-
Journal of Chromatography. A Oct 2023Ricin is a highly toxic protein toxin that poses a potential bioterrorism threat due to its potency and widespread availability. However, the accurate quantification of...
A protein standard absolute quantification strategy for enhanced absolute quantification of ricin in complex matrices using in vitro synthesized mutant holoprotein as internal standard by ultra-high-performance liquid chromatography-tandem mass spectrometry.
Ricin is a highly toxic protein toxin that poses a potential bioterrorism threat due to its potency and widespread availability. However, the accurate quantification of ricin through absolute mass spectrometry (MS) using a protein standard absolute quantification (PSAQ) strategy is not widely practiced. This limitation primarily arises from the presence of interchain disulfide bonds, which hinder the production of full-length isotope-labeled ricin as an internal standard (IS) in vitro. In this study, we have developed a novel approach for the absolute quantification of ricin in complex matrices using recombinant single-chain and full-length mutant ricin as the protein IS, instead of isotope-labeled ricin, in conjunction with ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The amino acid sequence of the ricin mutant internal standard (RMIS) was designed by introducing site mutations in specific amino acids of trypsin/Glu-C enzymatic digestion marker peptides of ricin. To simplify protein expression, the A-chain and B-chain of RMIS were directly linked to replace the original interchain disulfide bonds. The RMISs were synthesized using an Escherichia coli expression system. An appropriate RMIS was selected as the protein IS based on consistent digestion efficiency, UHPLC-MS/MS behavior, antibody recognition function, lectin activity, and proper depurination activity with intact ricin. The RMIS was utilized to simultaneously quantify A- and B-chain marker peptides of ricin through UHPLC-MS/MS. This method was thoroughly validated using a milk matrix. By employing internal protein standards, this quantitative strategy overcomes the challenges posed by variations in extraction recoveries, matrix effects, and digestion efficiency encountered when working with different matrices. Consequently, calibration curves generated from milk matrix-spiked samples were utilized to accurately and precisely quantify ricin in river water and plasma samples. Moreover, the established method successfully detected intact ricin in samples obtained from the sixth Organization for the Prohibition of Chemical Weapons (OPCW) exercise on biotoxin analysis. This study presents a novel PSAQ strategy that enables the accurate quantification of ricin in complex matrices.
Topics: Tandem Mass Spectrometry; Chromatography, High Pressure Liquid; Ricin; Amino Acid Sequence; Escherichia coli; Disulfides
PubMed: 37717454
DOI: 10.1016/j.chroma.2023.464373 -
Bioscience Reports Oct 2019Ricin interacts with the ribosomal P stalk to cleave a conserved adenine from the α-sarcin/ricin loop (SRL) of the rRNA. Ricin toxin A chain (RTA) uses Arg235 as the...
Ricin interacts with the ribosomal P stalk to cleave a conserved adenine from the α-sarcin/ricin loop (SRL) of the rRNA. Ricin toxin A chain (RTA) uses Arg235 as the most critical arginine for binding to the P stalk through electrostatic interactions to facilitate depurination. Structural analysis showed that a P2 peptide binds to a hydrophobic pocket on RTA and the last two residues form hydrogen bonds with Arg235. The importance of hydrophobic residues relative to Arg235 in the interaction with the P stalk in vivo and on the toxicity of RTA is not known. Here, we mutated residues in the hydrophobic pocket to analyze their contribution to toxicity and depurination activity in yeast and in mammalian cells. We found that Leu232, Tyr183 and Phe240 contribute cumulatively to toxicity, with Leu232 being the most significant. A quadruple mutant, Y183A/L232A/R235A/F240A, which combined mutations in critical hydrophobic residues with R235A completely abolished the activity of RTA, indicating that Arg235 and hydrophobic residues are required for full biological activity. Y183A and F240A mutants had reduced activity on RNA, but higher activity on ribosomes compared with R235A in vitro, suggesting that they could partially regain activity upon interaction with ribosomes. These results expand the region of interaction between RTA and the P stalk critical for cellular activity to include the hydrophobic pocket and provide the first evidence that interaction of P stalk with the hydrophobic pocket promotes a conformational rearrangement of RTA to correctly position the active site residues for catalytic attack on the SRL.
Topics: Binding Sites; Hydrophobic and Hydrophilic Interactions; Leucine; Ribosomes; Ricin; Saccharomyces cerevisiae
PubMed: 31548364
DOI: 10.1042/BSR20192022 -
AMB Express May 2024Ribosome-inactivating proteins (RIPs) are highly active N-glycosidases that depurinate both bacterial and eukaryotic rRNAs, halting protein synthesis during translation....
