The Experts below are selected from a list of 273 Experts worldwide ranked by ideXlab platform
Nilgun E. Tumer - One of the best experts on this subject based on the ideXlab platform.
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ribosome Depurination by ricin leads to inhibition of endoplasmic reticulum stress induced hac1 mrna splicing on the ribosome
Journal of Biological Chemistry, 2019Co-Authors: Michael Pierce, Diana Vengsarkar, John E Mclaughlin, Jennifer Nielsen Kahn, Nilgun E. TumerAbstract: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 HAC1u (u 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 HAC1u mRNA splicing. A ribosome-binding mutant, which showed reduced binding to ribosomes but retained Depurination activity, inhibited HAC1u 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 HAC1u mRNA at the splice site junctions. Inhibition of the UPR correlated with the Depurination level, suggesting that ribosomes play a role in splicing of HAC1u mRNA. We show that HAC1u mRNA is associated with ribosomes and does not get processed on depurinated ribosomes, thereby inhibiting the UPR. These results demonstrate that RTA inhibits HAC1u mRNA splicing through its Depurination activity on the ribosome without directly affecting IRE1 oligomerization or the splicing reaction and provide evidence that IRE1 recognizes HAC1u mRNA that is associated with ribosomes.
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wild type rta and less toxic variants have distinct requirements for png1 for their Depurination activity and toxicity in saccharomyces cerevisiae
PLOS ONE, 2014Co-Authors: Xiaoping Li, Nilgun E. TumerAbstract:: Ricin A chain (RTA) undergoes retrograde trafficking and is postulated to use components of the endoplasmic reticulum (ER) associated degradation (ERAD) pathway to enter the cytosol to depurinate ribosomes. However, it is not known how RTA evades degradation by the proteasome after entry into the cytosol. We observed two distinct trafficking patterns among the precursor forms of wild type RTA and nontoxic variants tagged with enhanced green fluorescent protein (EGFP) at their C-termini in yeast. One group, which included wild type RTA, underwent ER-to-vacuole transport, while another group, which included the G83D variant, formed aggregates in the ER and was not transported to the vacuole. Peptide: N-glycanase (Png1), which catalyzes degradation of unfolded glycoproteins in the ERAD pathway affected Depurination activity and toxicity of wild type RTA and G83D variant differently. PreG83D variant was deglycosylated by Png1 on the ER membrane, which reduced its Depurination activity and toxicity by promoting its degradation. In contrast, wild type preRTA was deglycosylated by the free pool of Png1 in the cytosol, which increased its Depurination activity, possibly by preventing its degradation. These results indicate that wild type RTA has a distinct requirement for Png1 compared to the G83D variant and is deglycosylated by Png1 in the cytosol as a possible strategy to avoid degradation by the ERAD pathway to reach the ribosome.
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arginine residues on the opposite side of the active site stimulate the catalysis of ribosome Depurination by ricin a chain by interacting with the p protein stalk
Journal of Biological Chemistry, 2013Co-Authors: Xiaoping Li, Jennifer Nielsen Kahn, Peter C Kahn, Przemyslaw Grela, Nilgun E. TumerAbstract:Ricin inhibits protein synthesis by depurinating the α-sarcin/ricin loop (SRL). Ricin holotoxin does not inhibit translation unless the disulfide bond between the A (RTA) and B (RTB) subunits is reduced. Ricin holotoxin did not bind ribosomes or depurinate them but could depurinate free RNA. When RTA is separated from RTB, arginine residues located at the interface are exposed to the solvent. Because this positively charged region, but not the active site, is blocked by RTB, we mutated arginine residues at or near the interface of RTB to determine if they are critical for ribosome binding. These variants were structurally similar to wild type RTA but could not bind ribosomes. Their Km values and catalytic rates (kcat) for an SRL mimic RNA were similar to those of wild type, indicating that their activity was not altered. However, they showed an up to 5-fold increase in Km and up to 38-fold decrease in kcat toward ribosomes. These results suggest that the stalk binding stimulates the catalysis of ribosome Depurination by RTA. The mutated arginines have side chains behind the active site cleft, indicating that the ribosome binding surface of RTA is on the opposite side of the surface that interacts with the SRL. We propose that stalk binding stimulates the catalysis of ribosome Depurination by orienting the active site of RTA toward the SRL and thereby allows docking of the target adenine into the active site. This model may apply to the translation factors that interact with the stalk.
