Liver lipid metabolism disruption significantly contributes to excessive fat buildup in waterfowl. Research suggests that the supplementation of Threonine (Thr) in the diet can improve liver lipid metabolism disorder, while Thr deficiency can lead to such metabolic disorders in the liver. The mechanisms through which Thr regulates lipid metabolism remain unclear. STAT3 (signal transducer and activator of transcription 3), a crucial transcription factor in the JAK-STAT (Janus kinase-signal transducer and activator of transcription) pathway, participates in various biological processes, including lipid and energy metabolism. This research investigates the potential involvement of STAT3 in the increased lipid storage seen in primary duck hepatocytes as a result of a lack of Thr. Using small interfering RNA and Stattic, a specific STAT3 phosphorylation inhibitor, we explored the impact of STAT3 expression patterns on Thr-regulated lipid synthesis metabolism in hepatocytes. Through transcriptome sequencing, we uncovered pathways related to lipid synthesis and metabolism jointly regulated by Thr and STAT3. The results showed that Thr deficiency increases lipid deposition in primary duck hepatocytes (p < 0.01). The decrease in protein and phosphorylation levels of STAT3 directly caused this deposition (p < 0.01). Transcriptomic analysis revealed that Thr deficiency and STAT3 knockdown jointly altered the mRNA expression levels of pathways related to long-chain fatty acid synthesis and energy metabolism (p < 0.05). Thr deficiency, through mediating STAT3 inactivation, upregulated ELOVL7, PPARG, MMP1, MMP13, and TIMP4 mRNA levels, and downregulated PTGS2 mRNA levels (p < 0.01). In summary, these results suggest that Thr deficiency promotes lipid synthesis, reduces lipid breakdown, and leads to lipid metabolism disorders and triglyceride deposition by downregulating STAT3 activity in primary duck hepatocytes.

O-linked glycosylation is a complex post-translational modification (PTM) in human proteins that plays a critical role in regulating various cellular metabolic and signaling pathways. In contrast to N-linked glycosylation, O-linked glycosylation lacks specific sequence features and maintains an unstable core structure. Identifying O-linked threonine glycosylation sites (OTGs) remains challenging, requiring extensive experimental tests. While bioinformatics tools have emerged for predicting OTGs, their reliance on limited conventional features and absence of well-defined feature selection strategies limit their effectiveness. To address these limitations, we introduced HOTGpred (Human O-linked Threonine Glycosylation predictor), employing a multi-stage feature selection process to identify the optimal feature set for accurately identifying OTGs. Initially, we assessed 25 different feature sets derived from various pretrained protein language model (PLM)-based embeddings and conventional feature descriptors using nine classifiers. Subsequently, we integrated the top five embeddings linearly and determined the most effective scoring function for ranking hybrid features, identifying the optimal feature set through a process of sequential forward search. Among the classifiers, the extreme gradient boosting (XGBT)-based model, using the optimal feature set (HOTGpred), achieved 92.03 % accuracy on the training dataset and 88.25 % on the balanced independent dataset. Notably, HOTGpred significantly outperformed the current state-of-the-art methods on both the balanced and imbalanced independent datasets, demonstrating its superior prediction capabilities. Additionally, SHapley Additive exPlanations (SHAP) and ablation analyses were conducted to identify the features contributing most significantly to HOTGpred. Finally, we developed an easy-to-navigate web server, accessible at https://balalab-skku.org/HOTGpred/, to support glycobiologists in their research on glycosylation structure and function.

The nutritional benefits of mare milk are attracting increasing consumer interest. Limited availability due to low yield poses a challenge for widespread adoption. Although lysine and threonine are often used to enhance protein synthesis and muscle mass in horses, their impact on mare milk yield and nutrient composition remains underexplored. This study investigated the effects of lysine and threonine supplementation on 24 healthy Yili mares, mares at day 30 of lactation, over a 120-day period. The mares were divided into control and three experimental groups (six mares each) under pure grazing conditions. The control group received no amino acid supplementation, while experimental groups received varying daily doses of lysine and threonine: Group I (40 g lysine + 20 g threonine), Group II (60 g lysine + 40 g threonine), and Group III (80 g lysine + 60 g threonine). Supplementation in Group II notably increased milk yield, while Groups I and II showed higher milk fat percentages, and all experimental groups exhibited improved milk protein percentages. Additionally, blood levels of total protein, albumin, triglycerides, and glucose were reduced. Detailed analyses from Group II at peak lactation (day 60) included targeted metabolomics and microbial sequencing of milk, blood, and fecal samples. Amino acid metabolomics assessed amino acid content in mare milk and serum, while 16S rRNA gene sequencing evaluated rectal microbial composition. The results indicated that lysine and threonine supplementation significantly increased levels of threonine and creatine in the blood, and lysine, threonine, glutamine, and alanine in mare milk. Microbial analysis revealed a higher prevalence of certain bacterial families and genera, including Prevotellaceae, p_251_o5, and Rikenellaceae at the family level, and unclassified_p_251_o5, Prevotellaceae_UCG_001, and Rikenellaceae_RC9_gut_group at the genus level. Multi-omics analysis showed positive correlations between specific fecal genera and amino acids in mare milk. For instance, Prevotellaceae_UCG_003, unclassified Bacteroidetes_BS11_gut_group, and Corynebacterium were positively correlated with lysine, while unclassified Prevotellaceae was positively correlated with alanine and threonine, and Unclassified_Bacteroidales_BS11_gut_group was positively correlated with glutamine. In summary, lysine and threonine supplementation in grazing lactating mares enhanced milk production and improved milk protein and fat quality. It is recommended that herders, veterinarians, and technicians consider amino acid content in the diet of lactating mares. The optimal supplementation levels under grazing conditions for Yili horses were determined to be 60 g lysine and 40 g threonine per day. Future research should explore the molecular mechanisms by which these amino acids influence milk protein and lipid synthesis in mare mammary epithelial cells.

