Amino Acid Residues Research Articles

Carrier-Guided Proteome Analysis in a High Protein Background: An Improved Approach to Host Cell Protein Identification.

Many shotgun proteomics experiments are negatively influenced by highly abundant proteins, such as those measuring residual host cell proteins (HCP) amidst highly abundant recombinant biotherapeutic or plasma proteins amidst albumin and immunoglobulins. While western blotting and ELISAs can reveal the presence of specific low abundance proteins from highly abundant background proteins, mass spectrometry approaches are required to define the low abundance protein composition in these scenarios. The challenge in detecting low abundance proteins in a high protein background by standard shotgun approaches is that spectra are often not triggered on their peptides in data dependent acquisition methods but rather on the highly abundant background peptides. Here, we use tandem mass tags (TMT) to introduce a carrier proteome approach to enhance the detection of proteins, such as from residual host cell proteomes amidst a highly abundant background. Using a mixture of bovine serum albumin (BSA) and E. coli as a mock high background/low abundance target protein formulation, we demonstrate proof-of-principle experiments allowing the improved detection of target proteins amidst a high protein background. While we observed significant coisolation interference, we mitigated it by using a spike-in interference detection TMT channel. Finally, we use the approach to identify 300 residual E. coli proteins from a protein A pulldown of a human IgG antibody, demonstrating that it may be applicable to analysis of HCPs in biotherapeutic protein formulations.

Cysteine-Persulfide Sulfane Sulfur-Ligated Zn Complex of Sulfur-Carrying SufU in the SufCDSUB System for Fe-S Cluster Biosynthesis.

SufU, a component of the SufCDSUB Fe-S cluster biosynthetic system, serves as a Zn-dependent sulfur-carrying protein that delivers inorganic sulfur in the form of cysteine persulfide from SufS to SufBCD. To understand this sulfur delivery mechanism, we studied the X-ray crystal structure of SufU and its sulfur-carrying state (persulfurated SufU) and performed functional analysis of the conserved amino acid residues around the Zn sites. Interestingly, sulfur-carrying SufU with Cys41-persulfide (Cys41-Sγ-Sδ-) exhibited a unique Zn coordination structure, in which electrophilic Sγ is ligated to Zn and nucleophilic/anionic Sδ is bound to distally conserved Arg125. This structure is distinct from those of other Cys-persulfide-Sδ-ligated metals of metalloproteins, such as hybrid cluster proteins and SoxAX. Functional analysis of SufU variants with Zn-ligand and Arg125 substitutions revealed that both Zn and Arg125 are critical for the function of SufU with SufS. The Zn-persulfide structure of SufU provides insight into the sulfur-transfer process, suggesting that persulfide-Sδ- is stabilized via bridging by Zn and Arg125 of SufU.

The O-GlcNAc database: introducing new features and tools developed from community feedback.

O-GlcNAc is a reversible post-translational modification found on serine and threonine residues of nucleocytoplasmic proteins. Four years ago, we released the O-GlcNAc Database( oglcnac.mcw.edu ), a comprehensive catalog of O-GlcNAcylated proteins that has become one of the most cited resources in the field, with hundreds of unique users per month. We are now presenting an updated O-GlcNAc Database, which includes nearly 20,000 O-GlcNAcylated proteins and 48 species, marking substantial growth in data volume and scope. This paper presents the most noteworthy features implemented over the last year, often originating from feedback from the O-GlcNAc community. Among these features, we provide a brief overview of the database content, introduce our new protein viewer mode, and discuss the implementation of subcellular localization information and its applications in the O-GlcNAc score. We also provide an interface to use CytOVS, a tool designed to evaluate and sort O-GlcNAcome datasets derived from MS experiments. In conclusion, this new and improved O-GlcNAc Database represents a significant advancement in providing a comprehensive and expanded resource for researchers in the field of O-GlcNAc biology.

Cryptosporidium PI(4)K inhibitor EDI048 is a gut-restricted parasiticidal agent to treat paediatric enteric cryptosporidiosis.

