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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 -
SLAS Discovery : Advancing Life... Mar 2021Saporin, a type I ribosome-inactivating protein from soapwort plant, is a potent protein synthesis inhibitor. Catalytically, saporin is a characteristic -glycosidase,...
Saporin, a type I ribosome-inactivating protein from soapwort plant, is a potent protein synthesis inhibitor. Catalytically, saporin is a characteristic -glycosidase, and it depurinates a specific adenine residue from a universally conserved loop of the major ribosomal RNA (rRNA) of eukaryotic cells. It is well-known that saporin induces apoptosis through different pathways, including ribotoxic stress response, cell signal transduction, genomic DNA fragmentation and RNA abasic lyase (RAlyase) activity, and NAD depletion by poly-(ADP)-ribose polymerase hyperactivation. Saporin's high enzymatic activity, high stability, and resistance to conjugation procedures make it a well-suited tool for immunotherapy approaches.In the present study, we focus on saporin-based targeted toxins that may be efficacious therapeutic agents for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Our discussed points suggest that saporin may be a strategic molecule for therapeutic knockout treatments and a powerful candidate for novel drugs in the struggle against coronavirus 2019 (COVID-19).
Topics: Antiviral Agents; Apoptosis; Humans; Immunotoxins; NAD; Poly(ADP-ribose) Polymerases; Saporins; Signal Transduction; COVID-19 Drug Treatment
PubMed: 33155515
DOI: 10.1177/2472555220970911 -
Journal of Medicinal Chemistry Oct 2021Ricin toxin A subunit (RTA) is the catalytic subunit of ricin, which depurinates an adenine from the sarcin/ricin loop in eukaryotic ribosomes. There are no approved...
Ricin toxin A subunit (RTA) is the catalytic subunit of ricin, which depurinates an adenine from the sarcin/ricin loop in eukaryotic ribosomes. There are no approved inhibitors against ricin. We used a new strategy to disrupt RTA-ribosome interactions by fragment screening using surface plasmon resonance. Here, using a structure-guided approach, we improved the affinity and inhibitory activity of small-molecular-weight lead compounds and obtained improved compounds with over an order of magnitude higher efficiency. Four advanced compounds were characterized by X-ray crystallography. They bind at the RTA-ribosome binding site as the original compound but in a distinctive manner. These inhibitors bind remotely from the catalytic site and cause local conformational changes with no alteration of the catalytic site geometry. Yet they inhibit depurination by ricin holotoxin and inhibit the cytotoxicity of ricin in mammalian cells. They are the first agents that protect against ricin holotoxin by acting directly on RTA.
Topics: Animals; Binding Sites; Chlorocebus aethiops; Crystallography, X-Ray; Dose-Response Relationship, Drug; Drug Design; Enzyme Inhibitors; Models, Molecular; Molecular Structure; Ribosomes; Ricin; Small Molecule Libraries; Structure-Activity Relationship; Surface Plasmon Resonance; Vero Cells
PubMed: 34648707
DOI: 10.1021/acs.jmedchem.1c01370 -
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 -
Chemical Research in Toxicology Apr 2020Nitrogen mustards (NM) are an important class of chemotherapeutic drugs used in the treatment of malignant tumors. The accepted mechanism of action of NM is through the...
Nitrogen mustards (NM) are an important class of chemotherapeutic drugs used in the treatment of malignant tumors. The accepted mechanism of action of NM is through the alkylation of DNA bases. NM-adducts block DNA replication in cancer cells by forming cytotoxic DNA interstrand cross-links. We previously characterized several adducts formed by reaction of bis(2-chloroethyl)ethylamine (NM) with calf thymus (CT) DNA and the MDA-MB-231 mammary tumor cell line. The monoalkylated N7-guanine (NM-G) adduct and its cross-link (G-NM-G) were major lesions. The cationic NM-G undergoes a secondary reaction through depurination to form an apurinic (AP) site or reacts with hydroxide to yield the stable ring-opened -substituted formamidopyrimidine (NM-Fapy-G) adduct. Both of these lesions are mutagenic and may contribute to secondary tumor development, a major clinical limitation of NM chemotherapy. We established a kinetic model with NM-treated female mice and measured the rates of formation and removal of NM-DNA adducts and AP sites. We employed liquid chromatography-mass spectrometry (LC-MS) to measure NM-G, G-NM-G, and NM-Fapy-G adducts in liver, lung, and spleen over 168 h. NM-G reached a maximum level within 6 h in all organs and then rapidly declined. The G-NM-G cross-link and NM-FapyG were more persistent with half-lives over three-times longer than NM-G. We quantified AP site lesions in the liver and showed that NM treatment increased AP site levels by 3.7-fold over the basal levels at 6 h. The kinetics of AP site repair closely followed the rate of removal of NM-G; however, AP sites remained 1.3-fold above basal levels 168 h post-treatment with NM. Our data provide new insights into NM-induced DNA damage and biological processing . The quantitative measurement of the spectrum of NM adducts and AP sites can serve as biomarkers in the design and assessment of the efficacy of novel chemotherapeutic regimens.