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a relatively low level of ribosome Depurination by mutant forms of ricin toxin a chain can trigger protein synthesis inhibition cell signaling and apoptosis in mammalian cells
The International Journal of Biochemistry & Cell Biology, 2012Co-Authors: Amanda E Jetzt, Nilgun E. Tumer, Jushun Cheng, Xiaoping Li, Wendie S CohickAbstract:The A chain of the plant toxin ricin (RTA) is an N-glycosidase that inhibits protein synthesis by removing a specific adenine from the 28S rRNA. RTA also induces ribotoxic stress, which activates stress-induced cell signaling cascades and apoptosis. However, the mechanistic relationship between Depurination, protein synthesis inhibition and apoptosis remains an open question. We previously identified two RTA mutants that suggested partial independence of these processes in a yeast model. The goals of this study were to establish an endogenous RTA expression system in mammalian cells and utilize RTA mutants to examine the relationship between Depurination, protein synthesis inhibition, cell signaling and apoptosis in mammalian cells. The non-transformed epithelial cell line MAC-T was transiently transfected with plasmid vectors encoding precursor (pre) or mature forms of wild-type (WT) RTA or mutants. PreRTA was glycosylated indicating that the native signal peptide targeted RTA to the ER in mammalian cells. Mature RTA was not glycosylated and thus served as a control to detect changes in catalytic activity. Both pre- and mature WT RTA induced ribosome Depurination, protein synthesis inhibition, activation of cell signaling and apoptosis. Analysis of RTA mutants showed for the first time that Depurination can be reduced by 40% in mammalian cells with minimal effects on inhibition of protein synthesis, activation of cell signaling and apoptosis. We further show that protein synthesis inhibition by RTA correlates more linearly with apoptosis than ribosome Depurination.
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shiga toxin 1 is more dependent on the p proteins of the ribosomal stalk for Depurination activity than shiga toxin 2
The International Journal of Biochemistry & Cell Biology, 2011Co-Authors: Jiachi Chiou, Xiaoping Li, Miguel Remacha, Juan P G Ballesta, Nilgun E. TumerAbstract:Abstract Shiga toxins produced by Escherichia coli O157:H7 are responsible for food poisoning and hemolytic uremic syndrome (HUS). The A subunits of Shiga toxins (Stx1A and Stx2A) inhibit translation by depurinating a specific adenine in the large rRNA. To determine if Stx1A and Stx2A require the ribosomal stalk for Depurination, their activity and cytotoxicity were examined in the yeast P protein deletion mutants. Stx1A and Stx2A were less toxic and depurinated ribosomes less in a strain lacking P1/P2 on the ribosome and in the cytosol (ΔP2) than in a strain lacking P1/P2 on the ribosome, but containing free P2 in the cytosol (ΔP1). To determine if cytoplasmic P proteins facilitated Depurination, Stx1A and Stx2A were expressed in the P0ΔAB mutant, in which the binding sites for P1/P2 were deleted on the ribosome, and P1/P2 accumulated in the cytosol. Stx1A was less toxic and depurinated ribosomes less in P0ΔAB, suggesting that intact binding sites for P1/P2 were critical. In contrast, Stx2A was toxic and depurinated ribosomes in P0ΔAB as in wild type, suggesting that it did not require the P1/P2 binding sites. Depurination of ΔP1, but not P0ΔAB ribosomes increased upon addition of purified P1α/P2β in vitro, and the increase was greater for Stx1 than for Stx2. We conclude that cytoplasmic P proteins stimulate Depurination by Stx1 by facilitating the access of the toxin to the ribosome. Although ribosomal stalk is important for Stx1 and Stx2 to depurinate the ribosome, Stx2 is less dependent on the stalk proteins for activity than Stx1 and can depurinate ribosomes with an incomplete stalk better than Stx1.