Diarrhoeal disease caused by Cryptosporidium is a major cause of morbidity and mortality in young and malnourished children from low- and middle-income countries, with no vaccine or effective treatment. Here we describe the discovery of EDI048, a Cryptosporidium PI(4)K inhibitor, designed to be active at the infection site in the gastrointestinal tract and undergo rapid metabolism in the liver. By using mutational analysis and crystal structure, we show that EDI048 binds to highly conserved amino acid residues in the ATP-binding site. EDI048 is orally efficacious in an immunocompromised mouse model despite negligible circulating concentrations, thus demonstrating that gastrointestinal exposure is necessary and sufficient for efficacy. In neonatal calves, a clinical model of cryptosporidiosis, EDI048 treatment resulted in rapid resolution of diarrhoea and significant reduction in faecal oocyst shedding. Safety and pharmacological studies demonstrated predictable metabolism and low systemic exposure of EDI048, providing a substantial safety margin required for a paediatric indication. EDI048 is a promising clinical candidate for the treatment of life-threatening paediatric cryptosporidiosis.

Revealing the Structure of Sheer N-Acetylglucosamine, an Essential Chemical Scaffold in Glycobiology.

We explored the conformational landscape of N-acetyl-α-d-glucosamine (α-GlcNAc), a fundamental chemical scaffold in glycobiology. Solid samples were vaporized by laser ablation, expanded in a supersonic jet, and characterized by broadband chirped pulse Fourier transform microwave spectroscopy. In the isolation conditions of the jet, three different structures of GlcNAc have been discovered. These are conclusively identified by comparing the experimental values of the rotational constants with those predicted by theoretical calculations. The conformational preferences are controlled by intramolecular hydrogen bond networks formed between the polar groups in the acetamido group and the hydroxyl groups and dominated in all cases by a strong OH···O═C interaction. We reported an exception to the gauche effect due to the enhanced stability observed for the Tg+ conformer. All the structures present the same disposition of the acetamido group, which explains the highly selective binding of N-acetylglucosamine with different amino acid residues. Thus, the comprehensive structural data provided here shall help to shed some light on the biological role of this relevant amino sugar.

Monoclonal Antibody against Porcine LAG3 Inhibits Porcine Reproductive and Respiratory Syndrome Virus Infection

Lymphocyte activation gene 3 (LAG3) is an inhibitory receptor and the interaction between fibrinogen-like protein 1 and LAG3 can inhibit the anti-tumor effect of T cells both in vivo and in vitro, which was regarded as a new immune evasion mechanism. Porcine reproductive and respiratory syndrome (PRRS), caused by PRRSV, is an infectious disease characterized by reproductive disorders in pregnant sows and gilts and respiratory problems in pigs of all ages, seriously impacting the pig industry worldwide. In this study, monoclonal antibodies (mAbs) against porcine LAG3 (pLAG3) were developed, and one mAb (1C2) showed good reactivity with pLAG3 on PHA-activated porcine peripheral blood lymphocytes. Epitope mapping showed the epitope recognized by mAb 1C2 was located at amino acid residues 214–435 of pLAG3. LAG3 expression in the tissues of PRRSV-infected pigs was detected, using mAb 1C2 as the primary antibody, and the results revealed that PRRSV infection caused a marked increase in LAG3 expression compared to the control group. Interference of LAG3 expression on PHA-activated lymphocytes promoted PRRSV replication in the co-culture system of monocyte-derived dendritic cells and lymphocytes, whereas overexpression of LAG3 or blocking of the LAG3 signal with mAb 1C2 inhibited PRRSV replication, indicating that PRRSV infection activates the LAG3-signaling pathway, suggesting that this pathway plays an important role in PRRSV pathogenesis. The results obtained lay the foundation for subsequent research on the role of LAG3 in PRRS and other diseases with persistent infection characteristics.

