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Journal of Biochemistry Jul 2022An extreme thermophile, Thermus thermophilus grows at an optimum temperature of around 70°C and produces 16 different polyamines including long-chain and branched-chain...
An extreme thermophile, Thermus thermophilus grows at an optimum temperature of around 70°C and produces 16 different polyamines including long-chain and branched-chain polyamines. We found that the composition of polyamines in the thermophile cells changes with culture temperature. Long-chain and branched-chain polyamines (unusual polyamines) were increased in the cells grown at high temperature such as 80°C, but they were minor components in the cells grown at relatively lower temperature such as 60°C. The effects of polyamines on cell growth were studied using T. thermophilus HB8 ΔspeA deficient in arginine decarboxylase. Cell growth of this mutant strain was significantly decreased at 70°C. This mutant strain cannot produce polyamines and grows poorly at 75°C. It was also determined whether polyamines are directly involved in protecting DNA from DNA double-strand breaks (DSBs) induced by heat. Polyamines protected DNA against double-strand breaks. Therefore, polyamines play essential roles in cell growth at extremely high temperature through maintaining a functional conformation of DNA against DSBs and depurination.
Topics: DNA; Hot Temperature; Polyamines; Temperature; Thermus thermophilus
PubMed: 35639548
DOI: 10.1093/jb/mvac048 -
Toxicology in Vitro : An International... Mar 2023Enterohemorrhagic or Shiga toxin-producing Escherichia coli is a food-poisoning bacterium that grows in the intestine to produce Shiga toxin (Stx). In this study, the...
Enterohemorrhagic or Shiga toxin-producing Escherichia coli is a food-poisoning bacterium that grows in the intestine to produce Shiga toxin (Stx). In this study, the effects of 20 polyphenols on the cytotoxicity of Stx1 and Stx2 in Vero cells were investigated. Among these, epigallocatechin gallate, butein, isorhapontigenin, hesperetin, morin, luteolin, resveratrol, and rhapontigenin showed inhibitory effects on the cytotoxicity of Stxs at 0.4 mmol/L. Furthermore, Vero cells pre-treated with these polyphenols were resistant to Stx at 0.4 mmol/L. However, luteolin showed the most potent inhibitory and cytoprotective effect against Stxs at 0.08 mmol/L or more. This inhibitory mechanism of luteolin was determined using a cell-free protein synthesis system and quantitative reverse transcription PCR assay to detect depurination of 28S rRNA in Vero cells. Luteolin did not inhibit the cell-free protein synthesis by Stxs, suggesting that the enzymatic activity of the Stx A subunit was not inhibited by luteolin. The depurination of 28S rRNA by Stxs was also investigated in Vero cells. The 28S rRNA depurination by Stxs was suppressed in Vero cells treated with Stxs which had been pretreated with luteolin. These results suggest that luteolin inhibits the incorporation of Stxs into Vero cells. This is the first report to show that luteolin inhibits the cytotoxicity of both Stx1 and Stx2 by inhibiting the incorporation of Stxs into Vero cells.
Topics: Animals; Chlorocebus aethiops; Vero Cells; Shiga Toxin; Shiga Toxin 1; Shiga Toxin 2; Luteolin; RNA, Ribosomal, 28S
PubMed: 36535555
DOI: 10.1016/j.tiv.2022.105537 -
Biochemistry Oct 2022Positively charged N-terminal histone tails play important roles in maintaining the nucleosome (and chromatin) structure and function. Charge alteration, including those...
Positively charged N-terminal histone tails play important roles in maintaining the nucleosome (and chromatin) structure and function. Charge alteration, including those imposed by post-translational modifications, impacts chromatin dynamics, protein binding, and the fate of DNA damage. There is evidence that N-terminal histone tails affect the local ionic environment within a nucleosome core particle (NCP), but this phenomenon is not well understood. Determining the modulation of the local ionic environment within an NCP by histone tails could help uncover the underlying mechanisms of their functions and effects. Utilizing bottom-up syntheses of NCPs containing wild-type or mutated histones and a fluorescent probe that is sensitive to the local ionic environment, we show that interaction with positively charged N-terminal tails increases the local ionic strength near nucleosomal DNA. The effect is diminished by replacing positively charged residues with neutral ones or deleting a tail in its entirety. Replacing the fluorescent probe with the major DNA methylation product, 7-methyl-2'-deoxyguanosine (MdG), revealed changes in the depurination rate constant varying inversely with local ionic strength. These data indicate that the MdG hydrolysis rates depend on and also inform on local ionic strength in an NCP. Overall, histone tail charge contributes to the complexity of the NCP structure and function by modulating the local ionic strength.
