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Plant Physiology May 2020Jasmonate-induced protein 60 (JIP60) is a ribosome-inactivating protein (RIP) from barley () and is involved in the plant immune response dependent on jasmonate...
Jasmonate-induced protein 60 (JIP60) is a ribosome-inactivating protein (RIP) from barley () and is involved in the plant immune response dependent on jasmonate hormones. Here, we demonstrate in that transient expression of the N-terminal domain of JIP60, from which the inhibitor domain (amino acids 163-185) is removed, initiates cell death, leading to extensive necrosis of leaf tissues. We used structure prediction of JIP60 to identify potential catalytic amino acids in the active site and tested these by mutagenesis and in planta assays of necrosis induction by expression in , as well as through an in vitro translation-inactivation assay. We found that Tyr 96, Glu 201, Arg 204, and Trp 234 in the presumptive active site of JIP60 are conserved in 815 plant RIPs in the Pfam database that were identified by HUMMR as containing a RIP domain. When these amino acid residues are individually mutated, the necrosis-inducing activity is completely abolished. We therefore propose that the role of these amino acids in JIP60 activity is to depurinate adenosine in ribosomes. This study provides insight into the catalytic mechanism of JIP60.
Topics: Catalytic Domain; Hordeum; Plant Leaves; Plant Proteins; Ribosomes; Nicotiana
PubMed: 32123042
DOI: 10.1104/pp.19.01029 -
International Journal of Molecular... Feb 2021Ribosome-inactivating proteins (RIPs) are a class of cytotoxic enzymes that can inhibit protein translation by depurinating rRNA. Most plant RIPs are synthesized with a...
Ribosome-inactivating proteins (RIPs) are a class of cytotoxic enzymes that can inhibit protein translation by depurinating rRNA. Most plant RIPs are synthesized with a leader sequence that sequesters the proteins to a cell compartment away from the host ribosomes. However, several rice RIPs lack these signal peptides suggesting they reside in the cytosol in close proximity to the plant ribosomes. This paper aims to elucidate the physiological function of two nucleocytoplasmic RIPs from rice, in particular, the type 1 RIP referred to as OsRIP1 and a presumed type 3 RIP called nuRIP. Transgenic rice lines overexpressing these RIPs were constructed and studied for developmental effects resulting from this overexpression under greenhouse conditions. In addition, the performance of transgenic seedlings in response to drought, salt, abscisic acid and methyl jasmonate treatment was investigated. Results suggest that both RIPs can affect methyl jasmonate mediated stress responses.
Topics: Abscisic Acid; Acetates; Cyclopentanes; Cytosol; Gene Expression Regulation, Plant; Green Fluorescent Proteins; Oryza; Oxylipins; Phenotype; Plant Proteins; Plants, Genetically Modified; Protein Biosynthesis; Ribosomes; Salts; Saporins; Seedlings; Stress, Physiological
PubMed: 33535383
DOI: 10.3390/ijms22031434 -
Molecules (Basel, Switzerland) Oct 2019A wide range of endogenous and exogenous alkylating agents attack DNA to generate various alkylation adducts. N7-methyl-2-deoxyguanosine (Fm7dG) is the most abundant...
A wide range of endogenous and exogenous alkylating agents attack DNA to generate various alkylation adducts. N7-methyl-2-deoxyguanosine (Fm7dG) is the most abundant alkylative DNA lesion. If not repaired, Fm7dG can undergo spontaneous depurination, imidazole ring-opening, or bypass by translesion synthesis DNA polymerases. Human DNA polymerase η (polη) efficiently catalyzes across Fm7dG in vitro, but its structural basis is unknown. Herein, we report a crystal structure of polη in complex with templating Fm7dG and an incoming nonhydrolyzable dCTP analog, where a 2'-fluorine-mediated transition destabilization approach was used to prevent the spontaneous depurination of Fm7dG. The structure showed that polη readily accommodated the Fm7dG:dCTP base pair with little conformational change of protein and DNA. In the catalytic site, Fm7dG and dCTP formed three hydrogen bonds with a Watson-Crick geometry, indicating that the major keto tautomer of Fm7dG is involved in base pairing. The polη-Fm7dG:dCTP structure was essentially identical to the corresponding undamaged structure, which explained the efficient bypass of the major methylated lesion. Overall, the first structure of translesion synthesis DNA polymerase bypassing Fm7dG suggests that in the catalytic site of Y-family DNA polymerases, small N7-alkylguanine adducts may be well tolerated and form the canonical Watson-Crick base pair with dCTP through their keto tautomers.
