was determined by the method of fluorescence recovery. Pure compounds and their complexes underwent a sensory evaluation for bitterness. &#x0D; Chitosan and succinylated chitosan formed complexes with whey peptides and such proteinogenic amino acids as arginine, valine, leucine, methionine, and tryptophan. The equimolar binding of tryptophan, leucine, and valine occurred in an aqueous chitosan solution (in terms of glucosamine). Methionine appeared to be the least effective in chitosan interaction, while arginine failed to complex both with chitosan and succinylated chitosan. Chitosan and succinylated chitosan biocomposites with peptides and leucine, methionine, and valine proved to be less bitter that the original substances. The samples with arginine maintained the same sensory properties. Chitosan complexes with tryptophan and peptides increased their antioxidant activity by 1.7 and 2.0 times, respectively, while their succinylated chitosan complexes demonstrated a 1.5 fold increase.&#x0D; Chitosan and succinylated chitosan biocomplexes with tryptophan and whey protein peptides had excellent antioxidant and sensory properties. However, chitosan proved more effective than succinylated chitosan, probably, because it was richer in protonated amino groups, which interacted with negatively charged amino acids groups.