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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., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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 (T m ) by >5 °C; one mutation, T83P, increased the T m value by 18.2 °C(T83P, T m  = 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., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest regarding the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

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 Glc 1 Man 9 GlcNAc 2 (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., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Yuto Nakamachi, Shinji Tanaka, Taiki Kuribara, Kiichiro Totani has patent #P7261366 issued to KH Neochem Co. Ltd. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Pulmonary fibrosis (PF) is the terminal manifestation of a type of pulmonary disease, which seriously affects the respiratory function of the body, and with no effective cure for treatment. This study evaluated the effect of sea cucumber peptides (SCP) on bleomycin-induced SD rat PF., Results: SCP can inhibit the PF induced by bleomycin. PF and SCP did not affect the food intake of rats, but PF reduced the body weight of rats, and SCP could improve the weight loss. SCP reduced lung index in PF rats in a dose-dependent manner. SCP significantly reduced IL-1β, IL-6, TNF-α, α-SMA and VIM expression levels in lung tissue (P < 0.05), significantly decreased TGF-β1 expression level in serum (P < 0.01) and the LSCP group and MSCP group had better inhibitory effects on PF than the HSCP group. Histomorphological results showed that SCP could ameliorate the structural damage of lung tissue, alveolar wall rupture, inflammatory cell infiltration, fibroblast proliferation and deposition of intercellular matrix and collagen fibers caused by PF. The improvement effect of the MSCP group was the most noteworthy in histomorphology. Metabolomics results showed that SCP significantly downregulated catechol, N-acetyl-l-histidine, acetylcarnitine, stearoylcarnitine, d-mannose, l-threonine, l-alanine, glycine, 3-guanidinopropionic acid, prostaglandin D2 and embelic acid d-(-)-β-hydroxybutyric acid expression levels in lung tissue., Conclusion: SCP ameliorate bleomycin-induced SD rat PF. KEGG pathway analysis proved that SCP intervened in PF mainly via the lysosome pathway, with d-mannose as the key factor. © 2023 Society of Chemical Industry., (© 2023 Society of Chemical Industry.)

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., (Copyright © 2024. Published by Elsevier Inc.)

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., (Copyright © 2024. Published by Elsevier Inc.)

Protein post-translational modifications play central roles in regulating protein functions. Lysine threonylation is a newly discovered reversible post-translational modification. However, the biological effect of lysine threonylation on proteins remains largely elusive. Here we report a chemical biology approach for site-specific incorporation of N ε-threonyllysine into proteins with high efficiency and investigate the biological effect of lysine threonylation on Aurora kinase A. Using this unnatural amino acid mutagenesis approach, we find that threonylation of Lys162 of Aurora kinase A inhibits its kinase activity both in vitro and in vivo and that the inhibitory effect can be reversed by the deacetylase Sirtuin 3, which removes the threonylated group from the lysine. Additionally, threonylation of Aurora kinase A makes its substrate p53 more stable in the cell. Therefore, our study demonstrates that site-specific lysine threonylation is a powerful method for probing the biological effect of protein threonylation.

Here, we report a novel α chain hemoglobin (Hb) variant found during routine thalassemia screening. This Hb variant can be detected by capillary electrophoresis (CE) but cannot be recognized by high performance liquid chromatography (HPLC). Sanger sequencing revealed a heterozygous missense substitution at nucleotide 373 on the HBA2 gene, which results in the replacement of serine by threonine at codon 124 [α124(H7)Ser→Thr ( T CC> A CC), HBA2 : c.373T>A]. It is the first report of this variant, named Hb Huadu for the birthplace of the proband. In addition, the proband coinherited the heterozygous codons 41/42 (-TTCT) ( HBB : c126&#95;129delCTTT) on the β-globin gene.

Soluble pyridine nucleotide transhydrogenases (STHs) are flavoenzymes involved in the redox homeostasis of the essential cofactors NAD(H) and NADP(H). They catalyze the reversible transfer of reducing equivalents between the two nicotinamide cofactors. The soluble transhydrogenase from Escherichia coli (SthA) has found wide use in both in vivo and in vitro applications to steer reducing equivalents toward NADPH-requiring reactions. However, mechanistic insight into SthA function is still lacking. In this work, we present a biochemical characterization of SthA, focusing for the first time on the reactivity of the flavoenzyme with molecular oxygen. We report on oxidase activity of SthA that takes place both during transhydrogenation and in the absence of an oxidized nicotinamide cofactor as an electron acceptor. We find that this reaction produces the reactive oxygen species hydrogen peroxide and superoxide anion. Furthermore, we explore the evolutionary significance of the well-conserved CXXXXT motif that distinguishes STHs from the related family of flavoprotein disulfide reductases in which a CXXXXC motif is conserved. Our mutational analysis revealed the cysteine and threonine combination in SthA leads to better coupling efficiency of transhydrogenation and reduced reactive oxygen species release compared to enzyme variants with mutated motifs. These results expand our mechanistic understanding of SthA by highlighting reactivity with molecular oxygen and the importance of the evolutionarily conserved sequence motif., Competing Interests: Conflict of interest The authors declare that there are no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

β-Hydroxynorvaline (βHNV) is unnatural amino acid structurally identical to the threonine amino acid with branched ethyl group instead of threonine's methyl. It is a known competitive inhibitor that readily bind to Threonyl-tRNA synthetase's (ThrRS) catalytic site and blocks its function. In this work, we utilized a combination of Molecular Dynamics simulation (MD) and Quantum Mechanics/Molecular Mechanics (QM/MM) methodologies to provide mechanistic insights into its inhibition reaction for ThrRS. Due to the presence of Zn(II) with its Lewis acidity character, only the ionized form of βHNV gives an enzymatically feasible energy barrier. Furthermore, in consistence with the homochirality behavior of this active site, we observed only one conformation of βHNV that could be acylated in the active site of ThrRS. Considering these new findings together with the recent search for new antibacterial agents, our findings should guide pharmaceutical scientists with further knowledge regarding the chemical nature of this drug. Moreover, benchmarking analysis of the utilized DFT functional has also been performed to identify the impact of various DFT functionals on representing the geometry and kinetics of our system. Notably, our Zn(II) containing chemical models are found to be responsive to the %HF contribution included together with the dispersion correction. Importantly, the BP86(0%HF)-D3 functional is found to display the greatest impact on the rate-limiting step kinetically. The crucial role played by Zn(II) is further enriched when its mutation with the chemically similar Cd(II) led to dramatic difference via obtaining less feasible reaction mechanism from thermodynamic and kinetic perspectives., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Intramolecular S -acylation of a thiol-installed threonine with a thioester unit, followed by S-O acyl transfer and subsequent desulphurisation, allows the synthesis of lactone peptides. A protocol has been developed enabling the cyclisation of a linear peptide, a reaction which has not been achieved by conventional methods.