Aminoacyl-tRNA synthetases (ARSs) are ubiquitously expressed, essential enzymes that complete the first step of protein translation: ligation of amino acids to cognate tRNAs. Genes encoding ARSs have been implicated in myriad dominant and recessive phenotypes, the latter often affecting multiple tissues but with frequent involvement of the central and peripheral nervous systems, liver, and lungs. Threonyl-tRNA synthetase (TARS1) encodes the enzyme that ligates threonine to tRNATHR in the cytoplasm. To date, TARS1 variants have been implicated in a recessive brittle hair phenotype. To better understand TARS1-related recessive phenotypes, we engineered three TARS1 missense variants at conserved residues and studied these variants in Saccharomyces cerevisiae and Caenorhabditis elegans models. This revealed two loss-of-function variants, including one hypomorphic allele (R433H). We next used R433H to study the effects of partial loss of TARS1 function in a compound heterozygous mouse model (R432H/null). This model presents with phenotypes reminiscent of patients with TARS1 variants and with distinct lung and skin defects. This study expands the potential clinical heterogeneity of TARS1-related recessive disease, which should guide future clinical and genetic evaluations of patient populations.

Killer whales (Orcinus orca) occur seasonally in the eastern Canadian Arctic (ECA), where their range expansion associated with declining sea ice have raised questions about the impacts of increasing killer whale predation pressure on Arctic-endemic prey. We assessed diet and distribution of ECA killer whales using bulk and compound-specific stable isotope analysis (CSIA) of amino acids (AA) of 54 skin biopsies collected from 2009 to 2020 around Baffin Island, Canada. Bulk ECA killer whale skin δ15N and δ13C values did not overlap with potential Arctic prey after adjustment for trophic discrimination, and instead reflected foraging history in the North Atlantic prior to their arrival in the ECA. Adjusted killer whale stable isotope (SI) values primarily overlapped with several species of North Atlantic baleen whales or tuna. Amino acid (AA)-specific δ15N values indicated the ECA killer whales fed primarily on marine mammals, having similar glutamic acid δ15N-phenylalanine δ15N (δ15NGlx-Phe) and threonine δ15N (δ15NThr) as mammal-eating killer whales from the eastern North Pacific (ENP) that served as a comparative framework. However, one ECA whale grouped with the fish-eating ENP ecotype based δ15NThr. Distinctive essential AA δ13C of ECA killer whale groups, along with bulk SI similarity to killer whales from different regions of the North Atlantic, indicates different populations converge in Arctic waters from a broad source area. Generalist diet and long-distance dispersal capacity favour range expansions, and integration of these insights will be critical for assessing ecological impacts of increasing killer whale predation pressure on Arctic-endemic species.

The thermal stability of trimeric lectin BC2L-CN was investigated and found to be considerably altered when mutating residue 83, originally a threonine, located at the fucose-binding loop. Mutants were analyzed using differential scanning calorimetry and isothermal microcalorimetry. Although most mutations decreased the affinity of the protein for oligosaccharide H type 1, six mutations increased the melting temperature (Tm) by >5 °C; one mutation, T83P, increased the Tm value by 18.2 °C(T83P, Tm = 96.3 °C). In molecular dynamic simulations, the investigated thermostable mutants, T83P, T83A, and T83S, had decreased fluctuations in the loop containing residue 83. In the T83S mutation, the side-chain hydroxyl group of serine formed a hydrogen bond with a nearby residue, suggesting that the restricted movement of the side-chain resulted in fewer fluctuations and enhanced thermal stability. Residue 83 is located at the interface and near the upstream end of the equivalent loop in a different protomer; therefore, fluctuations by this residue likely propagate throughout the loop. Our study of the dramatic change in thermal stability by a single amino acid mutation provides useful insights into the rational design of protein structures, especially the structures of oligomeric proteins.

