-
Se Pu = Chinese Journal of... Mar 2021Type Ⅱ ribosome-inactivating proteins (RIPs) are an important class of protein toxins that consist of A and B chains linked by an interchain disulfide bond. The... (Review)
Review
Type Ⅱ ribosome-inactivating proteins (RIPs) are an important class of protein toxins that consist of A and B chains linked by an interchain disulfide bond. The B-chain with lectin-like activity is responsible for binding to the galactose-containing receptors on eukaryotic cell surfaces, which is essential for A-chain internalization by endocytosis. The A-chain has -glycosidase activity that irreversibly depurinates a specific adenine from 28S ribosomal RNA (28S rRNA) and terminates protein synthesis. The synergistic effect of the A-B chain inactivates the ribosome, inhibits protein synthesis, and exhibits high cytotoxicity. Ricin and abrin that are expressed by the plants and , respectively, are typical type Ⅱ RIPs. The toxicity of ricin and abrin are 385 times and 2885 times, respectively, more that of the nerve agent VX. Owing to their ease of preparation, wide availability, and potential use as a bioterrorism agent, type Ⅱ RIPs have garnered increasing attention in recent years. Ricin is listed as a prohibited substance under schedule 1A of the Chemical Weapons Convention (CWC). The occurrence of ricin-related bioterrorism incidents in recent years has promoted the development of accurate, sensitive, and rapid detection and identification technology for type Ⅱ RIPs. Significant progress has been made in the study of toxicity mechanisms and detection methods of type Ⅱ RIPs, which primarily involve qualitative and quantitative analysis methods including immunological assays, mass spectrometry analysis methods, and toxin activity detection methods based on depurination and cytotoxicity. Immunoassays generally involve the specific recognition of antigens and antibodies, which is based on oligonucleotide molecular recognition elements called aptamers. These methods are fast and highly sensitive, but for highly homologous proteins in complex samples, they provide false positive results. With the rapid development of biological mass spectrometry detection technology, techniques such as electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) are widely used in the identification of proteins. These methods not only provide accurate information on molecular weight and structure of proteins, but also demonstrate accurate quantification. Enzyme digestion combined with mass spectrometry is the predominantly used detection method. Accurate identification of protein toxins can be achieved by fingerprint analysis of enzymatically digested peptides. For analysis of protein toxins in complex samples, abundant peptide markers are obtained using a multi-enzyme digestion strategy. Targeted mass spectrometry analysis of peptide markers is used to obtain accurate qualitative and quantitative information, which effectively improves the accuracy and sensitivity of the identification of type Ⅱ RIP toxins. Although immunoassay and mass spectrometry detection methods can provide accurate identification of type Ⅱ RIPs, they cannot determine whether the toxins will retain potency. The widely used detection methods for activity analysis of type Ⅱ RIPs include depurination assay based on -glycosidase activity and cytotoxicity assay. Both the methods provide simple, rapid, and sensitive analysis of type Ⅱ RIP toxicity, and complement other detection methods. Owing to the importance of type Ⅱ RIP toxins, the Organization for the Prohibition of Chemical Weapons (OPCW) has proposed clear technical requirements for the identification and analysis of relevant samples. We herein reviewed the structural characteristics, mechanism of action, and the development and application of type Ⅱ RIP detection methods; nearly 70 studies on type Ⅱ RIP toxins and their detection methods have been cited. In addition to the technical requirements of OPCW for the unambiguous identification of biotoxins, the trend of future development of type Ⅱ RIP-based detection technology has been explored.
Topics: Abrin; Plant Proteins; Ribosome Inactivating Proteins; Ribosomes; Ricin; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
PubMed: 34227307
DOI: 10.3724/SP.J.1123.2020.10001 -
Journal of Chromatography. B,... Jan 2023The exact levels of some DNA adducts, like N7-deoxyguanosine (N7-dG), can be under-calculated since these adducts may depurinate due to their chemical instability,...