Topics: Animals; DNA Adducts; Female; Kinetics; Mass Spectrometry; Mechlorethamine; Mice; Mice, Inbred C57BL; Molecular Structure; Tissue Distribution
PubMed: 32174110
DOI: 10.1021/acs.chemrestox.0c00012 -
Chemical Communications (Cambridge,... May 2023A practical strategy for the total stepwise solid-phase synthesis of peptide-oligonucleotide conjugates was developed. In this strategy, the Boc/Bu protecting groups are...
A practical strategy for the total stepwise solid-phase synthesis of peptide-oligonucleotide conjugates was developed. In this strategy, the Boc/Bu protecting groups are utilized for the side chains of Trp, His, Arg, Asp, and Glu, and is deprotected in borate buffer at 90 °C to avoid depurination of the oligonucleotide caused by strong acid treatment. The advantage of this strategy is that the abovementioned amino acids are readily available in the market and the side reaction of deguanidination of the Arg residue can be avoided. This side-chain Boc/Bu protection strategy will expand the applicability of total stepwise synthesis in the preparation of peptide-oligonucleotide conjugates.
Topics: Amino Acid Sequence; Oligonucleotides; Solid-Phase Synthesis Techniques; Peptides; Amino Acids
PubMed: 37039333
DOI: 10.1039/d3cc00868a -
International Journal of Legal Medicine Jan 2023The aim of this study was to identify artificial single-nucleotide variants (SNVs) in degraded trace DNA samples. In a preliminary study, blood samples were stored for...
The aim of this study was to identify artificial single-nucleotide variants (SNVs) in degraded trace DNA samples. In a preliminary study, blood samples were stored for up to 120 days and whole-genome sequencing was performed using the Snakemake workflow dna-seq-gatk-variant-calling to identify positions that vary between the time point 0 sample and the aged samples. In a follow-up study on blood and saliva samples stored under humid and dry conditions, potential marker candidates for the estimation of the age of a blood stain (= time since deposition) were identified. Both studies show that a general decrease in the mean fragment size of the libraries over time was observed, presumably due to the formation of abasic sites during DNA degradation which are more susceptible to strand breaks by mechanical shearing of DNA. Unsurprisingly, an increase in the number of failed genotype calls (no coverage) was detected over time. Both studies indicated the presence of artificial SNVs with the majority of changes happening at guanine and cytosine positions. This confirms previous studies and can be explained by depurination through hydrolytic attacks which more likely deplete guanine while deamination leads to cytosine to thymine variants. Even complete genotype switches from homozygote 0/0 genotypes to the opposite 1/1 genotypes were observed. While positions with such drastic changes might provide suitable candidate markers for estimating short-term time since deposition (TsD), 11 markers were identified which show a slower gradual change of the relative abundance of the artificial variant in both blood and saliva samples, irrespective of storage conditions.
Topics: Humans; Aged; Follow-Up Studies; Genotype; Whole Genome Sequencing; DNA; Nucleotides; High-Throughput Nucleotide Sequencing; Polymorphism, Single Nucleotide
PubMed: 36352329
DOI: 10.1007/s00414-022-02911-0 -
Biomedicines Apr 2023Saporin is a type 1 ribosome-inactivating protein widely used as toxic payload in the construction of targeted toxins, chimeric molecules formed by a toxic portion...