Acetate, a promising yet underutilized carbon source for biological production, was explored for the efficient production of homoserine and threonine in Escherichia coli W. A modular metabolic engineering approach revealed the crucial roles of both acetate assimilation pathways (AckA/Pta and Acs), optimized TCA cycle flux and glyoxylate shunt activity, and enhanced CoA availability, mediated by increased pantothenate kinase activity, for efficient homoserine production. The engineered strain W-H22/pM2/pR1P exhibited a high acetate assimilation rate (5.47 mmol/g cell/h) and produced 44.1 g/L homoserine in 52 h with a 53% theoretical yield (0.18 mol/mol) in fed-batch fermentation. Similarly, strain W-H31/pM2/pR1P achieved 45.8 g/L threonine in 52 h with a 65% yield (0.22 mol/mol). These results represent the highest reported levels of amino acid production using acetate, highlighting its potential as a valuable and sustainable feedstock for biomanufacturing.

High-mannose-type glycan structure of N-glycoproteins plays important roles in the proper folding of proteins in sorting glycoprotein secretion and degradation of misfolded proteins in the endoplasmic reticulum (ER). The Glc1Man9GlcNAc2 (G1M9)-type N-glycan is one of the most important signaling molecules in the ER. However, current chemical synthesis strategies are laborious, warranting more practical approaches for G1M9-glycopeptide development. Wang et al. reported the procedure to give G1M9-Asn-Fmoc through chemical modifications and purifications from 40 chicken eggs, but only 3.3 mg of G1M9-glycopeptide was obtained. Therefore, better methods are needed to obtain more than 10 mg of G1M9-glycopeptide. In this study, we report the preparation of G1M9-glycopeptide (13.2 mg) linking Asn-Gly-Thr triad as consensus sequence from 40 chicken eggs. In this procedure, λ-carrageenan treatment followed by papain treatment was used to separate the Fc region of IgY antibody that harbors high-mannose glycans. Moreover, cotton hydrophilic interaction liquid chromatography was adapted for easy purification. The resulting G1M9-Asn(Fmoc)-Gly-Thr was identified by nuclear magnetic resonance and mass spectroscopy. G1M9-Asn(Fmoc)-Gly, G1M9-Asn(Fmoc), and G1M9-OH were also detected by mass spectroscopy. Here, our developed G1M9-tripeptide might be useful for the elucidation of glycoprotein functions as well as the specific roles of the consensus sequence.

Fluorine (F) is an important element in the synthesis of molecules broadly used in medicine, agriculture, and materials. F addition to organic structures represents a unique strategy for tuning molecular properties, yet this atom is rarely found in Nature and approaches to produce fluorometabolites (such as fluorinated amino acids, key building blocks for synthesis) are relatively scarce. This chapter discusses the use of L-threonine aldolase enzymes (LTAs), a class of enzymes that catalyze reversible aldol addition to the α-carbon of glycine. The C-C bond formation ability of LTAs, together with their known substrate promiscuity, make them ideal for in vitro F biocatalysis. Here, we describe protocols to harness the activity of the low-specificity LTAs isolated from Escherichia coli and Pseudomonas putida on 2-fluoroacetaldehyde to efficiently synthesize 4-fluoro-L-threonine in vitro. This chapter also provides a comprehensive account of experimental protocols to implement these activities in vivo. These methods are illustrative and can be adapted to produce other fluorometabolites of interest.

β-Hydroxy-α-amino acids (βHAAs) are an essential class of building blocks of therapeutically important compounds and complex natural products. They contain two chiral centers at Cα and Cβ positions, resulting in four possible diastereoisomers. Many innovative asymmetric syntheses have been developed to access structurally diverse βHAAs. The main challenge, however, is the control of the relative and absolute stereochemistry of the asymmetric carbons in a sustainable way. In this respect, there has been considerable attention focused on the chemoenzymatic synthesis of βHAAs via a one-step process. Nature has evolved different enzymatic routes to produce these valuable βHAAs. Among these naturally occurring transformations, L-threonine transaldolases present potential biocatalysts to generate βHAAs in situ. 4-Fluorothreonine transaldolase from Streptomyces sp. MA37 (FTaseMA) catalyzes the cross-over transaldolation reaction between L-Thr and fluoroacetaldehyde to give 4-fluorothreonine and acetaldehyde (Ad). It has been demonstrated that FTaseMA displays considerable substrate plasticity toward structurally diverse aldehyde acceptors, leading to the production of various βHAAs. In this chapter, we describe methods for the preparation of FTaseMA, and the chemoenzymatic synthesis of βHAAs from various aldehydes and L-Thr using FTaseMA.