The exact levels of some DNA adducts, like N7-deoxyguanosine (N7-dG), can be under-calculated since these adducts may depurinate due to their chemical instability, leading to corresponding nucleobase adducts being released into the cytoplasm. To accurately quantify the levels of DNA adducts, it is necessary to consider those modified nucleobases. However, high levels and diversity of cytoplasmic small molecule metabolites (SMMs) can strongly interfere with the detection of adducts, and it is almost impossible to remove them with nucleobase adducts being well retained. Therefore, we aimed to establish an optimized enrichment method based on solid-phase extraction (SPE) to reduce the co-elution of SMMs with target analytes. In this vein, we employed three bisphenols (BPA, BPF, and BPAF) as examples, prepared corresponding N7-guanine (N7-Gua) adducts, loaded on an Oasis hydrophilic-lipophilic balance ® (HLB) cartridge, used a series of mobile phases containing different percentage of methanol for elution, and evaluated the levels of these adducts in each eluent. First, we found that neutral samples led to the best retention for all three adducts compared with acidified or basified ones. We next employed normal distribution fitting model to characterize the elution of analytes from HO/methanol with different pHs and observed that neutral mobile phases resulted in more hydrophobic elution for all three adducts. Besides, N7-BPA-Gua and N7-BPF-Gua obtained narrow fitted peaks at neutral pH, while N7-BPAF-Gua had minimized elution windows at low pH. After optimization, we exposed 293T cells to the aforementioned bisphenols and quantified the N7-Gua adducts in the cytoplasm and the corresponding N7-dG adducts in genomic DNA. The results revealed that with the same levels of BPs exposure, BPAF led to the highest levels of adducts in both cytoplasm and genomic DNA samples, followed by BPA and BPF in order. In summary, our research established an appropriate model to describe the elution of DNA adducts in the SPE, applied it to optimize the loading and elution conditions, and discussed the genotoxicity of bisphenols by accurate quantification of both cleaved and uncleaved N7-dG adducts.
Topics: DNA Adducts; Methanol; DNA; Guanine; Indicators and Reagents; Cytoplasm; Solid Phase Extraction; Genomics
PubMed: 36586340
DOI: 10.1016/j.jchromb.2022.123574 -
International Journal of Molecular... Sep 2022The application of oligonucleotides as drugs for different genetic diseases is increasing rapidly. Since 2016 they are used during spinal muscular atrophy treatment with...
The application of oligonucleotides as drugs for different genetic diseases is increasing rapidly. Since 2016 they are used during spinal muscular atrophy treatment with the use of nusinersen oligonucleotide. The purpose of this study was to improve methods for the analysis of serum samples of patients treated with nusinersen. The results showed that liquid-liquid extraction (with phenol/chloroform) is insufficient and an additional purification step using solid-phase extraction is necessary. The best results were obtained for microextraction by packed sorbents. Important parameters in the optimization of the method were mainly the type of amine in the mobile phase and the stationary phase. Both influenced the selectivity of metabolite separation and thus their correct identification; while amine type impacted also the intensity of signals. Finally, the highest resolution of separation and the highest peak areas were obtained for ,-dimethylbutylamine or ,-diisopropylthylamine with an octadecyl column with a terminal aryl group. Over a dozen of metabolites were successfully identified with the use of methods developed during the study. The 3' exonucleases and 5' exonucleases were mainly responsible for nusinersen metabolism, consequently, 3'end shortmers, and 5'end shortmers were observed, as well as metabolites with simultaneous loss of bases at both ends of the sequence. However, some depurination and depyrimidination products were also identified. To the best of our knowledge, this is the first report on nusinersen and its metabolite identification in serum samples by liquid chromatography and mass spectrometry.
Topics: Amines; Child; Exonucleases; Humans; Muscular Atrophy, Spinal; Oligonucleotides
PubMed: 36077568
DOI: 10.3390/ijms231710166 -
Extremophiles : Life Under Extreme... Dec 2022Archaea and bacteria in geothermal environments are predicted to suffer DNA depurination in vivo at high rates, which raises questions regarding the biological roles of...
