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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 -
Chemistry (Weinheim An Der Bergstrasse,... Jun 2022Ribosome-inactivating proteins, a family of highly cytotoxic proteins, interfere with protein synthesis by depurinating a specific adenosine residue within the conserved...
Ribosome-inactivating proteins, a family of highly cytotoxic proteins, interfere with protein synthesis by depurinating a specific adenosine residue within the conserved α-sarcin/ricin loop of eukaryotic ribosomal RNA. Besides being biological warfare agents, certain RIPs have been promoted as potential therapeutic tools. Monitoring their deglycosylation activity and their inhibition in real time have remained, however, elusive. Herein, we describe the enzymatic preparation and utility of consensus RIP hairpin substrates in which specific G residues, next to the depurination site, are surgically replaced with G and G, fluorescent G analogs. By strategically modifying key positions with responsive fluorescent surrogate nucleotides, RIP-mediated depurination can be monitored in real time by steady-state fluorescence spectroscopy. Subtle differences observed in preferential depurination sites provide insight into the RNA folding as well as RIPs' substrate recognition features.
Topics: Nucleosides; Plant Proteins; RNA; RNA, Ribosomal; Ribosome Inactivating Proteins; Ribosomes
PubMed: 35390188
DOI: 10.1002/chem.202200994 -
Philosophical Transactions of the Royal... Jan 1999The increase in proportion of the non-biological (D-) isomer of aspartic acid (Asp) relative to the L-isomer has been widely used in archaeology and geochemistry as a... (Review)
Review
The increase in proportion of the non-biological (D-) isomer of aspartic acid (Asp) relative to the L-isomer has been widely used in archaeology and geochemistry as a tool for dating. the method has proved controversial, particularly when used for bones. The non-linear kinetics of Asp racemization have prompted a number of suggestions as to the underlying mechanism(s) and have led to the use of mathematical transformations which linearize the increase in D-Asp with respect to time. Using one example, a suggestion that the initial rapid phase of Asp racemization is due to a contribution from asparagine (Asn), we demonstrate how a simple model of the degradation and racemization of Asn can be used to predict the observed kinetics. A more complex model of peptide bound Asx (Asn + Asp) racemization, which occurs via the formation of a cyclic succinimide (Asu), can be used to correctly predict Asx racemization kinetics in proteins at high temperatures (95-140 degrees C). The model fails to predict racemization kinetics in dentine collagen at 37 degrees C. The reason for this is that Asu formation is highly conformation dependent and is predicted to occur extremely slowly in triple helical collagen. As conformation strongly influences the rate of Asu formation and hence Asx racemization, the use of extrapolation from high temperatures to estimate racemization kinetics of Asx in proteins below their denaturation temperature is called into question. In the case of archaeological bone, we argue that the D:L ratio of Asx reflects the proportion of non-helical to helical collagen, overlain by the effects of leaching of more soluble (and conformationally unconstrained) peptides. Thus, racemization kinetics in bone are potentially unpredictable, and the proposed use of Asx racemization to estimate the extent of DNA depurination in archaeological bones is challenged.
Topics: Aspartic Acid; Bone and Bones; Collagen; DNA; Dentin; Fossils; In Vitro Techniques; Kinetics; Models, Chemical; Paleontology; Protein Denaturation; Protein Structure, Secondary; Proteins; Stereoisomerism
PubMed: 10091247
DOI: 10.1098/rstb.1999.0359 -
Cellular Microbiology Jan 2012Shiga toxin-producing bacteria cause widespread outbreaks of bloody diarrhoea that may progress to life-threatening systemic complications. Shiga toxins (Stxs), the main... (Review)
Review
Shiga toxin-producing bacteria cause widespread outbreaks of bloody diarrhoea that may progress to life-threatening systemic complications. Shiga toxins (Stxs), the main virulence factors expressed by the pathogens, are ribosome-inactivating proteins which inhibit protein synthesis by removing an adenine residue from 28S rRNA. Recently, Stxs were shown to activate multiple stress-associated signalling pathways in mammalian cells. The ribotoxic stress response is activated following the depurination reaction localized to the α-sarcin/ricin loop of eukaryotic ribosomes. The unfolded protein response (UPR) may be initiated by toxin unfolding within the endoplasmic reticulum, and maintained by production of truncated, misfolded proteins following intoxication. Activation of the ribotoxic stress response leads to signalling through MAPK cascades, which appears to be critical for activation of innate immunity and regulation of apoptosis. Precise mechanisms linking ribosomal damage with MAPK activation require clarification but may involve recognition of ribosomal conformational changes and binding of protein kinases to ribosomes, which activate MAP3Ks and MAP2Ks. Stxs appear capable of activating all ER membrane localized UPR sensors. Prolonged signalling through the UPR induces apoptosis in some cell types. The characterization of stress responses activated by Stxs may identify targets for the development of interventional therapies to block cell damage and disease progression.