Organotin(IV) complexes of tridentate (ONO) hydrazone ligands: synthesis, spectral characterization, antituberculosis, antimicrobial, anti-inflammatory, molecular docking and cytotoxicity studies.

Infectious diseases have a significant impact in the historical trajectory of humanity, exerting profound influence on societies, driving advancements in medical science, and significantly impacting individuals on a worldwide scale. Consequently, this research endeavours to identify potent agents combatting tuberculosis, inflammation, and microbial deformities. The investigation focuses on hydrazones (1,2) endowed eight organotin(IV) complexes, where hydrazones were derived from 2-acetyl-1H-indene-1,3(2H)-dione and 2-phenoxypropanehydrazide/2-(2,4-dichlorophenoxy)propanehydrazide. All compounds underwent thorough characterization utilizing a variety of spectral and analytical techniques including, multinuclear NMR, FT-IR, HRMS, UV-Vis, SEM-EDAX, TGA, XRD, molar conductance measurements, establishing the pentacoordinated environment around tin(IV) ion with tridentate (ONO) mode of chelation of hydrazones. Powder XRD revealed the ligand's crystalline and complexes' semi-crystalline nature, while thermal analysis indicated two-step decomposition leaving tin oxide residue. In vitro evaluations utilize microplate alamar blue assay for assessing anti-tuberculosis activity, serial dilution technique for antimicrobial efficacy, and bovine serum albumin method for evaluating anti-inflammatory properties. The complexes exhibited higher biological activities than their respective ligands and the activity of the complexes follow the order: Ph2SnL1-2 > Bu2SnL1-2 > Et2SnL1-2 > Me2SnL1-2. Among them, phenyl complex (10) [Ph2SnL2] displays superior efficacy against TB dysfunction (MIC: 0.0180 ± 0.009μmol/mL) and also demonstrates exceptional potency in combating inflammation (IC50: 7.27 ± 0.04μM), and microbial (MIC: 0.0045μmol/mL) infections, comparable to standard drugs. Additionally, cytotoxicity testing against vero cell line revealed decreased toxicity at lower concentrations, and attenuated by chelation. Phenyl complex (10) [Ph2SnL2] shows promising cytotoxicity at 3.12µg/mL (19.29 ± 0.09%). Further, The diphenyltin(IV) complex (10), identified as the most effective against TB, shows stronger binding to key 3PTY protein residues (-42.2kJ/mol) compared to ligand (2) (-33.4kJ/mol), correlating with its superior anti-tuberculosis potency in biological assays. This comprehensive approach aims to actively contribute to ongoing initiatives addressing infectious diseases, thereby advancing global health and overall well-being.

8410 Lipid modification enhances the calcemic and bone building effects of injected PTH(1-14) and PTH(1-11) fragment peptides in mice

Abstract Disclosure: J. Höppner: None. H. Noda: None. A. Khatri: None. H. Jüppner: None. T.J. Gardella: None. PTH analogs with improved actions in vivo are of interest as potential treatment options for bone and mineral ion diseases such as hypoparathyroidism and osteoporosis. The efficacies in vivo of conventional PTH peptides are generally limited by rapid rates of clearance from the circulation (t1/2 = ∼ 5-30 minutes) and relatively short-dwell times on the receptor (PTH1R). This is especially true for small fragment analogs, such as M (modified)-PTH(1-14), and M-PTH(1-11), which have only been shown to be active in vitro. We explored whether PTH fragment peptide efficacy in vivo could be enhanced by addition of a lipid chain (e.g., a C15 palmitoyl chain (palm)) to either the peptide C-terminus or to the side chain of the amino acid residue at position 11 or 13. The C-tail lipid modifications were specifically designed to promote binding to serum albumin, and hence extend circulation half-life, while the lipid chains at sidechains of residue positions 11 and 13 were designed, based on structural models, to anchor the ligand to the receptor in situ -- i.e. by projection of the acyl chain between two adjacent transmembrane helices and into the surrounding plasma membrane -- to thereby extend receptor dwell time. Assessment of cAMP signaling potencies in HEK293/hPTH1R/glosensor cells revealed that both types of lipid modifications can preserve agonist potency, and that the lipid extensions at position 11 or 13 can indeed prolong receptor dwell times, as shown by extended durations of cAMP signaling after initial binding of Palm 11 or 13-modified M-PTH(1-14) and M-PTH(1-11) peptide fragment. Single subcutaneous injection of either Palm-13-M-PTH(1-14) or Palm-11-M-PTH(1-11) into mice resulted in prolonged increases in blood serum calcium levels, while 14 days of daily injection resulted in profound increases in bone mass, as measured by uCT, and marked increases in blood levels of the bone turnover markers CTX1 and P1NP, whereas non-lipidated M-PTH(1-14) control peptides were inactive in vivo. Peptide lipidation thus holds promise as a strategy to employ in the design of new PTH analogs with improved efficacies in vivo. Presentation: 6/1/2024