Cellular function is based on protein-protein interactions. A large proportion of these interactions involves the binding of short linear motifs (SLiMs) by folded globular domains. These interactions are regulated by post-translational modifications, such as phosphorylation, that create and break motif binding sites or tune the affinity of the interactions. In addition, motif-based interactions are involved in targeting serine/threonine kinases and phosphatases to their substrate and contribute to the specificity of the enzymatic actions regulating which sites are phosphorylated. Here, we review how SLiM-based interactions assist in determining the specificity of serine/threonine kinases and phosphatases, and how phosphorylation, in turn, affects motif-based interactions. We provide examples of SLiM-based interactions that are turned on/off, or are tuned by serine/threonine phosphorylation and exemplify how this affects SLiM-based protein complex formation., (© 2022 The Author(s).)

Integrins require an activation step before ligand binding and signaling that is mediated by talin and kindlin binding to the β integrin cytosolic domain (β-tail). Conflicting reports exist about the contribution of phosphorylation of a conserved threonine motif in the β1-tail (β1-pT788/pT789) to integrin activation. We show that widely used and commercially available antibodies against β1-pT788/pT789 integrin do not detect specific β1-pT788/pT789 integrin signals in immunoblots of several human and mouse cell lysates but bind bi-phosphorylated threonine residues in numerous proteins, which were identified by mass spectrometry experiments. Furthermore, we found that fibroblasts and epithelial cells expressing the phospho-mimicking β1-TT788/789DD integrin failed to activate β1 integrins and displayed reduced integrin ligand binding, adhesion initiation and cell spreading. These cellular defects are specifically caused by the inability of kindlin to bind β1-tail polypeptides carrying a phosphorylated threonine motif or phospho-mimicking TT788/789DD substitutions. Our findings indicate that the double-threonine motif in β1-class integrins is not a major phosphorylation site but if phosphorylated would curb integrin function., (© 2022 Böttcher et al.)

Mucin-type O-glycosylation of serine or threonine residue in proteins is known to be one of the major post-translational modifications. In this study, two novel alkyl glycosides, N α -lauryl-O-(2-acetamido-2-deoxy-α-d-galactopyranosyl)-l-serineamide (GalNAc-Ser-C12) and N α -lauryl-O-(2-acetamido-2-deoxy-α-d-galactopyranosyl)-l-threonineamide (GalNAc-Thr-C12) were synthesized as saccharide primers to prime mucin-type O-glycan biosynthesis in cells. Upon incubating human gastric cancer MKN45 cells with the saccharide primers, 22 glycosylated products were obtained, and their structures were analyzed using liquid chromatography-mass spectrometry and enzyme digestion. The amounts of glycosylated products were dependent on the amino acid residues in the saccharide primers. For example, in vitro synthesis of T antigen (Galβ1-3GalNAc), fucosyl-T (Fucα1-2Galβ1-3GalNAc), and sialyl-T (NeuAcα2-3Galβ1-3GalNAc) preferred a serine residue, whereas sialyl-Tn (NeuAcα2-6GalNAc) preferred a threonine residue. Furthermore, the glycosylated products derived from GalNAc-Ser/Thr-C12 and Gal-GalNAc-Ser/Thr-C12 using cell-free synthesis showed the same amino acid selectivity as those in the cell experiments. These results indicate that glycosyltransferases involved in the biosynthesis of mucin-type O-glycans distinguish amino acid residues conjugated to GalNAc. The saccharide primers developed in this study might be useful for comparing mucin-type oligosaccharides in cells and constructing oligosaccharide libraries to study cell function., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Non-ribosomal cyclic peptides are abundant in natural sources, exhibiting attractive bioactivities and favorable pharmacological properties. Furthermore, their structural complexity renders them as attractive synthetic targets. A general task for cyclic peptide synthesis is the peptide cyclization. Compared to the traditional dehydration-based peptide macrolactamization, chemoselective peptide ligation provides an alternative, sometimes advantageous, strategy to cyclize peptides. Herein, we provide a series of structurally complex cyclic peptide examples whose total syntheses were achieved via peptide ligation-mediated peptide cyclization. The special features of these strategies for achieving the total synthesis are highlighted., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Constitutive- and immunoproteasomes are part of the ubiquitin-proteasome system (UPS), which is responsible for the protein homeostasis. Selective inhibition of the immunoproteasome offers opportunities for the treatment of numerous diseases, including inflammation, autoimmune diseases, and hematologic malignancies. Although several inhibitors have been reported, selective nonpeptidic inhibitors are sparse. Here, we describe two series of compounds that target both proteasomes. First, benzoxazole-2-carbonitriles as fragment-sized covalent immunoproteasome inhibitors are reported. Systematic substituent scans around the fragment core of benzoxazole-2-carbonitrile led to compounds with single digit micromolar inhibition of the β5i subunit. Experimental and computational reactivity studies revealed that the substituents do not affect the covalent reactivity of the carbonitrile warhead, but mainly influence the non-covalent recognition. Considering the small size of the inhibitors, this finding emphasizes the importance of the non-covalent recognition step in the covalent mechanism of action. As a follow-up series, bidentate inhibitors are disclosed, in which electrophilic heterocyclic fragments, i.e., 2-vinylthiazole, benzoxazole-2-carbonitrile, and benzimidazole-2-carbonitrile were linked to threonine-targeting ( R )-boroleucine moieties. These compounds were designed to bind both the Thr1 and β5i-subunit-specific residue Cys48. However, inhibitory activities against (immuno)proteasome subunits showed that bidentate compounds inhibit the β5, β5i, β1, and β1i subunits with submicromolar to low-micromolar IC 50 values. Inhibitory assays against unrelated enzymes showed that compounds from both series are selective for proteasomes. The presented nonpeptidic and covalent derivatives are suitable hit compounds for the development of either β5i-selective immunoproteasome inhibitors or compounds targeting multiple subunits of both proteasomes.

Eph receptor tyrosine kinases play a key role in cell-cell communication. Lack of structural information on the entire multi-domain intracellular region of any Eph receptor has hindered understanding of their signaling mechanisms. Here, we use integrative structural biology to investigate the structure and dynamics of the EphA2 intracellular region. EphA2 promotes cancer malignancy through a poorly understood non-canonical form of signaling involving serine/threonine phosphorylation of the linker connecting its kinase and SAM domains. We show that accumulation of multiple linker negative charges, mimicking phosphorylation, induces cooperative changes in the EphA2 intracellular region from more closed to more extended conformations and perturbs the EphA2 juxtamembrane segment and kinase domain. In cells, linker negative charges promote EphA2 oligomerization. We also identify multiple kinases catalyzing linker phosphorylation. Our findings suggest multiple effects of linker phosphorylation on EphA2 signaling and imply that coordination of different kinases is necessary to promote EphA2 non-canonical signaling., (© 2021. The Author(s).)