Topics: Adenine; Animals; Bacteria; Endoplasmic Reticulum; Humans; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Protein Synthesis Inhibitors; RNA, Ribosomal, 28S; Ribosomes; Shiga Toxins; Stress, Physiological; Unfolded Protein Response
PubMed: 21899699
DOI: 10.1111/j.1462-5822.2011.01684.x -
Toxins Mar 2018We wish to make the following correction to the published paper [1].[...].
We wish to make the following correction to the published paper [1].[...].
PubMed: 29494495
DOI: 10.3390/toxins10030107 -
Nucleic Acids Research Aug 1996Fully protected CPG-immobilized monomer, dimer and trimer oligonucleotides were used to study depurination during the chemical synthesis of oligonucleotides....
Fully protected CPG-immobilized monomer, dimer and trimer oligonucleotides were used to study depurination during the chemical synthesis of oligonucleotides. Disappearance of the oligonucleotide during acid exposure time relative to an internal thymidine standard not subject to depurination was monitored by reverse phase HPLC analysis. Depurination half-times obtained for dichloroacetic acid (DCA) and trichloroacetic acid (TCA) in methylene chloride were found to be 3% DCA >> 15% DCA > 3% TCA. In order to understand the implications of depurination during DNA synthesis, the detritylation kinetics of model compounds DMT-dG-pT dimer and DMT-[17mer] mixed-base sequence were also measured. These results improve our ability to properly balance the contradictory goals of obtaining maximum detritylation with minimum depurination in oligonucleotide synthesis.
Topics: Adenosine; Base Sequence; Guanosine; Kinetics; Molecular Sequence Data; Oligodeoxyribonucleotides; Purines; Trityl Compounds
PubMed: 8760893
DOI: 10.1093/nar/24.15.3053 -
Toxins Nov 2010Ribosome-inactivating proteins (RIPs) are EC3.2.32.22 N-glycosidases that recognize a universally conserved stem-loop structure in 23S/25S/28S rRNA, depurinating a... (Review)
Review
Ribosome-inactivating proteins (RIPs) are EC3.2.32.22 N-glycosidases that recognize a universally conserved stem-loop structure in 23S/25S/28S rRNA, depurinating a single adenine (A4324 in rat) and irreversibly blocking protein translation, leading finally to cell death of intoxicated mammalian cells. Ricin, the plant RIP prototype that comprises a catalytic A subunit linked to a galactose-binding lectin B subunit to allow cell surface binding and toxin entry in most mammalian cells, shows a potency in the picomolar range. The most promising way to exploit plant RIPs as weapons against cancer cells is either by designing molecules in which the toxic domains are linked to selective tumor targeting domains or directly delivered as suicide genes for cancer gene therapy. Here, we will provide a comprehensive picture of plant RIPs and discuss successful designs and features of chimeric molecules having therapeutic potential.
Topics: Animals; Antineoplastic Agents; Disease Models, Animal; Humans; Immunotoxins; Mice; Molecular Sequence Data; Molecular Targeted Therapy; Neoplasms; Plant Diseases; Plant Proteins; Protein Structure, Tertiary; Rats; Ribosome Inactivating Proteins; Ricin; Sequence Analysis, Protein
PubMed: 22069572
DOI: 10.3390/toxins2112699 -
The Journal of Steroid Biochemistry and... Jul 2011Among the numerous small molecules in the body, the very few aromatic ones include the estrogens and dopamine. In relation to cancer initiation, the estrogens should be... (Review)
Review
Among the numerous small molecules in the body, the very few aromatic ones include the estrogens and dopamine. In relation to cancer initiation, the estrogens should be considered as chemicals, not as hormones. Metabolism of estrogens is characterized by two major pathways. One is hydroxylation to form the 2- and 4-catechol estrogens, and the second is hydroxylation at the 16α position. In the catechol pathway, the metabolism involves further oxidation to semiquinones and quinones, including formation of the catechol estrogen-3,4-quinones, the major carcinogenic metabolites of estrogens. These electrophilic compounds react with DNA to form the depurinating adducts 4-OHE(1)(E(2))-1-N3Ade and 4-OHE(1)(E(2))-1-N7Gua. The apurinic sites obtained by this reaction generate the mutations that may lead to the initiation of cancer. Oxidation of catechol estrogens to their quinones is normally in homeostasis, which minimizes formation of the quinones and their reaction with DNA. When the homeostasis is disrupted, excessive amounts of catechol estrogen quinones are formed and the resulting increase in depurinating DNA adducts can lead to initiation of cancer. Substantial evidence demonstrates the mutagenicity of the estrogen metabolites and their ability to induce transformation of mouse and human breast epithelial cells, and tumors in laboratory animals. Furthermore, women at high risk for breast cancer or diagnosed with the disease, men with prostate cancer, and men with non-Hodgkin lymphoma all have relatively high levels of estrogen-DNA adducts, compared to matched control subjects. Specific antioxidants, such as N-acetylcysteine and resveratrol, can block the oxidation of catechol estrogens to their quinones and their reaction with DNA. As a result, the initiation of cancer can be prevented.