Ankaflavin and Monascin Prevent Fibrillogenesis of Hen Egg White Lysozyme: Focus on Noncovalent and Covalent Interactions.

Misfolding and amyloid fibrillogenesis of proteins have close relationships with several neurodegenerative diseases. The present work investigates the inhibitive activities of ankaflavin (AK) and monascin (MS), two yellow pigments separated from Monascus-fermented rice, on hen egg white lysozyme (HEWL) fibrillation. The results demonstrated that AK/MS suppressed HEWL fibrillation through interfering with the nucleation period and AK was more potent. Fluorescence quenching and in silico docking studies revealed that AK/MS bond to HEWL by the formation of noncovalent forces with some critical amino acid residues that tend to form fibrils. Compared to those of AK, hydrogen bonding interactions between MS and Asn46, Trp62, and Trp63 residues in HEWL were slightly weaker. Besides, the covalent interaction between MS and HEWL with the binding site of Arg68 was found. These observations offered reasonable explanations for the difference in the mechanisms of AK and MS inhibiting HEWL fibrillogenesis. In a word, all data acquired herein indicated AK/MS as potent candidates for the improvement and treatment of neurological disorders.

Computational Analyses of Amino Acid Molecules in PDE7A for Elucidating their Evolutionary Diversity and Protein Interactions in Multiple Mammalian Species

This study explores the protein interactions and evolutionary conservation of phosphodiesterase type 7A (PDE7A) across different species, to identify the species with the highest similarity to humans, which has important applications in many fields such as pharmacology, comparative biology, biochemistry, oncology, endocrinology, and reproductive sciences. Computational analyses of the amino acid sequence of this enzyme revealed a common evolutionary relationship between the selected species and humans, but camels show the most variable evolutionary lineage in their PDE7A protein over time. In searches for homologous proteins, identity analysis results for four returned amino acid sequences were as follows: 91.81 ± 4.25% (mean ± SD), while the similarity between the sequences was 94.66% ± 4.55, and analysis of BLOSUM62 (Blocks Substitution Matrix 62) yielded a minimum value of 0.901 ± 0.055. All species have closely related physicochemical properties. The computational analyses revealed conserved amino acid residues of the gene across the studied species, which likely contribute to the similar expression patterns of the PDE7A gene in various animals. The findings also suggest that rats are a suitable model for gaining deeper insights into human biology.