Background The neonatal heart maintains its entire regeneration capacity within days after birth. Using quantitative phosphoproteomics technology, we identified that SGK3 (serine/threonine-protein kinase 3) in the neonatal heart is highly expressed and activated after myocardial infarction. This study aimed to uncover the function and related mechanisms of SGK3 on cardiomyocyte proliferation and cardiac repair after apical resection or ischemia/reperfusion injury. Methods and Results The effect of SGK3 on proliferation and oxygen glucose deprivation/reoxygenation- induced apoptosis in isolated cardiomyocytes was evaluated using cardiomyocyte-specific SGK3 overexpression or knockdown adenovirus5 vector. In vivo, gain- and loss-of-function experiments using cardiomyocyte-specific adeno-associated virus 9 were performed to determine the effect of SGK3 in cardiomyocyte proliferation and cardiac repair after apical resection or ischemia/reperfusion injury. In vitro, overexpression of SGK3 enhanced, whereas knockdown of SGK3 decreased, the cardiomyocyte proliferation ratio. In vivo, inhibiting the expression of SGK3 shortened the time window of cardiac regeneration after apical resection in neonatal mice, and overexpression of SGK3 significantly promoted myocardial repair and cardiac function recovery after ischemia/reperfusion injury in adult mice. Mechanistically, SGK3 promoted cardiomyocyte regeneration and myocardial repair after cardiac injury by inhibiting GSK-3β (glycogen synthase kinase-3β) activity and upregulating β-catenin expression. SGK3 also upregulated the expression of cell cycle promoting genes G1/S-specific cyclin-D1, c-myc (cellular-myelocytomatosis viral oncogene), and cdc20 (cell division cycle 20), but downregulated the expression of cell cycle negative regulators cyclin kinase inhibitor P 21 and cyclin kinase inhibitor P 27. Conclusions Our study reveals a key role of SGK3 on cardiac repair after apical resection or ischemia/reperfusion injury, which may reopen a novel therapeutic option for myocardial infarction.

The attachment of a single O-linked β-N-acetylglucosamine (O-GlcNAc) to serine and threonine residues of numerous proteins in the nucleus, cytoplasm, and mitochondria is a reversible post-translational modification (PTM) and plays an important role as a regulator of various cellular processes in both healthy and disease states. Advances in strategies and tools that allow for the detection of dynamic O-GlcNAcylation on cellular proteins have helped to enhance our initial and ongoing understanding of its dynamic effects on cellular stimuli and given insights into its link to the pathogenesis of several chronic diseases. Furthermore, chemical genetic strategies and related tools have been successfully applied to a myriad of biological systems with a new level of spatiotemporal and molecular precision. These strategies have started to be used in studying and controlling O-GlcNAcylation both in vivo and in vitro. In this minireview, overviews of recent advances in molecular tools being applied to the detection and identification of O-GlcNAcylation on cellular proteins as well as on individual proteins are provided. In addition, chemical genetic strategies that have already been applied or are potentially usable in O-GlcNAc functional are also discussed., (© 2021 Wiley-VCH GmbH.)

We have been developing bacterial expression systems for human mucin-type O-glycosylation on therapeutic proteins, which is initiated by the addition of α-linked GalNAc to serine or threonine residues by enzymes in the GT-27 family of glycosyltransferases. Substrate preference across different isoforms of this enzyme is influenced by isoform-specific amino acid sequences at the site of glycosylation, which we have exploited to engineer production of Core 1 glycan structures in bacteria on human therapeutic proteins. Using RP-HPLC with a novel phenyl bonded phase to resolve intact protein glycoforms, the effect of sequon mutation on O-glycosylation initiation was examined through in vitro modification of the naturally O-glycosylated human interferon α-2b, and a sequon engineered human growth hormone. As part of the development of our glycan engineering in the bacterial expression system we are surveying various orthologues of critical enzymes to ensure complete glycosylation. Here we present an in vitro enzyme kinetic profile of three related GT-27 orthologues on natural and engineered sequons in recombinant human interferon α2b and human growth hormone where we show a significant change in kinetic properties with the amino acid changes. It was found that optimizing the protein substrate amino acid sequence using Isoform Specific O-Glycosylation Prediction (ISOGlyP, http://isoglyp.utep.edu/index.php) resulted in a measurable increase in kcat/KM, thus improving glycosylation efficiency. We showed that the Drosophila orthologue showed superior activity with our human growth hormone designed sequons compared with the human enzyme., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

CASK (Ca 2+ /calmodulin-dependent Ser/Thr kinase) is a member of the MAGUK (membrane-associated guanylate kinase) family that functions as neurexin kinases with roles implicated in neuronal synapses and trafficking. The lack of a canonical DFG motif, which is altered to GFG in CASK, led to the classification as a pseudokinase. However, functional studies revealed that CASK can still phosphorylate substrates in the absence of divalent metals. CASK dysfunction has been linked to many diseases, including colorectal cancer, Parkinson's disease, and X-linked mental retardation, suggesting CASK as a potential drug target. Here, we exploited structure-based design for the development of highly potent and selective CASK inhibitors based on 2,4-diaminopyrimidine-5-carboxamides targeting an unusual pocket created by the GFG motif. The presented inhibitor design offers a more general strategy for the development of pseudokinase ligands that harbor unusual sequence motifs. It also provides a first chemical probe for studying the biological roles of CASK.

MAP4K4 is a Ste20 member and reported to play important roles in various pathologies, including in cancer. However, the mechanism by which MAP4K4 promotes pancreatic cancer is not fully understood. It is suggested that MAP4K4 might function as a cancer promoter via specific downstream target(s) in an organ-specific manner. Here we identified MLK3 as a direct downstream target of MAP4K4. The MAP4K4 and MLK3 associates with each other, and MAP4K4 phosphorylates MLK3 on Thr738 and increases MLK3 kinase activity and downstream signaling. The phosphorylation of MLK3 by MAP4K4 promotes pancreatic cancer cell proliferation, migration, and colony formation. Moreover, MAP4K4 is overexpressed in human pancreatic tumors and directly correlates with the disease progression. The MAP4K4-specific pharmacological inhibitor, GNE-495, impedes pancreatic cancer cell growth, migration, induces cell death, and arrests cell cycle progression. Additionally, the GNE-495 reduced the tumor burden and extended survival of the KPC mice with pancreatic cancer. The MAP4K4 inhibitor also reduced MAP4K4 protein expression, tumor stroma, and induced cell death in murine pancreatic tumors. These findings collectively suggest that MLK3 phosphorylation by MAP4K4 promotes pancreatic cancer, and therefore therapies targeting MAP4K4 might alleviate the pancreatic cancer tumor burden in patients., (© 2021. The Author(s).)