Saporin is a type 1 ribosome-inactivating protein widely used as toxic payload in the construction of targeted toxins, chimeric molecules formed by a toxic portion linked to a carrier moiety. Among the most used carriers, there are large molecules (mainly antibodies) and small molecules (such as neurotransmitters, growth factors and peptides). Some saporin-containing targeted toxins have been used for the experimental treatment of several diseases, giving very promising results. In this context, one of the reasons for the successful use of saporin lies in its resistance to proteolytic enzymes and to conjugation procedures. In this paper, we evaluated the influence of derivatization on saporin using three heterobifunctional reagents, namely 2-iminothiolane (2-IT), N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) and 4-succinimidyloxycarbonyl-α-methyl-α-[2-pyridyldithio]toluene (SMPT). In order to obtain the highest number of inserted -SH groups with the lowest reduction of saporin biological activities, we assessed the residual ability of saporin to inhibit protein synthesis, to depurinate DNA and to induce cytotoxicity after derivatization. Our results demonstrate that saporin maintains an excellent resistance to derivatization processes, especially with SPDP, and permit us to define reaction conditions, in which saporin biological properties may not be altered. Therefore, these findings provide useful information for the construction of saporin-based targeted toxins, especially with small carriers.
PubMed: 37189832
DOI: 10.3390/biomedicines11041214 -
International Journal of Molecular... Oct 2021Ribosome-inactivating proteins (RIPs) hydrolyze the N-glycosidic bond and depurinate a specific adenine residue (A-4324 in rat 28S ribosomal RNA, rRNA) in the conserved...
Ribosome-inactivating proteins (RIPs) hydrolyze the N-glycosidic bond and depurinate a specific adenine residue (A-4324 in rat 28S ribosomal RNA, rRNA) in the conserved α-sarcin/ricin loop (α-SRL) of rRNA. In this study, we have purified and characterized lyophyllin, an unconventional RIP from , an edible mushroom. The protein resembles peptidase M35 domain of peptidyl-Lys metalloendopeptidases. Nevertheless, protein either from the mushroom or in recombinant form possessed N-glycosidase and protein synthesis inhibitory activities. A homology model of lyophyllin was constructed. It was found that the zinc binding pocket of this protein resembles the catalytic cleft of a classical RIP, with key amino acids that interact with the adenine substrate in the appropriate positions. Mutational studies showed that E122 may play a role in stabilizing the positively charged oxocarbenium ion and H121 for protonating N-3 of adenine. The tyrosine residues Y137 and Y104 may be used for stacking the target adenine ring. This work first shows a protein in the peptidase M35 superfamily based on conserved domain search possessing N-glycosidase activity.
Topics: Agaricales; Amino Acid Sequence; Animals; Base Sequence; Endoribonucleases; Fungal Proteins; HeLa Cells; Hep G2 Cells; Humans; Peptide Hydrolases; Protein Binding; RNA, Ribosomal, 28S; Rats; Ribosome Inactivating Proteins; Ricin
PubMed: 34769028
DOI: 10.3390/ijms222111598 -
The Journal of Biological Chemistry Apr 2022During ricin intoxication in mammalian cells, ricin's enzymatic (RTA) and binding (RTB) subunits disassociate in the endoplasmic reticulum. RTA is then translocated into...
During ricin intoxication in mammalian cells, ricin's enzymatic (RTA) and binding (RTB) subunits disassociate in the endoplasmic reticulum. RTA is then translocated into the cytoplasm where, by virtue of its ability to depurinate a conserved residue within the sarcin-ricin loop (SRL) of 28S rRNA, it functions as a ribosome-inactivating protein. It has been proposed that recruitment of RTA to the SRL is facilitated by ribosomal P-stalk proteins, whose C-terminal domains interact with a cavity on RTA normally masked by RTB; however, evidence that this interaction is critical for RTA activity within cells is lacking. Here, we characterized a collection of single-domain antibodies (VHs) whose epitopes overlap with the P-stalk binding pocket on RTA. The crystal structures of three such VHs (V9E1, V9F9, and V9B2) in complex with RTA revealed not only occlusion of the ribosomal P-stalk binding pocket but also structural mimicry of C-terminal domain peptides by complementarity-determining region 3. In vitro assays confirmed that these VHs block RTA-P-stalk peptide interactions and protect ribosomes from depurination. Moreover, when expressed as "intrabodies," these VHs rendered cells resistant to ricin intoxication. One VH (V9F6), whose epitope was structurally determined to be immediately adjacent to the P-stalk binding pocket, was unable to neutralize ricin within cells or protect ribosomes from RTA in vitro. These findings are consistent with the recruitment of RTA to the SRL by ribosomal P-stalk proteins as a requisite event in ricin-induced ribosome inactivation.
Topics: Animals; Epitopes; Mammals; Peptides; RNA, Ribosomal, 28S; Ribosomal Proteins; Ribosomes; Ricin; Single-Domain Antibodies
PubMed: 35182523
DOI: 10.1016/j.jbc.2022.101742