Topics: Chromatin; DNA; Deoxyguanosine; Fluorescent Dyes; Histones; Nucleosomes; Osmolar Concentration
PubMed: 36136907
DOI: 10.1021/acs.biochem.2c00342 -
The Analyst Jun 2023The accurate and sensitive detection of active biotoxin proteins and the determination of their kinetics are vital for the upsurge of chemical attacks but still limited....
The accurate and sensitive detection of active biotoxin proteins and the determination of their kinetics are vital for the upsurge of chemical attacks but still limited. Herein, we report a liquid chromatography-tunable ultraviolet spectroscopic-quadrupole mass spectrometric detection (LC-TUV-QDa) method of active ricin. This method has the advantage of the accurate quantification of active ricin in decreased oligonucleotide (oligo) substrates as well as the produced adenine, in which the QDa detection offers the confirmative evidence of oligo and adenine products. We invented a strong cation exchange (SCX)-tip sample pretreatment way to facilitate the requirement of clean product injection without any fouling proteins. After full-method validation, a wide linear range of 1-5000 ng mL was obtained with a high sensitivity of 1 ng mL active ricin based on the most preferable deoxynucleobase-hybrid RNA (Rd) substrate, Rd12, and without any enrichment. We also fully depicted the kinetic parameters of ricin and its six Rd or RNA substrates and evaluated 11 nucleobase-modified oligos as substrates based on Rd12. Further, we fulfilled an improved molecular docking analysis and revealed that the binding of Rd12 to ricin was more likely to occur at pH 7.4 (typical and conditions) than at pH 4.0 (typical conditions). With the aid of SCX-tip as a microenzymatic reactor, we can exert the catalytic activity of ricin as -glycosidase in pH 7.4 toward its Rd12 substrate, with a comparable catalytic efficiency at pH 4.0. This is the first successful implementation of an experiment toward oligo substrates at neutral pH, standing on the shoulder of plenty of previously reported efforts all performed under acidic conditions. This method will provide a new and powerful way to detect active ricin when tackling relevant problems in public safety and security.
Topics: Ricin; Chromatography, Liquid; Kinetics; Molecular Docking Simulation; Tandem Mass Spectrometry; Adenine; RNA; Oligonucleotides
PubMed: 37195783
DOI: 10.1039/d3an00217a -
Biochimica Et Biophysica Acta.... Dec 2019The generally accepted model of ricin intoxication assumes that direct inactivation of ribosomes by depurination of a specific adenine residue within the...
The generally accepted model of ricin intoxication assumes that direct inactivation of ribosomes by depurination of a specific adenine residue within the sarcin-ricin-loop (SRL) on the 60S ribosomal subunit is a major source of its toxicity. The model proposes that SRL depurination leads to protein synthesis inhibition, evoking ribotoxic stress with concomitant induction of numerous metabolic pathways, which lead to cell death. However, the direct relationship between the depurination and its impact on the translational machinery in vivo has never been satisfactorily explained. In this work, we approached a long-standing question about the influence of SRL depurination on the functioning of the translational machinery in vivo. We have shown that an already low level of depurinated ribosomes exert an effect on cell metabolism, indicating that minute modification within the ribosomal pool is sufficient to elicit a toxic effect. Importantly, depurination does not affect notably any particular step of translation, and translational slowdown caused by ricin is not a direct consequence of depurination and cannot be considered as the sole source of cell death. Instead, SRL depurination in a small fraction of ribosomes blocks cell cycle progression with no effect on cell viability. In this work, we have provided a comprehensive picture of the impact of SRL depurination on the translational apparatus in vivo. We propose that ribosomes with depurinated SRL represent a small imprinted ribosomal pool, which generates a specific signal for the cell to halt the cell cycle.
