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Biochemistry Apr 2024Shiga toxin 2a (Stx2a) is the virulence factor of (STEC), which is associated with hemolytic uremic syndrome, the leading cause of pediatric kidney failure. The A1...
Shiga toxin 2a (Stx2a) is the virulence factor of (STEC), which is associated with hemolytic uremic syndrome, the leading cause of pediatric kidney failure. The A1 subunit of Stx2a (Stx2A1) binds to the conserved C-terminal domain (CTD) of the ribosomal P-stalk proteins to remove an adenine from the sarcin-ricin loop (SRL) in the 28S rRNA, inhibiting protein synthesis. There are no antidotes against Stx2a or any other ribosome-inactivating protein (RIP). The structural and functional details of the binding of Stx2A1 to the P-stalk CTD are not known. Here, we carry out a deletion analysis of the conserved P-stalk CTD and show that the last eight amino acids (P8) of the P-stalk proteins are the minimal sequence required for optimal affinity and maximal inhibitory activity against Stx2A1. We determined the first X-ray crystal structure of Stx2A1 alone and in complex with P8 and identified the exact binding site. The C-terminal aspartic acid of the P-stalk CTD serves as an anchor, forming key contacts with the conserved arginine residues at the P-stalk binding pocket of Stx2A1. Although the ricin A subunit (RTA) binds to the P-stalk CTD, the last aspartic acid is more critical for the interaction with Stx2A1, indicating that RIPs differ in their requirements for the P-stalk. These results demonstrate that the catalytic activity of Stx2A1 is inhibited by blocking its interactions with the P-stalk, providing evidence that P-stalk binding is an essential first step in the recruitment of Stx2A1 to the SRL for depurination.
Topics: Humans; Child; Shiga Toxin 2; Ribosomes; Ricin; Aspartic Acid; Binding Sites; Peptides; Escherichia coli
PubMed: 38467020
DOI: 10.1021/acs.biochem.3c00733 -
Biochimie Jul 2024Understanding the diversity of DNA structure and functions in biology requires tools to study this biomolecule selectively and thoroughly. Fluorescence methods are...
Understanding the diversity of DNA structure and functions in biology requires tools to study this biomolecule selectively and thoroughly. Fluorescence methods are powerful technique for non-invasive research. Due to the low quantum yield, the intrinsic fluorescence of nucleotides has not been considered for use in the detection and differentiation of nucleic acid bases. Here, we have studied the influence of protonation of nucleotides on their fluorescence properties. We show that protonation of ATP and GTP leads to enhanced intrinsic fluorescence. Fluorescence enhancement at acidic pH has been observed for double-stranded DNA and single-stranded oligonucleotides. The formation of G4 secondary structures apparently protected certain nucleotides from protonation, resulting in less pronounced fluorescence enhancement. Furthermore, acid-induced depurination under protonation was less noticeable in G4 structures than in double-stranded and single-stranded DNA. We show that changes in the intrinsic fluorescence of guanine can be used as a sensitive sensor for changes in the structure of the DNA and for the protonation of specific nucleotides.
Topics: Guanine; Protons; DNA; Guanosine Triphosphate; Hydrogen-Ion Concentration; Fluorescence; Spectrometry, Fluorescence; DNA, Single-Stranded; Adenosine Triphosphate; Nucleic Acid Conformation; G-Quadruplexes
PubMed: 38447859
DOI: 10.1016/j.biochi.2024.03.003 -
Bioorganic & Medicinal Chemistry Feb 2024Ricin, a category-B agent for bioterrorism, and Shiga toxins (Stxs), which cause food poisoning bind to the ribosomal P-stalk to depurinate the sarcin/ricin loop. No...
Ricin, a category-B agent for bioterrorism, and Shiga toxins (Stxs), which cause food poisoning bind to the ribosomal P-stalk to depurinate the sarcin/ricin loop. No effective therapy exists for ricin or Stx intoxication. Ribosome binding sites of the toxins have not been targeted by small molecules. We previously identified CC10501, which inhibits toxin activity by binding the P-stalk pocket of ricin toxin A subunit (RTA) remote from the catalytic site. Here, we developed a fluorescence polarization assay and identified a new class of compounds, which bind P-stalk pocket of RTA with higher affinity and inhibit catalytic activity with submicromolar potency. A lead compound, RU-NT-206, bound P-stalk pocket of RTA with similar affinity as a five-fold larger P-stalk peptide and protected cells against ricin and Stx2 holotoxins for the first time. These results validate the P-stalk binding site of RTA as a critical target for allosteric inhibition of the active site.