8369 Overexpression of Neurosecretory Protein GL, a Novel Small Secretory Protein, Induces Fat Accumulation in Mice

Abstract Disclosure: K. Ukena: None. Y. Narimatsu: None. A. Ogasawara: None. M. Furumitsu: None. E. Iwakoshi-Ukena: None. Hypothalamic neuropeptides and peripheral factors influence food intake, body mass, and energy expenditure. For instance, neuropeptide Y (NPY)/agouti-related peptide (AgRP) are potent orexigenic factors and α-melanocyte-stimulating hormone (α-MSH) is an anorexigenic factor, respectively. In addition, leptin and insulin derived from peripheral tissues act on the hypothalamus to influence energy homeostasis. Precise hypothalamic control of energy balance affects puberty, thermoregulation, energy storage, and survival during different life-history stages. We have previously discovered a novel cDNA encoding the precursor of a neurosecretory small protein in the hypothalamus of birds and rodents (1, 2). The precursor protein contained a signal peptide sequence, a mature peptide sequence with 80 amino acid residues, a glycine amidation signal, and a dibasic amino acid cleavage site. Because the predicted C-terminal amino acids of the peptide were Gly-Leu-NH2, the small protein was named neurosecretory protein GL (NPGL). Quantitative RT-PCR analysis demonstrated that NPGL mRNA was expressed exclusively in the mediobasal hypothalamus (MBH), including the arcuate nucleus, and the expression was elevated on food deprivation in rodents. This finding led us to predict that NPGL was involved in the regulation of energy homeostasis. Indeed, using NPGL administration and neutralizing antibody administration in rats, we established that NPGL increased lipid accumulation in white adipose tissue (3). In the present study, to investigate the physiological function of NPGL in mice, we induced its overexpression using an adeno-associated virus (AAV) vector in NPGL-Cre driver mice. We then monitored food intake and recorded body mass changes. Npgl overexpression increased body mass. We also found that the overexpression resulted in increased masses of white adipose tissue and interscapular brown adipose tissue. Taken together, these results provide evidence for NPGL as a novel regulator of fat deposition in adipose tissues. Reference: (1) Ukena et al., Biochem. Biophys. Res. Commun. 2014 446:298-303. (2) Ukena, Gen. Comp. Endocrinol. 2018 260:164-170. (3) Iwakoshi-Ukena et al., eLife 2017 6:e28527. Presentation: 6/3/2024

PocketDTA: An advanced multimodal architecture for enhanced prediction of drug-target affinity from 3D structural data of target binding pockets.

Accurately predicting the drug-target binding affinity (DTA) is crucial to drug discovery and repurposing. Although deep learning has been widely used in this field, it still faces challenges with insufficient generalization performance, inadequate use of three-dimensional (3D) information and poor interpretability. To alleviate these problems, we developed the PocketDTA model. This model enhances the generalization performance by pre-trained models ESM-2 and GraphMVP. It ingeniously handles the first three (top-3) target binding pockets and drug 3D information through customized GVP-GNN Layers and GraphMVP-Decoder. Additionally, it employs a bilinear attention network to enhance interpretability. Comparative analysis with state-of-the-art (SOTA) methods on the optimized Davis and KIBA datasets reveals that the PocketDTA model exhibits significant performance advantages. Further, ablation studies confirm the effectiveness of the model components, whereas cold-start experiments illustrate its robust generalization capabilities. In particular, the PocketDTA model has shown significant advantages in identifying key drug functional groups and amino acid residues via molecular docking and literature validation, highlighting its strong potential for interpretability. Code and data are available at: Https://github.com/zhaolongNCU/PocketDTA.

Toward Absolute Quantification of Soluble Proteins via Proton Nuclear Magnetic Resonance Spectroscopy: Total Protein Concentration in Blood Plasma.

For absolute protein quantification using nuclear magnetic resonance (NMR) spectroscopy, we considered proteins as homopolymers and effective amino acid (AA) residues (AAREff) as monomer units. For diverse classes of proteins, we determined the AAREff molecular weight as 111.5 ± 3.2 Da and the number of hydrogens per AA as 7.8 ± 0.2. Their ratio of 14.3 ± 0.3 (g/LP)/(mol/LH) remains constant across various protein classes and is equivalent to Kjeldahl's nitrogen-to-protein conversion constant of 5.78 ± 0.29 gN/gP. By analogy to the Kjeldahl method, we suggest that the total integral of a 1H NMR solution protein spectrum could be used for total protein quantification. We synthesized low-resolution protein spectra from the weighted sums of individual AA spectra and compared them with experimental spectra. In the methyl region, the ratio of the protein mass to the total number of protons in the synthetic spectra (corrected for the chemical shift mismatch) was ∼1 (mg/mL)/mM, which agrees with an earlier reported experimental ratio for urine (1.05 ± 0.06 (mg/mL)/mM). For human blood plasma, in the methyl region, we found empirical ratios of 1.115 ± 0.006 (mg/mL)/mM (using 96 patient samples) and 1.121 ± 0.011 (mg/mL)/mM for the NIST plasma standard. This numerical agreement points to universal conversion constants, i.e., protein mixtures with unknown compositions could be quantified without the need for calibration standards by measuring the millimolar proton concentration within the methyl region of the NMR spectrum using the same conversion constant.