Topics: Cell Survival; Protein Biosynthesis; Protein Processing, Post-Translational; RNA, Ribosomal; Ricin; Saccharomyces cerevisiae
PubMed: 31518597
DOI: 10.1016/j.bbamcr.2019.118554 -
Biochemistry Jun 2021is a Gram-negative bacterium that is responsible for gastric and duodenal ulcers. uses the unusual pathway with aminofutalosine (AFL) as an intermediate for...
is a Gram-negative bacterium that is responsible for gastric and duodenal ulcers. uses the unusual pathway with aminofutalosine (AFL) as an intermediate for menaquinone biosynthesis. Previous reports indicate that hydrolysis of AFL by 5'-methylthioadenosine nucleosidase (MTAN) is the direct path for producing downstream metabolites in the pathway. However, genomic analysis indicates is a candidate for encoding AFL deaminase (AFLDA), an activity for deaminating aminofutolasine. The product, futalosine, is not a known substrate for bacterial MTANs. Recombinant jhp0252 was expressed and characterized as an AFL deaminase (AFLDA). Its catalytic specificity includes AFL, 5'-methylthioadenosine, 5'-deoxyadenosine, adenosine, and -adenosylhomocysteine. The / value for AFL is 6.8 × 10 M s, 26-fold greater than that for adenosine. 5'-Methylthiocoformycin (MTCF) is a slow-onset inhibitor for AFLDA and demonstrated inhibitory effects on growth. Supplementation with futalosine partially restored growth under MTCF treatment, suggesting AFL deamination is significant for cell growth. The crystal structures of apo-AFLDA and with MTCF at the catalytic sites show a catalytic site Zn or Fe as the water-activating group. With bound MTCF, the metal ion is 2.0 Å from the sp hydroxyl group of the transition state analogue. Metabolomics analysis revealed that AFLDA has intracellular activity and is inhibited by MTCF. The pathway in bifurcates at aminofutalosine with MTAN producing adenine and depurinated futalosine and AFLDA producing futalosine. Inhibition of cellular MTAN or AFLDA decreased the cellular content of menaquinone-6, supporting roles for both enzymes in the pathway.
Topics: Catalytic Domain; Crystallography, X-Ray; Deoxyadenosines; Helicobacter pylori; Models, Molecular; N-Glycosyl Hydrolases; Nucleosides; Purine-Nucleoside Phosphorylase; Substrate Specificity; Thionucleosides; Vitamin K 2
PubMed: 34077175
DOI: 10.1021/acs.biochem.1c00215 -
Toxins Jul 2022Ricin is a toxin which enters cells and depurinates an adenine base in the sarcin-ricin loop in the large ribosomal subunit, leading to the inhibition of protein...
Ricin is a toxin which enters cells and depurinates an adenine base in the sarcin-ricin loop in the large ribosomal subunit, leading to the inhibition of protein translation and cell death. We postulated that this depurination event could be detected using Oxford Nanopore Technologies (ONT) direct RNA sequencing, detecting a change in charge in the ricin loop. In this study, A549 cells were exposed to ricin for 2-24 h in order to induce depurination. In addition, a novel software tool was developed termed RIPpore that could quantify the adenine modification of ribosomal RNA induced by ricin upon respiratory epithelial cells. We provided demonstrable evidence for the first time that this base change detected is specific to RIP activity using a neutralising antibody against ricin. We believe this represents the first detection of depurination in RNA achieved using ONT sequencers. Collectively, this work highlights the potential for ONT and direct RNA sequencing to detect and quantify depurination events caused by ribosome-inactivating proteins such as ricin. RIPpore could have utility in the evaluation of new treatments and/or in the diagnosis of exposure to ricin.
Topics: Adenine; Nanopores; RNA; Ribosomes; Ricin; Sequence Analysis, RNA
PubMed: 35878208
DOI: 10.3390/toxins14070470 -
Nanomaterials (Basel, Switzerland) Aug 2021Ubiquitous on Earth, DNA and other nucleic acids are being increasingly considered as promising biomass resources. Due to their unique chemical structure, which is...
