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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 -
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 -
Analytical Biochemistry Sep 2024Ricin is one of the most toxic substances known and a type B biothreat agent. Shiga toxins (Stxs) produced by E. coli (STEC) and Shigella dysenteriae are foodborne...
Ricin is one of the most toxic substances known and a type B biothreat agent. Shiga toxins (Stxs) produced by E. coli (STEC) and Shigella dysenteriae are foodborne pathogens. There is no effective therapy against ricin or STEC and there is an urgent need for inhibitors. Ricin toxin A subunit (RTA) and A1 subunit of Stx2a (Stx2A1) bind to the C-terminal domain (CTD) of the ribosomal P-stalk proteins to depurinate the sarcin/ricin loop. Modulation of toxin-ribosome interactions has not been explored as a strategy for inhibition. Therefore, development of assays that detect inhibitors targeting toxin-ribosome interactions remains a critical need. Here we describe a fluorescence anisotropy (FA)-based competitive binding assay using a BODIPY-TMR labeled 11-mer peptide (P11) derived from the P-stalk CTD to measure the binding affinity of peptides ranging from 3 to 11 amino acids for the P-stalk pocket of RTA and Stx2A1. Comparison of the affinity with the surface plasmon resonance (SPR) assay indicated that although the rank order was the same by both methods, the FA assay could differentiate better between peptides that show nonspecific interactions by SPR. The FA assay detects only interactions that compete with the labeled P11 and can validate inhibitor specificity and mechanism of action.
Topics: Ricin; Fluorescence Polarization; Ribosomes; Surface Plasmon Resonance; Shiga Toxin; Binding, Competitive; Protein Binding; Shiga Toxin 2
PubMed: 38825159
DOI: 10.1016/j.ab.2024.115580 -
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 -
Nucleosides, Nucleotides & Nucleic Acids May 2024The -methyladenosine (mA) epigenetic modification exists in many RNAs and is related to many human diseases. Chemically synthesized RNAs containing the modification are...
The -methyladenosine (mA) epigenetic modification exists in many RNAs and is related to many human diseases. Chemically synthesized RNAs containing the modification are required for projects aimed at studying biological processes, mechanisms, and pathogenesis related to mA. Existing methods for the synthesis of mA containing RNAs use tetrabutylammonium fluoride (TBAF) for the deprotection of the 2'-silyl protecting groups. Since TBAF is nonvolatile, and is relatively non-polar, its use in the desilylation of RNA requires repeated desalting, which is tedious and gives low yields. Here we report the use of the volatile and neat triethylamine hydrogen fluoride (TEA-HF) for desilylation of mA RNA synthesis. We found that the method is much simpler, and-in our hands-give significantly higher yield of RNA. Two major concerns for mA RNA synthesis are depurination and Dimroth rearrangement. HPLC and MALDI MS of the RNA indicated that depurination is not a problem for the new method. The absence of Dimroth rearrangement is proven by RNA digestion followed by HPLC analysis of the nucleosides.
PubMed: 38735066
DOI: 10.1080/15257770.2024.2353181 -
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 -
Biomolecular NMR Assignments Jun 2024Ricin is a potent plant toxin that targets the eukaryotic ribosome by depurinating an adenine from the sarcin-ricin loop (SRL), a highly conserved stem-loop of the rRNA....
Ricin is a potent plant toxin that targets the eukaryotic ribosome by depurinating an adenine from the sarcin-ricin loop (SRL), a highly conserved stem-loop of the rRNA. As a category-B agent for bioterrorism it is a prime target for therapeutic intervention with antibodies and enzyme blocking inhibitors since no effective therapy exists for ricin. Ricin toxin A subunit (RTA) depurinates the SRL by binding to the P-stalk proteins at a remote site. Stimulation of the N-glycosidase activity of RTA by the P-stalk proteins has been studied extensively by biochemical methods and by X-ray crystallography. The current understanding of RTA's depurination mechanism relies exclusively on X-ray structures of the enzyme in the free state and complexed with transition state analogues. To date we have sparse evidence of conformational dynamics and allosteric regulation of RTA activity that can be exploited in the rational design of inhibitors. Thus, our primary goal here is to apply solution NMR techniques to probe the residue specific structural and dynamic coupling active in RTA as a prerequisite to understand the functional implications of an allosteric network. In this report we present de novo sequence specific amide and sidechain methyl chemical shift assignments of the 267 residue RTA in the free state and in complex with an 11-residue peptide (P11) representing the identical C-terminal sequence of the ribosomal P-stalk proteins. These assignments will facilitate future studies detailing the propagation of binding induced conformational changes in RTA complexed with inhibitors, antibodies, and biologically relevant targets.