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

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

Aluminum fluorides as noncovalent Lewis acids in proteins: The case of phosphoryl transfer enzymes.

The Protein Data Bank (PDB) was scrutinized for the presence of noncovalent O···Al Triel Bonding (TrB) interactions, involving protein residues (e.g. GLU and GLN), adenosine/guanine diphosphate moieties (ADP and GDP), water molecules and two aluminum fluorides (AlF3 and AlF4-). The results were statistically analyzed, revealing a vast number of O···Al contacts in the active sites of phosphoryl transfer enzymes, with a marked directionality towards the Al σ-/π-hole. The physical nature of the TrBs studied herein was analyzed using Molecular Electrostatic Potential (MEP) maps, the Quantum Theory of Atoms in Molecules (QTAIM), the Non Covalent Interaction plot (NCIplot) visual index and Natural Bonding Orbital (NBO) studies. As far as our knowledge extends, it is the first time that O···Al TrBs are analyzed within a biological context, participating in protein trapping mechanisms related to phosphoryl transfer enzymes. Moreover, since they are involved in the stabilization of aluminum fluorides inside the protein's active site, we believe the results reported herein will be valuable for those scientists working in supramolecular chemistry, catalysis and rational drug design.

Altered dNTP pools accelerate tumor formation in mice.

Alterations in deoxyribonucleoside triphosphate (dNTP) pools have been linked to increased mutation rates and genome instability in unicellular organisms and cell cultures. However, the role of dNTP poolchanges in tumor development in mammals remains unclear. In this study, we present a mouse model with a point mutation at the allosteric specificity site of ribonucleotide reductase, RRM1-Y285A. This mutation reduced ribonucleotide reductase activity, impairing the synthesis of deoxyadenosine triphosphate (dATP) and deoxyguanosinetriphosphate (dGTP). Heterozygous Rrm1+/Y285A mice exhibited distinct alterations in dNTP pools across various organs, shorter lifespans and earlier tumor onset compared with wild-type controls. Mutational spectrum analysis of tumors revealed two distinct signatures, one resembling a signature extracted from a human cancer harboring a mutation of the same amino acid residue in ribonucleotide reductase, RRM1Y285C. Our findings suggest that mutations in enzymes involved in dNTP metabolism can serve as drivers of cancer development.

Enhanced emulsification properties of microalgae protein through gellan gum conjugation: Mechanistic insights and applications in curcumin encapsulation and delivery