Ribosome-inactivating proteins (RIPs) are highly active N-glycosidases that depurinate both bacterial and eukaryotic rRNAs, halting protein synthesis during translation. Found in a diverse spectrum of plant species and tissues, RIPs possess antifungal, antibacterial, antiviral, and insecticidal properties linked to plant defense. In this study, we investigated the physiochemical properties of RIP peptides from the Cucurbitaceae family through bioinformatics approaches. Molecular weight, isoelectric point, aliphatic index, extinction coefficient, and secondary structures were analyzed, revealing their hydrophobic nature. The novelty of this work lies in the comprehensive examination of RIPs from the Cucurbitaceae family and their potential therapeutic applications. The study also elucidated the binding interactions of Cucurbitaceae RIPs with key biological targets, including Interleukin-6 (IL-6). Strong hydrogen bond interactions between RIPs and these targets suggest potential for innovative insilico drug design and therapeutic applications, particularly in cancer treatment. Comprehensive analysis of bond lengths using Ligpolt + software provides insights for optimizing molecular interactions, offering a valuable tool for drug design and structural biology studies.
PubMed: 38801471
DOI: 10.1186/s13568-024-01718-z -
Chemical Science Dec 2019DNA-encoded compound libraries are a widely used technology for target-based small molecule screening. Generally, these libraries are synthesized by solution phase...
DNA-encoded compound libraries are a widely used technology for target-based small molecule screening. Generally, these libraries are synthesized by solution phase combinatorial chemistry requiring aqueous solvent mixtures and reactions that are orthogonal to DNA reactivity. Initiating library synthesis with readily available controlled pore glass-coupled DNA barcodes benefits from enhanced DNA stability due to nucleobase protection and choice of dry organic solvents for encoded compound synthesis. We screened the compatibility of solid-phase coupled DNA sequences with 53 metal salts and organic reagents. This screening experiment suggests design of encoded library synthesis. Here, we show the reaction optimization and scope of three sp-bond containing heterocyclic scaffolds synthesized on controlled pore glass-connected DNA sequences. A ZnCl-promoted aza-Diels-Alder reaction with Danishefsky's diene furnished diverse substituted DNA-tagged pyridones, and a phosphoric acid organocatalyst allowed for synthesis of tetrahydroquinolines by the Povarov reaction and pyrimidinones by the Biginelli reaction, respectively. These three reactions caused low levels of DNA depurination and cover broad and only partially overlapping chemical space though using one set of DNA-coupled starting materials.
PubMed: 32055372
DOI: 10.1039/c9sc04708e -
Analytical Chemistry Sep 2019Hydrolysis of N3'-P5' phosphoramidate and thiophosphoramidate oligonucleotides with 0.1% formic acid leads to the cleavage of the 3' N-P bond and generates two products,...
Hydrolysis of N3'-P5' phosphoramidate and thiophosphoramidate oligonucleotides with 0.1% formic acid leads to the cleavage of the 3' N-P bond and generates two products, one of which contains a 5'-phosphate. Analysis of the hydrolytic products by liquid chromatography, coupled with mass spectrometry, reveals the mass ladder from both termini, which is used to determine the sequence. While acid hydrolysis does not result in depurination, internal fragments especially in the low mass range are detected. The method is applied to DNA and RNA analogues with and without modifications at the 2'-position. This approach enables rapid sequence confirmation of synthetic phosphoramidate oligonucleotides for quality control as well as denovo sequencing.
Topics: Amides; Formates; Hydrolysis; Mass Spectrometry; Oligonucleotides; Phosphoric Acids
PubMed: 31386344
DOI: 10.1021/acs.analchem.9b01858 -
Ecology and Evolution Jan 2021Ancient DNA research has developed rapidly over the past few decades due to improvements in PCR and next-generation sequencing (NGS) technologies, but challenges still...
Ancient DNA research has developed rapidly over the past few decades due to improvements in PCR and next-generation sequencing (NGS) technologies, but challenges still exist. One major challenge in relation to ancient DNA research is to recover genuine endogenous ancient DNA sequences from raw sequencing data. This is often difficult due to degradation of ancient DNA and high levels of contamination, especially homologous contamination that has extremely similar genetic background with that of the real ancient DNA. In this study, we collected whole-genome sequencing (WGS) data from 6 ancient samples to compare different mapping algorithms. To further explore more effective methods to separate endogenous DNA from homologous contaminations, we attempted to recover reads based on ancient DNA specific characteristics of deamination, depurination, and DNA fragmentation with different parameters. We propose a quick and improved pipeline for separating endogenous ancient DNA while simultaneously decreasing homologous contaminations to very low proportions. Our goal in this research was to develop useful recommendations for ancient DNA mapping and for separation of endogenous DNA to facilitate future studies of ancient DNA.
PubMed: 33437437
DOI: 10.1002/ece3.7056 -
Biomolecular NMR Assignments Apr 2020Trichobakin (TBK) is a type-I ribosome-inactivating protein (RIP-I), acting as an extremely potent inhibitor of protein synthesis in the cell-free translation system of...