Dissecting the function of proteins' post-translational modifications (PTMs) is seriously hindered by the difficulty in obtaining the homogeneous protein with the PTMs of interest. Chemical protein synthesis offers a great potential to overcome this limitation. Here, a detailed protocol is introduced for chemical synthesis of HMGA1a protein with site-specific modifications via Ser/Thr ligation strategy, by which we can systematically study the function of the triple phosphorylation (3pSer) in the HMGA1a acidic tail. For complete details on the use and execution of this protocol, please refer to Wei et al. (2021)., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

γ-Glutamylpeptides have been identified as potential biomarkers for a number of diseases including cancer, diabetes, and liver disease. In this study, we developed and validated a novel quantitative analytical strategy for measuring γ-glutamylisoleucine, γ-glutamylthreonine, and γ-glutamylvaline, all of which have been previously reported as potential biomarkers for prostate cancer in HeLa cells using ultra-high-performance liquid chromatography-tandem mass spectrometry. A BEH C18 column was used as the stationary phase. Mobile phase A was 99:1 water:formic acid and mobile phase B was acetonitrile. Chemical isotope labeling using benzoyl chloride was used as the internal standardization strategy. Sample preparation consisted of the addition of water to a frozen cell pellet, sonication, derivatization, centrifugation, and subsequent addition of an internal standard solution. The method was validated for selectivity, accuracy, precision, linearity, and stability. The determined concentrations of γ-glutamylisoleucine, γ-glutamylthreonine, and γ-glutamylvaline in HeLa cells were 1.92 ± 0.06, 10.8 ± 0.4, and 1.96 ± 0.04 pmol/mg protein, respectively. In addition, the qualitative analysis of these analytes in human serum was achieved using a modified sample preparation strategy. To the best of our knowledge, this is the first report of the use of benzoyl chloride for chemical isotope labeling for metabolite quantitation in cells., (© 2021 Wiley-VCH GmbH.)

XynR is a thermophilic and alkaline GH10 xylanase, identified in the culture broth of alkaliphilic and thermophilic Bacillus sp. strain TAR-1. We previously selected S92E as a thermostable variant from a site saturation mutagenesis library. Here, we attempted to select the alkaliphilic XynR variant from the library and isolated T315N. In the hydrolysis of beechwood xylan, T315N and S92E/T315N exhibited a broader bell-shaped pH-dependent activity than the wild-type (WT) XynR and S92E. The optimal pH values of T315N and S92E/T315N were 6.5-9.5 while those of WT and S92E were 6.5-8.5. On the other hand, T315N and S92E/T315N exhibited a narrower bell-shaped pH dependence of stability: the pHs at which the activity was stable after the incubation at 37 °C for 24 h were 6.0-8.5 for T315N and S92E/T315N, but 6.0-10.0 for WT and S92E. These results indicated that the mutation of Thr315 to Asn increased the alkaliphily but decreased the alkaline resistance., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Accumulating data suggest that the downregulation of DHCR24 is linked to the pathological risk factors of AD, denoting a potential role of DHCR24 in AD pathogenesis. However, it remains unclear whether the downregulation of DHCR24 affects the abnormal heper-phosphorylation of tau protein, which is involved in tauopathy. In present papers, immunofluorescence and Filipin III fluorescence results showed that DHCR24 knockdown significantly lowered the level of plasma membrane cholesterol and expression level of membrane lipid-raft structural protein caveolin-1; and overexpression of DHCR24 could increase the plasma membrane cholesterol levels and facilitating caveolae structure through increase the expression of caveolin-1. PP2A is the key phosphatase involving in tau phosphorylation, which is localized in cholesterol-dependent caveola/raft lipid domains. Here, the PP2A activity was detected by western blot assay. Interestingly, the level of p-PP2Ac at Y307 (inactive) and p-GSK3β at Y216 (active) in the downstream of the PP2A signal pathway were both significantly increased in silencing DHCR24 SH-SY5Y cells, which denoted an inhibition of the PP2A and activation of GSK3β signaling. Conversely, overexpression of DHCR24 blunted the inhibition effect of PP2A and activation of GSK3β. Besides, in the SH-SY5Y cell lines we demonstrated that DHCR24 knockdown obviously induced hyperphosphorylation of tau at Thr181, Thr231, Ser262, Ser396, and Ser422 Sites. In contrast, DHCR24 overexpression protects neuronal SH-SY5Y cells against the hyperphosphorylation of tau at Thr181, Thr231, Ser262, Ser396, and Ser422 Sites. Furthermore, PP2A activator D-erythro-Sphingosine (DES) also obviously inhibited the hyperphosphorylation of tau induced by DHCR24 knockdown. Collectively, our findings firstly confirmed that DHCR24 knockdown obviously induced abnormal hyperphosphorylation of tau by a novel lipid raft-dependent PP2A signaling. We propose that DHCR24 downregulation led to altered cholesterol synthesis as a potential mechanism in the progression of tau hyperphosphorylation involving in AD and other tauopathies.

Prostate cancer (PCa) is the second most leading cause of death in males. Our previous studies have demonstrated that δ-catenin plays an important role in prostate cancer progression. However, the molecular mechanism underlying the regulation of δ-catenin has not been fully explored yet. In the present study, we found that δ-catenin could induce phosphorylation of p21 Waf and stabilize p21 in the cytoplasm, thus blocking its nuclear accumulation for the first time. We also found that δ-catenin could regulate the interaction between AKT and p21, leading to phosphorylation of p21 at Thr-145 residue. Finally, EGF was found to be a key factor upstream of AKT/δ-catenin/p21 for promoting proliferation and metastasis in prostate cancer. Our findings provide new insights into molecular controls of EGF and the development of potential therapeutics targeting δ-catenin to control prostate cancer progression.

Herein, we report the total and semisyntheses of a series of polymyxin analogues with 2-Thr and 10-Thr modifications to reveal the structure-activity relationship (SAR), which has not been fully elucidated previously. We employed two total-synthetic strategies to facilitate the diversified replacements on 2-Thr or 10-Thr, respectively. Moreover, semisynthetic approaches were utilized to achieve selective esterification of 2-Thr or dual esterification of both 2- and 10-Thr. Based on the results of in vitro antibacterial assays, SAR analysis implicated that the replacement of 2-/10-Thr with amino acids carrying hydrophobic side chains can maintain the activity against Pseudomonas aeruginosa but had varied effects on other tested Gram-negative bacteria. The aminoacetyl esterification on 2-/10-Thr achieved excellent antibacterial activity, and the compound 76 exhibited 2-8-fold higher activity against different strains and lower toxicity toward the HK-2 cell line. This work explored the SAR of polymyxin 2-/10-Thr and provided a promising strategy for the development of novel polymyxin derivatives.

Cyanobactins comprise a widespread group of peptide metabolites produced by cyanobacteria that are often diversified by post-translational prenylation. Several enzymes have been identified in cyanobactin biosynthetic pathways that carry out chemically diverse prenylation reactions, representing a resource for the discovery of post-translational alkylating agents. Here, genome mining was used to identify orphan cyanobactin prenyltransferases, leading to the isolation of tolypamide from the freshwater cyanobacterium Tolypothrix sp. The structure of tolypamide was confirmed by spectroscopic methods, degradation, and enzymatic total synthesis. Tolypamide is forward-prenylated on a threonine residue, representing an unprecedented post-translational modification. Biochemical characterization of the cognate enzyme TolF revealed a prenyltransferase with strict selectivity for forward O-prenylation of serine or threonine but with relaxed substrate selectivity for flanking peptide sequences. Since cyanobactin pathways often exhibit exceptionally broad substrate tolerance, these enzymes represent robust tools for synthetic biology., (© 2021 Wiley-VCH GmbH.)