Topics: Alkylation; Base Pairing; Catalytic Domain; DNA; DNA Damage; DNA-Directed DNA Polymerase; Deoxycytosine Nucleotides; Deoxyguanosine; Humans; Kinetics; Metals; Models, Molecular; Nucleic Acid Conformation
PubMed: 31683505
DOI: 10.3390/molecules24213928 -
Analytical Chemistry Dec 2019DNA-DNA crosslinks, especially interstrand crosslinks (ICLs), cause cytotoxicity via blocking replication and transcription. Most measurements of ICLs lack sensitivity...
DNA-DNA crosslinks, especially interstrand crosslinks (ICLs), cause cytotoxicity via blocking replication and transcription. Most measurements of ICLs lack sensitivity and structural information. Here, a high resolution, accurate mass spectrometry (HRMS) method was developed to comprehensively determine the untargeted, totality of DNA crosslinks, a.k.a. DNA crosslinkomics. Two novel features were introduced into this method: the accurate mass neutral losses of both two 2-deoxyribose (dR) and one dR groups will screen for ICLs as modified dinucleosides; the accurate mass neutral losses of both of the two nucleobases and one nucleobase will detect unstable DNA crosslinks, that could undergo depurination. Our crosslinkomics approach was tested by screening for crosslinks in formaldehyde- and chlorambucil-treated calf thymus DNA. The results showed that all expected drug-bridged crosslinks were detected successfully, along with various unexpected crosslinks. Using HRMS, the molecular formula and chemical structures of these unexpected crosslinks were determined. The formation of apurinic/apyrimidinic (AP) site-derived crosslinks, at levels comparable to those for drug-bridged crosslinks, highlighted their novel, potential role in cytotoxicity. Our new crosslinkomics approach can detect expected and unexpected environmental and drug-induced crosslinks in biological samples. This broadens the existing cellular DNA adductome and offers the potential to become a powerful tool in precision medicine.
Topics: Animals; Cattle; Chlorambucil; Chromatography, Liquid; Cross-Linking Reagents; DNA; Formaldehyde; Mass Spectrometry
PubMed: 31670503
DOI: 10.1021/acs.analchem.9b04068 -
Computational and Structural... 2021Ribonucleotides (rNTPs) are predicted to be incorporated into the genome at a rate of up to 3 million times per cell division, making rNTPs the most common non-standard...
Ribonucleotides (rNTPs) are predicted to be incorporated into the genome at a rate of up to 3 million times per cell division, making rNTPs the most common non-standard nucleotide in the human genome. Typically, misinserted ribonucleotides are repaired by the ribonucleotide excision repair (RER) pathway, which is initiated by RNase H2 cleavage. However, rNTPs are susceptible to spontaneous depurination generating abasic ribonucleotides (rAPs), which are unable to be processed by RNase H2. Additionally, rAPs have been found in nascent RNA and coupled to R-loops. Recent work identified that base excision repair (BER) protein AP-Endonuclease 1 (APE1) is responsible for the initial processing of rAPs embedded in DNA and in R-loops. APE1 is a well characterized AP endonuclease that cleaves 5' of abasic sites, but its ability to cleave at rAPs remains poorly understood. Here, we utilize enzyme kinetics, X-ray crystallography, and molecular dynamics simulations to provide insight into rAP processing by APE1. Enzyme kinetics were used to determine pre-steady-state rates of APE1 cleavage on DNA substrates containing rAP, revealing a decrease in activity compared to cleavage at a canonical deoxy-AP substrate. Using X-ray crystallography, we identified novel contacts between the rAP and the APE1 active site. We demonstrate that the rAP sugar pucker is accommodated in the active site in a C3'-endo conformation, influencing its position and contributing to a decrease in activity compared to the deoxy-AP site. Together, this work provides molecular level insights into rAP processing by APE1 and advances our understanding of ribonucleotide processing within genomic DNA.