Ubiquitous on Earth, DNA and other nucleic acids are being increasingly considered as promising biomass resources. Due to their unique chemical structure, which is different from that of more common carbohydrate biomass polymers, materials based on nucleic acids may exhibit new, attractive characteristics. In this study, fluorescent nanoparticles (biodots) were prepared by a hydrothermal (HT) method from various nucleic acids (DNA, RNA, nucleotides, and nucleosides) to establish the relationship between the structure of precursors and fluorescent properties of biodots and to optimize conditions for preparation of the most fluorescent product. HT treatment of nucleic acids results in decomposition of sugar moieties and depurination/depyrimidation of nucleobases, while their consequent condensation and polymerization gives fluorescent nanoparticles. Fluorescent properties of DNA and RNA biodots are drastically different from biodots synthesized from individual nucleotides. In particular, biodots synthesized from purine-containing nucleotides or nucleosides show up to 50-fold higher fluorescence compared to analogous pyrimidine-derived biodots. The polymeric nature of a precursor disfavors formation of a bright fluorescent product. The reported effect of the structure of the nucleic acid precursor on the fluorescence properties of biodots should help designing and synthesizing brighter fluorescent nanomaterials with broader specification for bioimaging, sensing, and other applications.
PubMed: 34578581
DOI: 10.3390/nano11092265 -
Methods in Molecular Biology (Clifton,... 2022The specificity and strength of protein-DNA complexes rely on tight interactions between side- and main chain atoms of amino acid residues and phosphates, sugars, and...
The specificity and strength of protein-DNA complexes rely on tight interactions between side- and main chain atoms of amino acid residues and phosphates, sugars, and base-specific groups. Various (in-gel) footprinting methods (for more information, see Chapter 11 ) allow the identification of the global-binding region but do not provide details on the contribution to complex formation of individual sequence-specific constituents of the DNA-binding site. Here, we describe how various chemicals can be used to randomly and sparingly modify specific bases or phosphates and allow the identification of those residues that are specifically protected against modification upon protein binding (protection studies) or interfere with complex formation when modified or removed prior to protein binding (premodification-binding interference). Each one of these complementary approaches has its advantages and shortcomings and results have to be interpreted with caution, having in mind the precise chemistry of the modification. However, used in combination, these methods provide an accurate and high-resolution image of the protein-DNA contacts.
Topics: Base Sequence; Binding Sites; DNA; Phosphates; Protein Binding
PubMed: 35922629
DOI: 10.1007/978-1-0716-2413-5_12 -
The Journal of Biological Chemistry Nov 2019Ricin undergoes retrograde transport to the endoplasmic reticulum (ER), and ricin toxin A chain (RTA) enters the cytosol from the ER. Previous reports indicated that RTA...
Ricin undergoes retrograde transport to the endoplasmic reticulum (ER), and ricin toxin A chain (RTA) enters the cytosol from the ER. Previous reports indicated that RTA inhibits activation of the unfolded protein response (UPR) in yeast and in mammalian cells. Both precursor (preRTA) and mature form of RTA (mRTA) inhibited splicing of ( for uninduced) mRNA, suggesting that UPR inhibition occurred on the cytosolic face of the ER. Here, we examined the role of ribosome binding and depurination activity on inhibition of the UPR using mRTA mutants. An active-site mutant with very low depurination activity, which bound ribosomes as WT RTA, did not inhibit mRNA splicing. A ribosome-binding mutant, which showed reduced binding to ribosomes but retained depurination activity, inhibited mRNA splicing. This mutant allowed separation of the UPR inhibition by RTA from cytotoxicity because it reduced the rate of depurination. The ribosome-binding mutant inhibited the UPR without affecting IRE1 oligomerization or cleavage of mRNA at the splice site junctions. Inhibition of the UPR correlated with the depurination level, suggesting that ribosomes play a role in splicing of mRNA. We show that mRNA is associated with ribosomes and does not get processed on depurinated ribosomes, thereby inhibiting the UPR. These results demonstrate that RTA inhibits mRNA splicing through its depurination activity on the ribosome without directly affecting IRE1 oligomerization or the splicing reaction and provide evidence that IRE1 recognizes mRNA that is associated with ribosomes.
Topics: Basic-Leucine Zipper Transcription Factors; Endoplasmic Reticulum Stress; Gene Expression Regulation, Fungal; Membrane Glycoproteins; Models, Biological; Mutation; Protein Multimerization; Protein Serine-Threonine Kinases; Purines; RNA Processing, Post-Transcriptional; RNA Splicing; RNA, Messenger; Repressor Proteins; Ribosomes; Ricin; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Unfolded Protein Response
PubMed: 31624149
DOI: 10.1074/jbc.RA119.009128