Topics: Ricin; Nuclear Magnetic Resonance, Biomolecular; Nitrogen Isotopes; Protein Subunits; Amino Acid Sequence
PubMed: 38642265
DOI: 10.1007/s12104-024-10172-8 -
Molecules (Basel, Switzerland) Mar 2024The plant-derived toxin ricin is classified as a type 2 ribosome-inactivating protein (RIP) and currently lacks effective clinical antidotes. The toxicity of ricin is...
The plant-derived toxin ricin is classified as a type 2 ribosome-inactivating protein (RIP) and currently lacks effective clinical antidotes. The toxicity of ricin is mainly due to its ricin toxin A chain (RTA), which has become an important target for drug development. Previous studies have identified two essential binding pockets in the active site of RTA, but most existing inhibitors only target one of these pockets. In this study, we used computer-aided virtual screening to identify a compound called RSMI-29, which potentially interacts with both active pockets of RTA. We found that RSMI-29 can directly bind to RTA and effectively attenuate protein synthesis inhibition and rRNA depurination induced by RTA or ricin, thereby inhibiting their cytotoxic effects on cells in vitro. Moreover, RSMI-29 significantly reduced ricin-mediated damage to the liver, spleen, intestine, and lungs in mice, demonstrating its detoxification effect against ricin in vivo. RSMI-29 also exhibited excellent drug-like properties, featuring a typical structural moiety of known sulfonamides and barbiturates. These findings suggest that RSMI-29 is a novel small-molecule inhibitor that specifically targets ricin toxin A chain, providing a potential therapeutic option for ricin intoxication.
Topics: Animals; Mice; Ricin; Ribosome Inactivating Proteins, Type 2; Drug Development; Hydrolases; Liver
PubMed: 38611715
DOI: 10.3390/molecules29071435 -
RNA Biology Jan 2024RNA modifications, including -7-methylguanosine (mG), are pivotal in governing RNA stability and gene expression regulation. The accurate detection of internal mG...
RNA modifications, including -7-methylguanosine (mG), are pivotal in governing RNA stability and gene expression regulation. The accurate detection of internal mG modifications is of paramount significance, given recent associations between altered mG deposition and elevated expression of the methyltransferase METTL1 in various human cancers. The development of robust mG detection techniques has posed a significant challenge in the field of epitranscriptomics. In this study, we introduce two methodologies for the global and accurate identification of mG modifications in human RNA. We introduce borohydride reduction sequencing (Bo-Seq), which provides base resolution mapping of mG modifications. Bo-Seq achieves exceptional performance through the optimization of RNA depurination and scission, involving the strategic use of high concentrations of NaBH, neutral pH and the addition of 7-methylguanosine monophosphate (mGMP) during the reducing reaction. Notably, compared to NaBH-based methods, Bo-Seq enhances the mG detection performance, and simplifies the detection process, eliminating the necessity for intricate chemical steps and reducing the protocol duration. In addition, we present an antibody-based approach, which enables the assessment of mG relative levels across RNA molecules and biological samples, however it should be used with caution due to limitations associated with variations in antibody quality between batches. In summary, our novel approaches address the pressing need for reliable and accessible methods to detect RNA mG methylation in human cells. These advancements hold the potential to catalyse future investigations in the critical field of epitranscriptomics, shedding light on the complex regulatory roles of mG in gene expression and its implications in cancer biology.
Topics: Humans; RNA; Nucleotides; Methylation; Methyltransferases; RNA Processing, Post-Transcriptional; Guanosine
PubMed: 38566310
DOI: 10.1080/15476286.2024.2337493 -
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