Archaea and bacteria in geothermal environments are predicted to suffer DNA depurination in vivo at high rates, which raises questions regarding the biological roles of their abasic-site-repair enzymes. Gene deletion and enzymatic assay demonstrated that the saci_0015 gene of Sulfolobus acidocaldarius encodes an AP endonuclease (Apn) accounting for as much as 95% of the assayable activity in cell extracts and is not essential for viability. To identify genetic functions of this enzyme, deletion (ΔApn) strains were examined with respect to growth, spontaneous mutation, transformation by ssDNA containing an abasic site, and conjugation. Relative to its isogenic control, the ΔApn strain did not exhibit any change in growth rate or final cell density, rate or spectrum of spontaneous mutation, transformation by DNA containing an abasic site, or efficiency of DNA transfer and recombination. The apparent lack of genetic impact of removing the major AP endonuclease was unexpected and indicated that abasic sites are rarely bypassed directly by DNA polymerases in S. acidocaldarius. AP endonuclease deficiency had no obvious effect on survival of S. acidocaldarius under several test conditions, but it accelerated the death of cells at 4º C under illumination. Our results suggest that the normal level of AP endonuclease in S. acidocaldarius is well above the minimum required for growth and cell division but not for recovery from prolonged exposure to certain low-temperature conditions. This situation illustrates a biological challenge that has not been emphasized in experimental studies of extremophiles, i.e., the problem of long-term survival under "non-extreme" conditions.
Topics: Archaea; Endonucleases; DNA-(Apurinic or Apyrimidinic Site) Lyase; Extremophiles; Cell Division
PubMed: 36456889
DOI: 10.1007/s00792-022-01286-9 -
Scientific Reports Sep 2020Ribosome inactivating proteins (RIPs) are RNA N-glycosidases that depurinate a specific adenine residue in the conserved sarcin/ricin loop of 28S rRNA. These enzymes...
Ribosome inactivating proteins (RIPs) are RNA N-glycosidases that depurinate a specific adenine residue in the conserved sarcin/ricin loop of 28S rRNA. These enzymes are widely distributed among plants and bacteria. Previously, we have described for the first time RIP genes in mosquitoes belonging to the Culicidae family. We showed that these genes are derived from a single event of horizontal gene transfer (HGT) from a prokaryotic donor. Mosquito RIP genes are evolving under purifying selection, strongly suggesting that these toxins have acquired a functional role. In this work, we show the existence of two RIP encoding genes in the genome of the whitefly Bemisia tabaci, a hemiptera species belonging to the Aleyrodidae family distantly related to mosquitoes. Contamination artifacts were ruled out analyzing three independent B. tabaci genome databases. In contrast to mosquito RIPs, whitefly genes harbor introns and according to transcriptomic evidence are transcribed and spliced. Phylogeny and the taxonomic distribution strongly support that whitefly RIP genes are derived from an independent HGT event from a plant source. These results, along with our previous description of RIPs in Diptera, suggest that the acquired genes are functional in these insects and confer some fitness advantage.
Topics: Animals; Gene Expression Profiling; Gene Transfer, Horizontal; Genes, Insect; Genes, Plant; Genome, Insect; Hemiptera; Phylogeny; RNA, Ribosomal, 28S; Ribosome Inactivating Proteins; Selection, Genetic; Sequence Alignment; Sequence Analysis, DNA
PubMed: 32968092
DOI: 10.1038/s41598-020-72267-1 -
Analytical Biochemistry Apr 2020A new method is presented for the automated determination of early eluting (E.E.) oligonucleotide impurities analyzed by IP-RP HPLC HRMS. E.E. are impurities shorter...
A new method is presented for the automated determination of early eluting (E.E.) oligonucleotide impurities analyzed by IP-RP HPLC HRMS. E.E. are impurities shorter than the main drug component and are primarily formed by the mechanisms of coupling failure, and depurination. The method is based on the detection of the theoretically derived most abundant mass of an impurity in the experimental data. An exhaustive list of candidate impurities and their formulas is automatically generated using the parent sequence and the known mechanisms of impurity formation. The approach accounts for possible modifications in the individual oligonucleotide sequence moieties (e.g., linkage, sugar, and base, 3', and 5' ends). The detected ion signal is summed to provide four nested levels of impurity breakdown information. The approach allows for the rapid determination of relationships and trends of impurities in samples generated by different manufacturing processes and conditions. Representative examples are given to illustrate the capabilities and utility of the approach in synthesis and purification process optimization applications.