Some bacterial flagellins are O-glycosylated on surface-exposed serine/threonine residues with nonulosonic acids such as pseudaminic acid, legionaminic acid and their derivatives by flagellin nonulosonic acid glycosyltransferases, also called motility-associated factors (Maf). We report here two new glycosidic linkages previously unknown in any organism, serine/threonine-O-linked N-acetylneuraminic acid (Ser/Thr-O-Neu5Ac) and serine/threonine-O-linked 3-deoxy-D-manno-octulosonic acid or keto-deoxyoctulosonate (Ser/Thr-O-KDO), both catalyzed by Geobacillus kaustophilus Maf and Clostridium botulinum Maf. We identified these novel glycosidic linkages in recombinant G. kaustophilus and C. botulinum flagellins that were coexpressed with their cognate recombinant Maf protein in Escherichia coli strains producing the appropriate nucleotide sugar glycosyl donor. Our finding that both G. kaustophilus Maf (putative flagellin sialyltransferase) and C. botulinum Maf (putative flagellin legionaminic acid transferase) catalyzed Neu5Ac and KDO transfer on to flagellin indicates that Maf glycosyltransferases display donor substrate promiscuity. Maf glycosyltransferases have the potential to radically expand the scope of neoglycopeptide synthesis and posttranslational protein engineering., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Protein kinase D2 belongs to a family of evolutionarily conserved enzymes regulating several biological processes. In a forward genetic screen for zebrafish cardiovascular mutants, we identified a mutation in the prkd2 gene. Homozygous mutant embryos develop as wild type up to 36 h post-fertilization and initiate blood flow, but fail to maintain it, resulting in a complete outflow tract stenosis. We identified a mutation in the prkd2 gene that results in a T757A substitution at a conserved residue in the kinase domain activation loop (T714A in human PRKD2) that disrupts catalytic activity and drives this phenotype. Homozygous mutants survive without circulation for several days, allowing us to study the extreme phenotype of no intracardiac flow, in the background of a functional heart. We show dysregulation of atrioventricular and outflow tract markers in the mutants and higher sensitivity to the Calcineurin inhibitor, Cyclosporin A. Finally we identify TBX5 as a potential regulator of PRKD2. Our results implicate PRKD2 catalytic activity in outflow tract development in zebrafish.This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Phosphoprotein phosphatase-1 (PP1) is a key player in the regulation of phospho-serine (pSer) and phospho-threonine (pThr) dephosphorylation and is involved in a large fraction of cellular signaling pathways. Aberrant activity of PP1 has been linked to many diseases, including cancer and heart failure. Besides a well-established activity control by regulatory proteins, an inhibitory function for phosphorylation (p) of a Thr residue in the C-terminal intrinsically disordered tail of PP1 has been demonstrated. The associated phenotype of cell-cycle arrest was repeatedly proposed to be due to autoinhibition of PP1 through either conformational changes or substrate competition. Here, we use PP1 variants created by mutations and protein semisynthesis to differentiate between these hypotheses. Our data support the hypothesis that pThr exerts its inhibitory function by mediating protein complex formation rather than by a direct mechanism of structural changes or substrate competition., (© 2020 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

Organic crystals are emerging as mechanically compliant, light-weight and chemically versatile alternatives to the commonly used silica and polymer waveguides. However, the previously reported organic crystals were shown to be able to transmit visible light, whereas actual implementation in telecommunication devices requires transparency in the near-infrared spectral range. Here we demonstrate that single crystals of the amino acid L-threonine could be used as optical waveguides and filters with high mechanical and thermal robustness for transduction of signals in the telecommunications range. On their (00[Formula: see text]) face, crystals of this material have an extraordinarily high Young's modulus (40.95 ± 1.03 GPa) and hardness (1.98 ± 0.11 GPa) for an organic crystal. First-principles density functional theory calculations, used in conjunction with analysis of the energy frameworks to correlate the structure with the anisotropy in the Young's modulus, showed that the high stiffness arises as a consequence of the strong charge-assisted hydrogen bonds between the zwitterions. The crystals have low optical loss in the O, E, S and C bands of the spectrum (1250-1600 nm), while they effectively block infrared light below 1200 nm. This property favors these and possibly other related organic crystals as all-organic fiber-optic waveguides and filters for transduction of information.

The use of mass spectrometry has dramatically increased the research pace in the life sciences. The influence of the technique is enormous and its results can have far-reaching consequences such as jail time when applied in forensics. Therefore, analytical chemists trained in proper procedure know that they must validate their experiments. However, those quality measures have not been adopted in a similar manner in the omics technologies even though the stakes are equally high. Reasons are, among others, the segregation of the data generation and data mining functions and an undue belief in software capabilities. In this article, problematic issues such as false or overinterpretation of data are discussed, and assistance is provided for mass spectrometry laymen to evaluate the quality of their results; a quick guide to mass spectral data interpretation of peptide fragmentation experiments, the basis of bottom-up proteomics, is offered. Good science can only be generated in tight collaboration of principal investigator, analytical chemist, and bioinformatician so that the limits and the potential of each method and approach can be responsibly communicated., (© 2020 The Authors. Journal of Mass Spectrometry published by John Wiley & Sons Ltd.)

BolA has been characterized as an important transcriptional regulator, which is induced in stationary phase of growth, and in response to several stresses. In Escherichia coli, its cellular function is associated with cell wall synthesis and division, morphology, permeability, motility and biofilm formation. Phosphorylation has been widely described as one of the most important events involved in the modulation of the activity of many transcription factors. In the present work, we have demonstrated in vivo and by mass spectrometry that BolA is phosphorylated in four highly conserved protein positions: S26, S45, T81 and S95. S95 is located in the C terminus unstructured region of the protein, and the other three sites are in the DNA-binding domain. These positions were mutated to nonphosphorylated residues, and their effects were investigated on different known BolA functions. Using northern blot experiments, we showed that the regulation of the expression of these Ser/Thr BolA mutants is performed at the post-translational level. Western blot results revealed that the stability/turnover of the mutated BolA proteins is differently affected depending on the dephosphorylated residue. Moreover, we provide evidences that phosphorylation events are crucial in the modulation of BolA activity as a transcription factor and as a regulator of cell morphology and biofilm development. Here, we propose that phosphorylation affects BolA downstream functions and discuss the possible significance of these phosphoresidues in the protein structure, stability, dimerization and function as a transcription factor., (© 2020 Federation of European Biochemical Societies.)