Topics: Binding Sites; Peptides; Protein Binding; Ribosomes; Ricin
PubMed: 38340640
DOI: 10.1016/j.bmc.2024.117614 -
Nucleosides, Nucleotides & Nucleic Acids 2024This work catalogued oligonucleotide sequences and sequence compositions based on the overall yield of full-length product obtained by the phosphoramidite...
This work catalogued oligonucleotide sequences and sequence compositions based on the overall yield of full-length product obtained by the phosphoramidite chemistry-based solid phase synthesis. In total, 76 sequences with different dinucleotide and trinucleotide repeats were synthesized, and the fully-deprotected products were analyzed by denaturing anion exchange HPLC. Overall, sequences containing more 2'-deoxyadenosine residues were obtained in relatively lower yields, likely due to the relative ease of 2'-deoxyadenosine to undergo depurination during the detritylation reaction. Furthermore, dinucleotide steps, such as d(CG)/d(GC) and d(AG)/d(GA), likely contribute the overall lower yields of full-length products as well.
Topics: Solid-Phase Synthesis Techniques; Organophosphorus Compounds; Deoxyribonucleotides; Base Sequence; Oligonucleotides; Chromatography, High Pressure Liquid
PubMed: 38116988
DOI: 10.1080/15257770.2023.2295478 -
Food and Chemical Toxicology : An... Nov 2023Safrole oxide (SAFO), a metabolite of naturally occurring hepatocarcinogen safrole, is implicated in causing DNA adduct formation. Our previous study first detected the...
Dose-response formation of N7-(3-benzo[1,3]dioxol-5-yl-2-hydroxypropyl)guanine in liver and urine correlates with micronucleated reticulocyte frequencies in mice administered safrole oxide.
Safrole oxide (SAFO), a metabolite of naturally occurring hepatocarcinogen safrole, is implicated in causing DNA adduct formation. Our previous study first detected the most abundant SAFO-induced DNA adduct, N7-(3-benzo[1,3] dioxol-5-yl-2-hydroxypropyl)guanine (N7γ-SAFO-G), in mouse urine using a well-developed isotope-dilution high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (ID-HPLC-ESI-MS/MS) method. This study further elucidated the genotoxic mode of action of SAFO in mice treated with SAFO 30, 60, 90, or 120 mg/kg for 28 days. The ID-HPLC-ESI-MS/MS method detected N7γ-SAFO-G with excellent sensitivity and specificity in mouse liver and urine of SAFO-treated mice. Our data provide the first direct evidence of SAFO-DNA adduct formation in rodent tissues. N7γ-SAFO-G levels in liver were significantly increased by SAFO 120 mg/kg compared with SAFO 30 mg/kg, suggesting rapid spontaneous or enzymatic depurination of N7γ-SAFO-G in tissue DNA. Urinary N7γ-SAFO-G exhibited a sublinear dose response. Moreover, the micronucleated peripheral reticulocyte frequencies increased dose-dependently and significantly correlated with N7γ-SAFO-G levels in liver (r = 0.8647; p < 0.0001) and urine (r = 0.846; p < 0.0001). Our study suggests that safrole-mediated genotoxicity may be caused partly by its metabolic activation to SAFO and that urinary N7γ-SAFO-G may serve as a chemically-specific cancer risk biomarker for safrole exposure.
Topics: Mice; Animals; DNA Adducts; Safrole; Tandem Mass Spectrometry; Spectrometry, Mass, Electrospray Ionization; Guanine; Reticulocytes; Liver; Chromatography, High Pressure Liquid
PubMed: 37739051
DOI: 10.1016/j.fct.2023.114056 -
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 -
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 -
Nucleic Acids Research Oct 2023Xeno-nucleic acids (XNAs) have gained significant interest as synthetic genetic polymers for practical applications in biomedicine, but very little is known about their...