The emulsification properties of microalgae protein (MP) are poor, especially under acidic and neutral conditions, which may limit the broad applications of MP in food processing. This study aims to explore the effects of gellan gum (GG) on the emulsification properties of MP. Firstly, MP-GG complexes were prepared and their structures characterized. Subsequently, MP-GG complexes stabilized emulsions were prepared and their stability evaluated. Finally, these emulsions were employed for the encapsulation and delivery of curcumin to evaluate their potential as an efficient nutrient delivery medium. Results indicated that MP-GG complexes were formed under various pH conditions, with pH 6 identified as optimal for complexes stability (zeta-potential value was −31 mV). UV–vis and fluorescence spectroscopy demonstrated that GG did not significantly alter the MP's structure but induced slight conformational changes, leading to the burial of some amino acid residues. Zeta potential measurements confirmed that MP-GG complexes were stabilized by strong electrostatic repulsions. The increase of GG content was conducive to providing more negative charge and promoting the dissolution and dispersion of the MP-GG complexes (MP: GG = 1: 1). Emulsions stabilized by MP-GG complexes exhibited smaller droplet sizes and improved stability compared to those stabilized by MP alone, especially at oil phase volume fractions of 60 % and 70 %. Rheological analysis indicated that GG enhanced emulsion stability by increasing viscosity, and higher oil phase volume fractions facilitated better MP-GG complexes adsorption on oil droplets, strengthening network structures of emulsions. During in vitro simulated gastrointestinal digestion, emulsions with a 70 % oil phase exhibited higher curcumin retention rate (31.09 %) and lower curcumin bioaccessibility (13.23 %) compared to those with a 60 % oil phase. This suggests that emulsions with higher oil phase volume fractions may be more suitable for colon-targeted curcumin delivery, with potential applications in promoting colon health. These findings confirm that the complexation of MP and GG was an effective way to improve the emulsification properties of MP. Emulsions stabilized by MP-GG complexes can serve as stable nutritional delivery systems for fat-soluble bioactive compounds.

Xanthine oxidase inhibitors: Virtual screening and mechanism of inhibition studies

Xanthine oxidase (XO), which plays a key role in purine metabolism, is an important target enzyme for the prevention and treatment of hyperuricemia. Inhibitory activity against XO is a common criterion for the screening of compounds with potential anti-hyperuricemic activity. In this study, 22 XO inhibitors were used to construct a 3D-QSAR pharmacophore model. Subsequently, molecular docking and in vitro activity evaluations were used to identify strong XO inhibitors from a list of 2000 natural compounds. The interaction mechanisms of these compounds with XO were analyzed based on inhibition kinetics and multi-spectral analyses. The pharmacophore model was composed of three hydrogen bond receptors and a hydrophobic center. The screened compounds — Diosmetin, Fisetin, and Genistein — all showed good XO inhibitory activity, with IC50 values of 1.86 ± 0.11 μM, 5.83 ± 0.08 μM, and 7.56 ± 0.10 μM, respectively. Kinetic analysis, fluorescence quenching assays, and molecular docking experiments showed that Diosmetin, Fisetin, and Genistein docked near the same active site of XO, mainly affecting the microenvironment of tryptophan residues. These molecules showed static binding to XO via hydrogen bonds, hydrophobic interactions, and van der Waals forces. Diosmetin and Genistein were competitive inhibitors, whereas Fisetin was a mixed inhibitor. Infrared spectroscopy showed that Diosmetin, Fisetin, and Genistein increased the α-helix content of XO from 7.4 % to 16.6 %, 21.4 %, and 11.2 %, respectively, thereby enhancing its stability. In summary, the pharmacophore model constructed in this study was accurate. The flavonoids Diosmetin, Fisetin, and Genistein effectively inhibited the activity of XO, and the amino acid residues LEU257, ILE353, and VAL259 played a key role in the interaction between the flavonoids and XO. These findings are of great significance for the screening and development of new XO inhibitors.

Functional characterization of CYP96T1-like cytochrome P450 from Lycoris aurea catalyzing para-para′ and para-ortho′ oxidative coupling in Amaryllidaceae alkaloids biosynthesis