This study was conducted to investigate the impact of genetic variants of immune checkpoint genes on the treatment outcome in small cell lung cancer (SCLC). In the present study, 261 platinum doublet-treated SCLC patients were enrolled. A total of 96 polymorphisms in 33 immune checkpoint-related genes were selected, and their association with chemotherapy response and survival outcomes were analyzed. Among the polymorphisms studied, CD155 rs1058402G > A (Ala67Thr, A67T) and CD226 rs763361C > T (Gly307Ser, G307S) were significantly associated with SCLC treatment outcome. The rs1058402G > A had a worse chemotherapy response and overall survival (under a dominant model, adjusted odds ratio [aOR] = 0.52, 95% confidence interval [CI] = 0.27-0.99, P = 0.05; adjusted hazard ratio [aHR] = 1.55, 95% CI = 1.12-2.14, P = 0.01, respectively). The rs763361C > T had better chemotherapy response and overall survival (under a dominant model, aOR = 2.03, 95% CI = 1.10-3.75, P = 0.02; aHR = 0.69, 95% CI = 0.51-0.94, P = 0.02, respectively). When the rs1058402GA/AA and rs763361CC genotypes were combined, the chemotherapy response and overall survival were significantly decreased as the number of bad genotypes increased (aOR = 0.52, 95% CI = 0.33-0.81, Ptrend = 0.004; aHR = 1.48, 95% CI = 1.19-1.84, Ptrend = 4 × 10 -4 , respectively). The 3-D structural model showed that CD155 A67T created a new hydrogen bond and structural change on CD155. These changes resulted in extending the distance and losing the hydrogen bonds between CD155 and CD226, thus weakening CD155/CD226 binding activity. In conclusion, CD155 rs1058402G > A and CD226 rs763361C > T may be useful for predicting the clinical outcomes of SCLC patients after chemotherapy.

l-Threonine transaldolases (lTTAs) are a poorly characterized class of pyridoxal-5'-phosphate (PLP) dependent enzymes responsible for the biosynthesis of diverse β-hydroxy amino acids. Here, we study the catalytic mechanism of ObiH, an lTTA essential for biosynthesis of the β-lactone natural product obafluorin. Heterologously expressed ObiH purifies as a mixture of chemical states including a catalytically inactive form of the PLP cofactor. Photoexcitation of ObiH promotes the conversion of the inactive state of the enzyme to the active form. UV-vis spectroscopic analysis reveals that ObiH catalyzes the retro-aldol cleavage of l-threonine to form a remarkably persistent glycyl quinonoid intermediate, with a half-life of ∼3 h. Protonation of this intermediate is kinetically disfavored, enabling on-cycle reactivity with aldehydes to form β-hydroxy amino acids. We demonstrate the synthetic potential of ObiH via the single step synthesis of (2 S ,3 R )-β-hydroxyleucine. To further understand the structural features underpinning this desirable reactivity, we determined the crystal structure of ObiH bound to PLP as the Schiff's base at 1.66 Å resolution. This high-resolution model revealed a unique active site configuration wherein the evolutionarily conserved Asp that traditionally H-bonds to the cofactor is swapped for a neighboring Glu. Molecular dynamics simulations combined with mutagenesis studies indicate that a structural rearrangement is associated with l-threonine entry into the catalytic cycle. Together, these data explain the basis for the unique reactivity of lTTA enzymes and provide a foundation for future engineering and mechanistic analysis.

This work reports on the concise total synthesis of eight natural products of the mugineic acid and avenic acid families (phytosiderophores). An innovative "east-to-west" assembly of the trimeric products resulted in a high degree of divergence enabling the formation of the final products in just 10 or 11 steps each with a minimum of overall synthetic effort. Chiral pool starting materials (l-malic acid, threonines) were employed for the outer building blocks while the middle building blocks were accessed by diastereo- and enantioselective methods. A highlight of this work consists in the straightforward preparation of epimeric hydroxyazetidine amino acids, useful building blocks on their own, enabling the first synthesis of 3''-hydroxymugineic acid and 3''-hydroxy-2'-deoxymugineic acid., (© 2020 The Authors. Published by Wiley-VCH GmbH.)

Phosphorylation is a ubiquitous type of post-translational modification (PTM) that occurs in both eukaryotic and prokaryotic cells where in a phosphate group binds with amino acid residues. These specific residues, i.e., serine (S), threonine (T), and tyrosine (Y), exhibit diverse functions at the molecular level. Recent studies have determined that some diseases such as cancer, diabetes, and neurodegenerative diseases are caused by abnormal phosphorylation. Based on its potential applications in biological research and drug development, the large-scale identification of phosphorylation sites has attracted interest. Existing wet-lab technologies for targeting phosphorylation sites are overpriced and time consuming. Thus, computational algorithms that can efficiently accelerate the annotation of phosphorylation sites from massive protein sequences are needed. Numerous machine learning-based methods have been implemented for phosphorylation sites prediction. However, despite extensive efforts, existing computational approaches continue to have inadequate performance, particularly in terms of overall ACC, MCC, and AUC. In this paper, we report a novel deep learning-based predictor to overcome these performance hurdles, DeepPPSite, which was constructed using a stacked long short-term memory recurrent network for predicting phosphorylation sites. The proposed technique expediently learns the protein representations from conjoint protein descriptors. The experimental results indicated that our model achieved superior performance on the training dataset for S, T and Y, with MCC values of 0.608, 0.602, and 0.558, respectively, using a 10-fold cross-validation test. We further determined the generalization efficacy of the proposed predictor DeepPPSite by conducting a rigorous independent test. The predictive MCC values were 0.358, 0.356, and 0.350 for the S, T, and Y phosphorylation sites, respectively. Rigorous cross-validation and independent validation tests for the three types of phosphorylation sites demonstrated that the designed DeepPPSite tool significantly outperforms state-of-the-art methods., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Mutations in voltage-gated sodium channels (Navs) can cause alterations in pain sensation, such as chronic pain diseases like inherited erythromelalgia. The mutation causing inherited erythromelalgia, Nav1.7 p.I848T, is known to induce a hyperpolarized shift in the voltage dependence of activation in Nav1.7. So far, however, the mechanism to explain this increase in voltage sensitivity remains unknown. In the present study, we show that phosphorylation of the newly introduced Thr residue explains the functional change. We expressed wildtype human Nav1.7, the I848T mutant, or other mutations in HEK293T cells and performed whole-cell patch-clamp electrophysiology. As the insertion of a Thr residue potentially creates a novel phosphorylation site for Ser/Thr kinases and because Nav1.7 had been shown in Xenopus oocytes to be affected by protein kinases C and A, we used different nonselective and selective kinase inhibitors and activators to test the effect of phosphorylation on Nav1.7 in a human system. We identify protein kinase C, but not protein kinase A, to be responsible for the phosphorylation of T848 and thereby for the shift in voltage sensitivity. Introducing a negatively charged amino acid instead of the putative phosphorylation site mimics the effect on voltage gating to a lesser extent. 3D modeling using the published cryo-EM structure of human Nav1.7 showed that introduction of this negatively charged site seems to alter the interaction of this residue with the surrounding amino acids and thus to influence channel function. These results could provide new opportunities for the development of novel treatment options for patients with chronic pain., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Chronic elevated free fatty (FFA) levels are linked to metabolic disorders and tumorigenesis. However, the molecular mechanism by which FFAs induce cancer remains poorly understood. Here, we show that the tumor suppressor PTEN protein levels were decreased in high fat diet (HFD) fed mice. As palmitic acid (PA, C16:0) showed a significant increase in the HFD fed mice, we further investigated its role in PTEN down regulation. Our studies revealed that exposure of cells to high doses of PA induced mTOR/S6K-mediated phosphorylation of PTEN at T366. The phosphorylation subsequently enhanced the interaction of PTEN with the E3 ubiquitin ligase WW domain-containing protein 2 (WWP2), which promoted polyubiquitination of PTEN and protein degradation. Consistent with PTEN degradation, exposure of cells to increased concentrations of PA also promoted PTEN-mediated AKT activation and cell proliferation. Significantly, a higher level of S6K activation, PTEN T366 phosphorylation, and AKT activation were also observed in the livers of the HFD fed mice. These results provide a molecular mechanism by which a HFD and elevated PA regulate cell proliferation through inactivation of tumor suppressor PTEN., (Published by Elsevier B.V.)