Topics: Automation; Carbohydrate Conformation; Chromatography, High Pressure Liquid; Chromatography, Reverse-Phase; Drug Contamination; Oligonucleotides
PubMed: 32067983
DOI: 10.1016/j.ab.2020.113623 -
The Analyst Jun 2024The study aimed to analyze nusinersen metabolites in the cerebrospinal fluid samples using ion-pair reversed-phase ultrahigh-performance liquid chromatography coupled...
Study of nusinersen metabolites in the cerebrospinal fluid of children with spinal muscular atrophy using ultra-high-performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry.
The study aimed to analyze nusinersen metabolites in the cerebrospinal fluid samples using ion-pair reversed-phase ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. Three different sample preparation methods were tested for extraction and purification, but solid phase extraction appeared to be the most suitable, allowing a significant sample enrichment (40-fold). This step was necessary to detect and identify metabolites of nusinersen in the cerebrospinal fluid. The developed and applied analytical procedure enabled the identification of nusinersen metabolites: sequences shorter by several nucleotides from the 3' end; shorter by several nucleotides from both the 3' and 5' ends; and some depurination products. To the best of our knowledge, this is the first report on the analysis and identification of nusinersen metabolites in cerebrospinal fluid samples taken from children with spinal muscular atrophy treated with Spinraza.
PubMed: 38828890
DOI: 10.1039/d4an00436a -
Toxicology Letters May 2024Ricin toxin (RT) is highly cytotoxic and can release a considerable amount of pro-inflammatory factors due to depurination, causing excessive inflammation that may...
Ricin toxin (RT) is highly cytotoxic and can release a considerable amount of pro-inflammatory factors due to depurination, causing excessive inflammation that may aggravate the harm to the body. Pyroptosis, a type of gasdermin-mediated cell death, is a contributor to the exacerbation of inflammation. Accumulating evidence indicate that pyroptosis plays a significant role in the pathogen infection and tissue injury, suggesting a potential correlation between pyroptosis and RT-induced inflammation. Here, we aim to demonstrate this correlation and explore its molecular mechanisms. Results showed that RT triggers mouse alveolar macrophage MH-S cells pyroptosis by activating caspase-3 and cleaving Gasgermin E (GSDME). In contrast, inhibition of caspase-3 with Z-DEVD-FMK (inhibitor of caspase-3) or knockdown of GSDME attenuates this process, suggesting the essential role of caspase-3/GSDME-mediated pyroptosis in contributing to RT-induced inflammation. Collectively, our study enhances our understanding of a novel mechanism of ricin cytotoxicity, which may emerge as a potential target in immunotherapy to control the RT-induced inflammation.
Topics: Pyroptosis; Ricin; Animals; Mice; Caspase 3; Inflammation; Cell Line; Macrophages, Alveolar; Gasdermins
PubMed: 38642674
DOI: 10.1016/j.toxlet.2024.04.007 -
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 -
Toxins Mar 2021International authorities classify ricin toxin present in castor seed as a potential agent for use in bioterrorism. Therefore, the detection, identification, and...
International authorities classify ricin toxin present in castor seed as a potential agent for use in bioterrorism. Therefore, the detection, identification, and characterization of ricin in various sample matrices are considered necessary actions for risk assessment during a suspected exposure. This study reports a portable electrochemical assay for detecting active ricin based on the adenine electro-oxidation released from herring sperm DNA substrate by its catalytic action. Also, kinetic parameters were calculated, and the values were of 3.14 µM and 2107 min. A linear response was found in optimized experimental conditions for ricin concentrations ranging from 8 to 120 ng/mL, and with a detection limit of 5.14 ng/mL. This proposed detection strategy emphasizes the possibility of field detection of active ricin in food matrices and can be applied to other endonucleolytic activities.
Topics: Adenine; Animals; DNA; Electrochemical Techniques; Fishes; Kinetics; Male; Reproducibility of Results; Ricin; Spermatozoa; Substrate Specificity
PubMed: 33810228
DOI: 10.3390/toxins13040238