Xeno-nucleic acids (XNAs) have gained significant interest as synthetic genetic polymers for practical applications in biomedicine, but very little is known about their biophysical properties. Here, we compare the stability and mechanism of acid-mediated degradation of α-l-threose nucleic acid (TNA) to that of natural DNA and RNA. Under acidic conditions and elevated temperature (pH 3.3 at 90°C), TNA was found to be significantly more resistant to acid-mediated degradation than DNA and RNA. Mechanistic insights gained by reverse-phase HPLC and mass spectrometry indicate that the resilience of TNA toward low pH environments is due to a slower rate of depurination caused by induction of the 2'-phosphodiester linkage. Similar results observed for 2',5'-linked DNA and 2'-O-methoxy-RNA implicate the position of the phosphodiester group as a key factor in destabilizing the formation of the oxocarbenium intermediate responsible for depurination and strand cleavage of TNA. Biochemical analysis indicates that strand cleavage occurs by β-elimination of the 2'-phosphodiester linkage to produce an upstream cleavage product with a 2'-threose sugar and a downstream cleavage product with a 3' terminal phosphate. This work highlights the unique physicochemical properties available to evolvable non-natural genetic polymers currently in development for biomedical applications.
PubMed: 37650628
DOI: 10.1093/nar/gkad716 -
National Science Review Aug 2023Current DNA base editors contain nuclease and DNA deaminase that enables deamination of cytosine (C) or adenine (A), but no method for guanine (G) or thymine (T) editing...
Current DNA base editors contain nuclease and DNA deaminase that enables deamination of cytosine (C) or adenine (A), but no method for guanine (G) or thymine (T) editing is available at present. Here we developed a deaminase-free glycosylase-based guanine base editor (gGBE) with G editing ability, by fusing Cas9 nickase with engineered N-methylpurine DNA glycosylase protein (MPG). By several rounds of MPG mutagenesis via unbiased and rational screening using an intron-split EGFP reporter, we demonstrated that gGBE with engineered MPG could increase G editing efficiency by more than 1500 fold. Furthermore, this gGBE exhibited high base editing efficiency (up to 81.2%) and high G-to-T or G-to-C (i.e. G-to-Y) conversion ratio (up to 0.95) in both cultured human cells and mouse embryos. Thus, we have provided a proof-of-concept of a new base editing approach by endowing the engineered DNA glycosylase the capability to selectively excise a new type of substrate.
PubMed: 37404457
DOI: 10.1093/nsr/nwad143 -
Gene Aug 2023Ribosome-inactivating proteins (RIPs) are RNA N-glycosidases that depurinate an adenine residue in the conserved alpha-sarcin/ricin loop (SRL) of rRNA, inhibiting...
Ribosome-inactivating proteins (RIPs) are RNA N-glycosidases that depurinate an adenine residue in the conserved alpha-sarcin/ricin loop (SRL) of rRNA, inhibiting protein synthesis. Previously, we reported the existence of these toxins in insects, whose presence is restricted to mosquitoes from the Culicinae subfamily (e.g., Aedes aegypti) and whiteflies from the Aleyrodidae family (e.g., Bemisia tabaci). Both groups of genes are derived from two independent horizontal gene transfer (HGT) events and are evolving under purifying selection. Here, we report and characterize the occurrence of a third HGT event in the Sciaroidea superfamily, which supports the recurrent acquisition of RIP genes by insects. Transcriptomic experiments, available in databases, allowed us to describe the temporal and spatial expression profiles for these foreign genes in these organisms. Furthermore, we found that RIP expression is induced after infection with pathogens and provided, for the first time, transcriptomic evidence of parasite SRL depurination. This evidence suggests a possible role of these foreign genes as immune effectors in insects.
Topics: Animals; Ribosome Inactivating Proteins; Gene Transfer, Horizontal; Insecta; Protein Biosynthesis; RNA, Ribosomal; Ricin; Hemiptera; Plant Proteins
PubMed: 37286020
DOI: 10.1016/j.gene.2023.147547