Water-soluble complexes are desirable for the aqueous detoxification of cyanide. Molybdenum complexes with α-amino acid and disulfide ligands with the formula K[(L)Mo 2 O 2 (μ-S) 2 (S 2 )] (L = leu ( 1 ), met ( 2 ), thr ( 3 ), and ser ( 4 )) were synthesized in a reaction of [(DMF) 3 MoO(μ-S) 2 (S 2 )] with deprotonated α-amino acids; leu, met, thr, and ser are the carboxylate anions of l-leucine, l-methionine, l-threonine, and l-serine, respectively. Potassium salts of α-amino acids (leu ( 1a ), met ( 2a ), thr ( 3a ), and ser ( 4a )) were prepared as precursors for complexes 1 - 4 , respectively, by employing a nonaqueous synthesis route. The ligand exchange reaction of [Mo 2 O 2 (μ-S) 2 (DMF) 6 ](I) 2 with deprotonated α-amino acids afforded bis -α-amino acid complexes, [(L) 2 Mo 2 O 2 (μ-S) 2 ] ( 6 - 8 ). A tris -α-amino acid complex, [(leu) 2 Mo 2 O 2 (μ-S) 2 (μ-leu + H)] ( 5 ; leu + H is the carboxylate anion of l-leucine with the amine protonated), formed in the reaction with leucine. 5 crystallized from methanol with a third weakly bonded leucine as a bridging bidentate carboxylate. An adduct of 8 with SCN - coordinated, 9 , crystallized and was structurally characterized. Complexes 1 - 4 are air stable and highly water-soluble chiral molecules. Cytotoxicity studies in the A549 cell line gave IC 50 values that range from 80 to 400 μM. Cyclic voltammetry traces of 1 - 8 show solvent-dependent irreversible electrochemical behavior. Complexes 1 - 4 demonstrated the ability to catalyze the reaction of thiosulfate and cyanide in vitro to exhaustively transform cyanide to thiocyanate in less than 1 h.

Ionic liquids (ILs) are useful in pharmaceutical industries and biotechnology as alternative solvents or sources for protein extraction and purification, preservation of biomolecules and for regulating the catalytic activity of enzymes. However, the binding mechanism, the non-covalent forces responsible for protein-IL interactions and dynamics of proteins in IL need to be investigated in depth for the effective use of ILs as alternatives. Herein, we disclose the molecular level understanding of the structural intactness and reactivity of a model protein cytochrome c (Cyt c) in biocompatible threonine-based ILs with the help of experimental techniques such as isothermal titration calorimetry (ITC), fluorescence spectroscopy, transmission electron microscopy (TEM) as well as molecular docking. Hydrophobic and electrostatic forces are responsible for the structural and conformational integrity of Cyt c in IL. The ITC experiments revealed the Cyt c-IL binding free energies are in the range of 10-14 kJ/mol and the molecular docking studies demonstrated that ILs interact at the surfaces of Cyt c. The results look promising as the ILs used here are non-toxic and biocompatible, and thus may find potential applications in structural biology and biotechnology., (© 2020 Wiley-VCH GmbH.)

Aminoacyl-tRNA synthetases (aaRSs) are an attractive class of antibacterial drug targets due to their essential roles in protein translation. While most traditional aaRS inhibitors target the binding pockets of substrate amino acids and/or ATP, we recently developed a class of novel tRNA-amino acid dual-site inhibitors including inhibitor 3 ((2S,3R)-2-amino-N-((E)-4-(6,7-dichloro-4-oxoquinazolin-3(4H)-yl)but-2-en-1-yl)-3-hydroxybutanamide) against threonyl-tRNA synthetase (ThrRS). Here, the binding modes and structure-activity relationships (SARs) of these inhibitors were analyzed by the crystal structures of Salmonella enterica ThrRS (SeThrRS) in complex with three of them. Based on the cocrystal structures, twelve quinazolinone-threonine hybrids were designed and synthesized, and their affinities, enzymatic inhibitory activities, and cellular potencies were evaluated. The best derivative 8g achieved a K d value of 0.40 μM, an IC 50 value of 0.50 μM against SeThrRS and MIC values of 16-32 μg/mL against the tested bacterial strains. The cocrystal structure of the SeThrRS-8g complex revealed that 8g induced a bended conformation for Met332 by forming hydrophobic interactions, which better mimicked the binding of tRNA Thr to ThrRS. Moreover, the inhibitory potency of 8g was less impaired than a reported ATP competitive inhibitor at high concentrations of ATP, supporting our hypothesis that tRNA site inhibitors are likely superior to ATP site inhibitors in vivo, where ATP typically reaches millimolar concentrations., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Thirty-six novel threoninamide carbamate derivatives were designed and synthesised using active fragment-based pharmacophore model. Antifungal activities of these compounds were tested against Oomycete fungi Phytophthora capsici in vitro and in vivo. Interestingly, compound I-1, I-2, I-3, I-6 and I-7 exhibited moderate control effect (>50%) against Pseudoperonospora cubensis in greenhouse at 6.25 μg/mL, which is better than that of control. Meanwhile most of these compounds exhibited significant inhibitory against P. capsici . The other nine fungi were also tested. More importantly, some compounds exhibited remarkably high activities against Sclerotinia sclerotiorum , P. piricola and R. solan in vitro with EC 50 values of 3.74-9.76 μg/mL. It is possible that the model is reliabile and this method can be used to discover lead compounds for the development of fungicides.