PubMed: 34188778
DOI: 10.1016/j.csbj.2021.05.035 -
PloS One 2022The Ricin toxin A chain (RTA), which depurinates an adenine base at a specific region of the ribosome leading to death, has two adjacent specificity pockets in its...
The Ricin toxin A chain (RTA), which depurinates an adenine base at a specific region of the ribosome leading to death, has two adjacent specificity pockets in its active site. Based on this structural information, many attempts have been made to develop small-molecule RTA inhibitors that simultaneously block the two pockets. However, no attempt has been successful. In the present study, we synthesized pterin-7-carboxamides with tripeptide pendants and found that one of them interacts with both pockets simultaneously to exhibit good RTA inhibitory activity. X-ray crystallographic analysis of the RTA crystal with the new inhibitor revealed that the conformational change of Tyr80 is an important factor that allows the inhibitors to plug the two pockets simultaneously.
Topics: Ricin; Pterins; Catalytic Domain; Crystallography, X-Ray; Ribosomes
PubMed: 36508422
DOI: 10.1371/journal.pone.0277770 -
Electrophoresis Mar 2020The long-term survival of RNA in postmortem tissues is a tricky topic. Many aged/forensic specimens show, in fact, high rates of null/inconclusive PCR-based results,...
The long-term survival of RNA in postmortem tissues is a tricky topic. Many aged/forensic specimens show, in fact, high rates of null/inconclusive PCR-based results, while reliable outcomes were sometimes achieved from archaeological samples. On the other hand, several data show that the RNA is a molecule that survives even to several physical-chemical stresses. In the present study, a simple protocol, which was already developed for the prolonged hydrolysis of DNA, was applied to a RNA sample extracted from blood. This protocol is based on the heat-mediated (70°C) hydrolysis for up to 36 h using ultrapure water and di-ethyl-pyro-carbonate-water as hydrolysis medium. Measurable levels of depurination were not found even if microfluidic devices showed a progressive pattern of degradation. The reverse transcription/quantitative PCR analysis of two (60 bp long) housekeeping targets (glyceraldehyde-3-phosphate dehydrogenase and porphobilinogen deaminase) showed that the percentage of amplifiable target (%AT) decreased in relation to the duration of the damaging treatment (r > 0.973). The comparison of the %AT in the degraded RNA and in the DNA samples that underwent the same damaging treatment showed that the %AT is always higher in RNA, reaching up to three orders of magnitude. Lastly, even the end-point PCR of blood-specific markers gave reliable results, which is in agreement with the body fluid origin of the sample. In conclusion, all the PCR-based results show that RNA maintains the ability to be retro-transcribed in short cDNA fragments even after 36 h of incubation at 70°C in mildly acidic buffers. It is therefore likely that the long-term survival of RNA samples depends mainly on the protection against RNAase attacks rather than on environmental factors (such as humidity and acidity) that are instead of great importance for the stability of DNA. As a final remark, our results suggest that the RNA analysis can be successfully performed even when DNA profiling failed.
Topics: DNA, Complementary; Forensic Genetics; Humans; Polymerase Chain Reaction; RNA; RNA Stability; Sensitivity and Specificity
PubMed: 31967656
DOI: 10.1002/elps.201900200 -
Frontiers in Pharmacology 2020Stenodactylin, a highly toxic type 2 ribosome-inactivating protein purified from the caudex of Harms, is a potential anticancer drug candidate. Previous studies...