Trichobakin (TBK) is a type-I ribosome-inactivating protein (RIP-I), acting as an extremely potent inhibitor of protein synthesis in the cell-free translation system of rabbit reticulocyte lysate (IC: 3.5 pM). In this respect, TBK surpasses the well-studied highly homologous RIP-I trichosanthin (IC: 20-27 pM), therefore creation of recombinant toxins based on it is of great interest. TBK needs to penetrate into cytosol through the cell membrane and specifically bind to α-sarcin/ricin loop of 28S ribosome RNA to perform the function of specific RNA depurination. At the moment, there is no detailed structural-dynamic information in solution about diverse states RIP-I can adopt at different stages on the way to protein synthesis inhibition. In this work, we report a near-complete assignment of H, C, and N TBK (27.3 kDa) resonances and analysis of the secondary structure based on the experimental chemical shifts data. This work will serve as a basis for further investigations of the structure, dynamics and interactions of the TBK with its molecular partners using NMR techniques.
Topics: Carbon-13 Magnetic Resonance Spectroscopy; N-Glycosyl Hydrolases; Nuclear Magnetic Resonance, Biomolecular; Plant Proteins; Protein Structure, Secondary; Proton Magnetic Resonance Spectroscopy; Ribosomes
PubMed: 31734904
DOI: 10.1007/s12104-019-09920-y -
ACS Infectious Diseases Jun 2024Shiga toxins are the main virulence factors of Shiga toxin producing (STEC) and . There is no effective therapy to counter the disease caused by these toxins. The A1...
Shiga toxins are the main virulence factors of Shiga toxin producing (STEC) and . There is no effective therapy to counter the disease caused by these toxins. The A1 subunits of Shiga toxins bind the C-termini of ribosomal P-stalk proteins to depurinate the sarcin/ricin loop. The ribosome binding site of Shiga toxin 2 has not been targeted by small molecules. We screened a fragment library against the A1 subunit of Shiga toxin 2 (Stx2A1) and identified a fragment, , which bound at the ribosome binding site and mimicked the binding mode of the P-stalk proteins. We synthesized analogs of and identified a series of molecules with similar affinity and inhibitory activity. These are the first compounds that bind at the ribosome binding site of Stx2A1 and inhibit activity. These compounds hold great promise for further inhibitor development against STEC infection.
PubMed: 38873918
DOI: 10.1021/acsinfecdis.4c00224 -
Journal of Hazardous Materials Jun 2022Methoxychlor (MXC) is an organopesticide classified as a "Proposed Persistent Organic Pollutant" in the Stockholm Convention, and recent studies revealed that MXC could...
Methoxychlor (MXC) is an organopesticide classified as a "Proposed Persistent Organic Pollutant" in the Stockholm Convention, and recent studies revealed that MXC could induce DNA strand breaks, whereas its underlying mechanisms were underinvestigated. Here, we first reported that hydroxymethoxychlor (HPTE), one of MXC's active metabolites, could be oxidized in vivo to form quinone intermediate, which attacked N7 position of 2'-deoxyguanosine to form N7-HPTE-deoxyguanosine (N7-HPTE-dG), followed by depurination to produce N7-HPTE-guanine (N7-HPTE-Gua) in MXC-treated mammalian cells and tissues from mice fed with MXC, employing an ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) method. We observed a positive correlation between the doses of MXC exposure and the levels of N7-HPTE-Gua and N7-HPTE-dG in cytoplasm and genomic DNA, respectively. Furthermore, after removal of exogenous MXC, the amount of genomic N7-HPTE-dG was significantly decreased during 24 h, while the level of cytoplasmic N7-HPTE-Gua was elevated during first 12 h, indicating the accumulation of the N7-HPTE-Gua in cells. Additionally, for animal experiment, genomic N7-HPTE-dG was observed in livers and cortexes from female C57BL/6 mice fed with MXC, suggesting a potential mechanism of its hepatoxicity and neurotoxicity. Overall, our study provides new understanding about the formation of MXC-induced DNA adducts in mammalian cells and animal models.
Topics: Animals; DNA Adducts; Deoxyguanosine; Female; Mammals; Methoxychlor; Mice; Mice, Inbred C57BL; Persistent Organic Pollutants; Phenols; Tandem Mass Spectrometry
PubMed: 35349845
DOI: 10.1016/j.jhazmat.2022.128741 -
IScience Sep 2023High-power screening (HPS) technologies, such as DNA-encoded library (DEL) technology, could exponentially increase the dimensions of the chemical space accessible for...
High-power screening (HPS) technologies, such as DNA-encoded library (DEL) technology, could exponentially increase the dimensions of the chemical space accessible for drug discovery. The intrinsic fragile nature of DNA is associated with cumbersome limitations and DNA durability (e.g., depurination, loss of phosphate groups, adduct formation) is compromised in numerous organic chemistry conditions that require empirical testing. An atlas of reaction conditions (temperature, pH, solvent/buffer, ligands, oxidizing reagents, catalysts, scavengers in function of time) that have been systematically tested in multiple combinations, indicates precisely limits useful for DEL construction. More importantly, this approach could be used broadly to effectively evaluate DNA-compatibility of any novel on-DNA chemical reaction, and it is compatible with different molecular methodologies. This atlas and the general approach presented, by allowing novel reaction conditions to be performed in presence of DNA, should greatly help in expanding the DEL chemical space as well as any field involving DNA durability.
PubMed: 37664608
DOI: 10.1016/j.isci.2023.107573