Protease-activated receptor 4 (PAR4) mediates sustained thrombin signaling in platelets and is required for a stable thrombus. PAR4 is activated by proteolysis of the N terminus to expose a tethered ligand. The structural basis for PAR4 activation and the location of its ligand binding site (LBS) are unknown. Using hydrogen/deuterium exchange (H/D exchange), computational modeling, and signaling studies, we determined the molecular mechanism for tethered ligand-mediated PAR4 activation. H/D exchange identified that the LBS is composed of transmembrane 3 (TM3) domain and TM7. Unbiased computational modeling further predicted an interaction between Gly48 from the tethered ligand and Thr153 from the LBS. Mutating Thr153 significantly decreased PAR4 signaling. H/D exchange and modeling also showed that extracellular loop 3 (ECL3) serves as a gatekeeper for the interaction between the tethered ligand and LBS. A naturally occurring sequence variant (P310L, rs2227376) and 2 experimental mutations (S311A and P312L) determined that the rigidity conferred by prolines in ECL3 are essential for PAR4 activation. Finally, we examined the role of the polymorphism at position 310 in venous thromboembolism (VTE) using the International Network Against Venous Thrombosis (INVENT) consortium multi-ancestry genome-wide association study (GWAS) meta-analysis. Individuals with the PAR4 Leu310 allele had a 15% reduction in relative risk for VTE (odds ratio, 0.85; 95% confidence interval, 0.77-0.94) compared with the Pro310 allele. These data are consistent with our H/D exchange, molecular modeling, and signaling studies. In conclusion, we have uncovered the structural basis for PAR4 activation and identified a previously unrecognized role for PAR4 in VTE., (© 2020 by The American Society of Hematology.)

Phage infection is common during the production of L-threonine by E. coli, and low L-threonine production and glucose conversion percentage are bottlenecks for the efficient commercial production of L-threonine. In this study, 20 antiphage mutants producing high concentration of L-threonine were obtained by atmospheric and room temperature plasma (ARTP) mutagenesis, and an antiphage E. coli variant was characterized that exhibited the highest production of L-threonine Escherichia coli ([E. coli] TRFC-AP). The elimination of fhuA expression in E. coli TRFC-AP was responsible for phage resistance. The biomass and cell growth of E. coli TRFC-AP showed no significant differences from those of the parent strain (E. coli TRFC), and the production of L-threonine (159.3 g L -1 ) and glucose conversion percentage (51.4%) were increased by 10.9% and 9.1%, respectively, compared with those of E. coli TRFC. During threonine production (culture time of 20 h), E. coli TRFC-AP exhibited higher activities of key enzymes for glucose utilization (hexokinase, glucose phosphate dehydrogenase, phosphofructokinase, phosphoenolpyruvate carboxylase, and PYK) and threonine synthesis (glutamate synthase, aspartokinase, homoserine dehydrogenase, homoserine kinase and threonine synthase) compared to those of E. coli TRFC. The analysis of metabolic flux distribution indicated that the flux of threonine with E. coli TRFC-AP reached 69.8%, an increase of 16.0% compared with that of E. coli TRFC. Overall, higher L-threonine production and glucose conversion percentage were obtained with E. coli TRFC-AP due to increased activities of key enzymes and improved carbon flux for threonine synthesis., (© 2020 American Institute of Chemical Engineers.)

Echinocandin B (ECB) is a key precursor of antifungal agent Anidulafungin, which has demonstrated clinical efficacy in patients with invasive candidiasis. In this study, the effects of microparticle-enhanced cultivation and methyl oleate on echinocandin B fermentation titer were investigated. The results showed that the titer was significantly influenced by the morphological type of mycelium, and mycelium pellet was beneficial to improve the titer of this secondary metabolism. First, different carbon sources were chosen for the fermentation, and methyl oleate achieved the highest echinocandin B titer of 2133 ± 50 mg/L, which was two times higher than that of the mannitol. The study further investigated the metabolic process of the fermentation, and the results showed that L-threonine concentration inside the cell could reach 275 mg/L at 168 h with methyl oleate, about 2.5 times higher than that of the mannitol. Therefore, L-threonine may be a key precursor of echinocandin B. In the end, a new method of adding microparticles for improving the mycelial morphology was used, and the addition of talcum powder (20 g/L, diameter of 45 µm) could make the maximum titer of echinocandin B reach 3148 ± 100 mg/L.

A family of polypeptide GalNAc-transferases (GalNAc-Ts) initiates mucin-type O-glycosylation, transferring GalNAc onto hydroxyl groups of Ser and Thr residues of target substrates. The 20 GalNAc-T isoenzymes in humans are classified into nine subfamilies according to sequence similarity. GalNAc-Ts select their sites of glycosylation based on weak and overlapping peptide sequence motifs, as well prior substrate O-GalNAc glycosylation at sites both remote (long-range) and neighboring (short-range) the acceptor. Together, these preferences vary among GalNAc-Ts imparting each isoenzyme with its own unique specificity. Studies on the first identified GalNAc-Ts showed Thr acceptors were preferred over Ser acceptors; however studies comparing Thr vs. Ser glycosylation across the GalNAc-T family are lacking. Using a series of identical random peptide substrates, with single Thr or Ser acceptor sites, we determined the rate differences (Thr/Ser rate ratio) between Thr and Ser substrate glycosylation for 12 isoenzymes (representing 7 GalNAc-T subfamilies). These Thr/Ser rate ratios varied across subfamilies, ranging from ~2 to ~18 (for GalNAc-T4/GalNAc-T12 and GalNAc-T3/GalNAc-T6, respectively), while nearly identical Thr/Ser rate ratios were observed for isoenzymes within subfamilies. Furthermore, the Thr/Ser rate ratios did not appreciably vary over a series of fixed sequence substrates of different relative activities, suggesting the ratio is a constant for each isoenzyme against single acceptor substrates. Finally, based on GalNAc-T structures, the different Thr/Ser rate ratios likely reflect differences in the strengths of the Thr acceptor methyl group binding to the active site pocket. With this work, another activity that further differentiates substrate specificity among the GalNAc-Ts has been identified., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Phosphorylation is a ubiquitous post-translational modification in proteins, and the phosphate group is present constitutively or transiently in most biological building blocks. These phosphorylated biomolecules are involved in many high-affinity binding/unbinding events that rely predominantly on electrostatic interactions. To build accurate models of these molecules, we need an improved description of the atomic partial charges for all relevant protonation states. In this work, we showed that the commonly used protocols to derive atomic partial charges using well-solvated molecules are inadequate to model the protonation equilibria in binding events. We introduced a protocol based on PB/MC calculations with a single representative conformation (of both protonation states) and used the resulting p K a estimations to help manually curate the atomic partial charges. The final charge set, which is fully compatible with the GROMOS 54A7 force field, proved to be very effective in modeling the protonation equilibrium in different phosphorylated peptides in the free (tetrapeptides, pentapeptides, and pY1021) and protein-complexed forms (pY1021/PLC-γ1 complex). This was particularly important in the case of the pY1021 bound to the SH2 domain of PLC-γ1, where only our curated charge set captured the correct protonation equilibrium at the neutral to slightly acidic pH range. The binding/unbinding phenomena in that pH range are biologically relevant, and to improve our models, we need to go beyond the commonly used protocols and obtain revised force field parameters for these molecules.