Stenodactylin, a highly toxic type 2 ribosome-inactivating protein purified from the caudex of Harms, is a potential anticancer drug candidate. Previous studies demonstrated that stenodactylin induces apoptosis and necroptosis in treated cells, involving the production of reactive oxygen species. We analyzed the effect of stenodactylin on Raji and Ramos (Human Burkitt's lymphoma cells) and MOLM-13 (acute myeloid leukemia cells). Moreover, we focused on the early events in MOLM-13 cells that characterize the cellular response to the toxin by whole-genome microarray analysis of gene expression. Treatment with stenodactylin induced the depurination of 28S rRNA within 4 h and increased the phosphorylation of p38 and JNK. A time-dependent activation of caspase 1, 2, 8, 9, 3/7 was also observed. Genome-wide gene expression microarray analysis revealed early changes in the expression of genes involved in the regulation of cell death, inflammation and stress response. After 4 h, a significant increase of transcript level was detectable for ATF3, BTG2, DUSP1, EGR1, and JUN. Increased upstream JUN signaling was also confirmed at protein level. The early response to stenodactylin treatment involves inflammatory and apoptotic signaling compatible with the activation of multiple cell death pathways. Because of the above described properties toward acute myeloid leukemia cells, stenodactylin may be a promising candidate for the design of new immunoconjugates for experimental cancer treatment.
PubMed: 32457623
DOI: 10.3389/fphar.2020.00630 -
Life (Basel, Switzerland) Jul 2019Polymerization of nucleotides under prebiotically plausible conditions has been a focus of several origins of life studies. Non-activated nucleotides have been shown to...
Polymerization of nucleotides under prebiotically plausible conditions has been a focus of several origins of life studies. Non-activated nucleotides have been shown to undergo polymerization under geothermal conditions when subjected to dry-wet cycles. They do so by a mechanism similar to acid-catalyzed ester-bond formation. However, one study showed that the low pH of these reactions resulted in predominantly depurination, thereby resulting in the formation of abasic sites in the oligomers. In this study, we aimed to systematically characterize the nature of the oligomers that resulted in reactions that involved one or more of the canonical ribonucleotides. All the reactions analyzed showed the presence of abasic oligomers, with purine nucleotides being affected the most due to deglycosylation. Even in the reactions that contained nucleotide mixtures, the presence of abasic oligomers was detected, which suggested that information transfer would be severely hampered due to losing the capacity to base pair via H-bonds. Importantly, the stability of the -glycosidic linkage, under conditions used for dry-wet cycling, was also determined. Results from this study further strengthen the hypothesis that chemical evolution in a pre-RNA World would have been vital for the evolution of informational molecules of an RNA World. This is evident in the high degree of instability displayed by -glycosidic bonds of canonical purine ribonucleotides under the same geothermal conditions that otherwise readily favors polymerization. Significantly, the resultant product characterization in the reactions concerned underscores the difficulty associated with analyzing complex prebiotically relevant reactions due to inherent limitation of current analytical methods.
PubMed: 31277469
DOI: 10.3390/life9030057 -
Bioorganic & Medicinal Chemistry Feb 2024Ricin, a category-B agent for bioterrorism, and Shiga toxins (Stxs), which cause food poisoning bind to the ribosomal P-stalk to depurinate the sarcin/ricin loop. No...
Ricin, a category-B agent for bioterrorism, and Shiga toxins (Stxs), which cause food poisoning bind to the ribosomal P-stalk to depurinate the sarcin/ricin loop. No effective therapy exists for ricin or Stx intoxication. Ribosome binding sites of the toxins have not been targeted by small molecules. We previously identified CC10501, which inhibits toxin activity by binding the P-stalk pocket of ricin toxin A subunit (RTA) remote from the catalytic site. Here, we developed a fluorescence polarization assay and identified a new class of compounds, which bind P-stalk pocket of RTA with higher affinity and inhibit catalytic activity with submicromolar potency. A lead compound, RU-NT-206, bound P-stalk pocket of RTA with similar affinity as a five-fold larger P-stalk peptide and protected cells against ricin and Stx2 holotoxins for the first time. These results validate the P-stalk binding site of RTA as a critical target for allosteric inhibition of the active site.
Topics: Binding Sites; Peptides; Protein Binding; Ribosomes; Ricin
PubMed: 38340640
DOI: 10.1016/j.bmc.2024.117614