Topics: Animals; Breast Neoplasms; DNA Adducts; Estrogens; Estrogens, Catechol; Female; Humans; Male; Neoplasms
PubMed: 21397019
DOI: 10.1016/j.jsbmb.2011.03.008 -
Frontiers in Plant Science 2018Ribosome-inactivating proteins (RIPs) are toxic -glycosidases that depurinate eukaryotic and prokaryotic rRNAs, thereby arresting protein synthesis during translation.... (Review)
Review
Ribosome-inactivating proteins (RIPs) are toxic -glycosidases that depurinate eukaryotic and prokaryotic rRNAs, thereby arresting protein synthesis during translation. RIPs are widely found in various plant species and within different tissues. It is demonstrated and in transgenic plants that RIPs have been connected to defense by antifungal, antibacterial, antiviral, and insecticidal activities. However, the mechanism of these effects is still not completely clear. There are a number of reviews of RIPs. However, there are no reviews on the biological functions of RIPs in defense against pathogens and insect pests. Therefore, in this report, we focused on the effect of RIPs from plants in defense against pathogens and insect pest attacks. First, we summarize the three different types of RIPs based on their physical properties. RIPs are generally distributed in plants. Then, we discuss the distribution of RIPs that are found in various plant species and in fungi, bacteria, algae, and animals. Various RIPs have shown unique bioactive properties including antibacterial, antifungal, antiviral, and insecticidal activity. Finally, we divided the discussion into the biological roles of RIPs in defense against bacteria, fungi, viruses, and insects. This review is focused on the role of plant RIPs in defense against bacteria, fungi, viruses, and insect attacks. The role of plant RIPs in defense against pathogens and insects is being comprehended currently. Future study utilizing transgenic technology approaches to study the mechanisms of RIPs will undoubtedly generate a better comprehending of the role of plant RIPs in defense against pathogens and insects. Discovering additional crosstalk mechanisms between RIPs and phytohormones or reactive oxygen species (ROS) against pathogen and insect infections will be a significant subject in the field of biotic stress study. These studies are helpful in revealing significance of genetic control that can be beneficial to engineer crops tolerance to biotic stress.
PubMed: 29479367
DOI: 10.3389/fpls.2018.00146 -
Pokeweed antiviral protein: its cytotoxicity mechanism and applications in plant disease resistance.Toxins Mar 2015Pokeweed antiviral protein (PAP) is a 29 kDa type I ribosome inactivating protein (RIP) found in pokeweed plants. Pokeweed produces different forms of PAP. This review... (Review)
Review
Pokeweed antiviral protein (PAP) is a 29 kDa type I ribosome inactivating protein (RIP) found in pokeweed plants. Pokeweed produces different forms of PAP. This review focuses on the spring form of PAP isolated from Phytolacca americana leaves. PAP exerts its cytotoxicity by removing a specific adenine from the α-sarcin/ricin loop of the large ribosomal RNA. Besides depurination of the rRNA, PAP has additional activities that contribute to its cytotoxicity. The mechanism of PAP cytotoxicity is summarized based on evidence from the analysis of transgenic plants and the yeast model system. PAP was initially found to be anti-viral when it was co-inoculated with plant viruses onto plants. Transgenic plants expressing PAP and non-toxic PAP mutants have displayed broad-spectrum resistance to both viral and fungal infection. The mechanism of PAP-induced disease resistance in transgenic plants is summarized.
Topics: Amino Acid Sequence; Disease Resistance; Molecular Sequence Data; Phytolacca americana; Plant Diseases; Plant Leaves; Plants, Genetically Modified; Ribosome Inactivating Proteins, Type 1; Saccharomyces cerevisiae; Sequence Alignment
PubMed: 25756953
DOI: 10.3390/toxins7030755