Amaryllidaceae alkaloids (AAs) are complex plant secondary metabolites possessing a wide range of biological activities. 4′-O-methylnorbelladine (4OMN) is the branchpoint intermediate for the entire AAs, and was the last common intermediate before AA pathway branches diverge. The cyclization of 4OMN by C-C oxidative coupling, which can afford para-para′, ortho-para′, and para-ortho′ scaffold, was catalyzed by cytochrome P450 96T (CYP96T) family enzymes. To clarify the mechanisms involved in this controversial step, four CYP96T homologs (LauCYP96T1, LauCYP96T1-like-1, LauCYP96T1-like-2 and LauCYP96T1-like-3) were cloned from the full-length transcriptome of Lycoris aurea. All the four LauCYP96T are localized to endoplasmic reticulum. Functional analysis reveals that LauCYP96T1 and LauCYP96T1-like proteins display inverted regioselectivity for oxidative coupling of 4OMN, in which LauCYP96T1 and LauCYP96T1-like-2 dominantly afford para-para′ scaffold, and LauCYP96T1-like-1 and LauCYP96T1-like-3 are responsible for para-ortho′ scaffold formation. Using molecular homology modeling and docking studies, we predicted models for the binding of 4OMN to LauCYP96T, and identified two amino acid residues that might be responsible for the dominant changes in generated products of para-ortho′ and para-para′ oxidative coupling. Our results highlight the functional diversity and promiscuity of LauCYP96T enzymes and might provide valuable information for Amaryllidaceae alkaloid production.

A-031 Development of novel ELISAs for total GDF-15 and H-specific GDF-15

Abstract Background To develop sensitive and specific ELISAs for the quantitative measurement of total human GDF-15 and H-specific (H202D mutant) GDF-15 in human serum, and other biological fluids. Growth/differentiation Factor 15 (GDF-15) is a divergent member of the TGF-β superfamily of growth factors. It is encoded in humans by a gene in chromosome 19. The human GDF-15 gene encodes for a protein of 308 amino acid residues which consists of a signal sequence (residues 1-29), pro-domain (30-194), and mature growth factor domain (195-308). The molecular weight of a mature GDF-15 dimer is 25 kDa. Approximately 25% of humans have a missense polymorphism in the GDF-15 gene resulting in mutation of histidine 202 to aspartate (histidine 6 in the mature domain), close to the N-terminus of the mature growth factor. This variant is associated with phenotypes in prostate cancer, hyperemesis gravidarum, (severe morning sickness in pregnancy), and rheumatoid arthritis. Methods Highly specific and reproducible total GDF-15 (AL-1014-r) and H-specific GDF-15 (AL-1018-r) ELISAs have been developed using specific monoclonal antibodies to help estimate the total and mutant (DD) concentration in serum in the respective assays. The ELISAs use 10 uL sample volume and are calibrated to recombinant human GDF-15 from R & D Systems (Biotechne, USA). These ELISAs were validated for their specificity using HH, DD, HD recombinant preparations and their circulating levels in male, female, 1st, and 2nd-trimester serum samples. Results The Total GDF-15 ELISA assay detects total GDF-15 including histidine 202 to aspartate mutation (HH, HD & DD variant) in the mature domain in equimolar proportions. The H-specific GDF-15 assay is specific to histidine at 202aa in the GDF-15 sequence and does not detect histidine 202 to aspartate mutation (DD variant). Median Levels of total GDF-15 in healthy males (n=18), females (n=17), 1st trimester (n=20), and 2nd trimester (n=20) were 847.9 pg/mL, 1182.4 pg/mL, 12759.7 pg/mL, and 12951.2 pg/mL. Median Levels of H-specific GDF-15 in healthy males (n=18), females (n=17), 1st trimester (n=20), and 2nd trimester (n=20) were 723.2 pg/mL, 641.3 pg/mL, 8381.4 pg/mL and 4652.1 pg/mL. Method comparison between total GDF-15 and commercial GDF-15 assay using serum samples (HH, wild type) in the range of 400-2500 pg/mL yielded a slope of 0.98 (r=0.97). The HD and DD mutant samples in the total assay recovered at twice the concentrations of the commercial assay. The total and intact assays are highly reproducible with the total coefficient of variation less than 7%. Conclusions Use of total GDF-15 assay in combination with the GDF-15 H-specific assay (DD nondetectable) ELISA can accurately estimate the mutant (DD) concentration in serum. This will enhance the knowledge of the GDF-15 measurements in the literature as the commercial assays were compromised for its immunoreactivity to the HD DD genotypes which is highly prevalent.