Katalin A. Hudak - One of the best experts on this subject based on the ideXlab platform.
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effect of Depurination on cellular and viral rna
2016Co-Authors: Kass A Jobst, Alexander Klenov, Kira C M Neller, Katalin A. HudakAbstract:Abasic sites in DNA arise from spontaneous damage and, if left unrepaired, contribute to mutations. Dedicated pathways have evolved to repair abasic DNA; however, the biological implications and cellular fate of RNA possessing these lesions are not well characterized. In this chapter, we review advances in the area of RNA Depurination, which produces the dominant form of abasic RNA in vivo. We discuss the biochemistry of RNA Depurination, the varied impacts of cellular and viral RNA Depurination, and the numerous methods available for detecting abasic RNA. Given the abundance of RNA in cells and the relative stability of abasic RNA compared with DNA, depurinated RNA likely has physiological significance. Recent work suggests a link between RNA Depurination and some age-related diseases as well as downstream signalling pathways that induce apoptosis. In addition, pathogens with RNA genomes are influenced by Depurination. For example, abasic sites of HIV-1 RNA may contribute to adaptation by mutation and recombination. Therefore, effects of RNA Depurination have medical relevance that warrants continued study.
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Depurination of brome mosaic virus rna3 inhibits its packaging into virus particles
Nucleic Acids Research, 2011Co-Authors: Rajita A Karran, Katalin A. HudakAbstract:Packaging of the segmented RNA genome of Brome mosaic virus (BMV) into discrete particles is an essential step in the virus life cycle; however, questions remain regarding the mechanism of RNA packaging and the degree to which the viral coat protein controls the process. In this study, we used a plant-derived glycosidase, Pokeweed antiviral protein, to remove 14 specific bases from BMV RNA3 to examine the effect of Depurination on virus assembly. Depurination of A771 within ORF3 and A1006 in the intergenic region inhibited coat protein binding and prevented RNA3 incorporation into particles. The disruption of interaction was not based on sequence identity, as mutation of these two purines to pyrimidines did not decrease coat protein-binding affinity. Rather, we suggest that base removal results in decreased thermodynamic stability of local RNA structures required for packaging, and that this instability is detected by coat protein. These results describe a new level of discrimination by coat protein, whereby it recognizes damage to specific viral RNA elements in the form of base removal and selects against incorporating the RNA into particles.
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homodimerization of pokeweed antiviral protein as a mechanism to limit Depurination of pokeweed ribosomes
Molecular Plant Pathology, 2010Co-Authors: Marina E Tourlakis, Rajita A Karran, Leroi V Desouza, Katalin A. HudakAbstract:SUMMARY Ribosome inactivating proteins are glycosidases synthesized by many plants and have been hypothesized to serve in defence against pathogens. These enzymes catalytically remove a conserved purine from the sarcin/ricin loop of the large ribosomal RNA, which has been shown in vitro to limit protein synthesis. The resulting toxicity suggests that plants may possess a mechanism to protect their ribosomes from Depurination during the synthesis of these enzymes. For example, pokeweed antiviral protein (PAP) is cotranslationally inserted into the lumen of the endoplasmic reticulum and travels via the endomembrane system to be stored in the cell wall. However, some PAP may retrotranslocate across the endoplasmic reticulum membrane to be released back into the cytosol, thereby exposing ribosomes to Depurination. In this work, we isolated and characterized a complexed form of the enzyme that exhibits substantially reduced activity. We showed that this complex is a homodimer of PAP and that dimerization involves a peptide that contains a conserved aromatic amino acid, tyrosine 123, located in the active site of the enzyme. Bimolecular fluorescence complementation demonstrated that the homodimer may form in vivo and that dimerization is prevented by the substitution of tyrosine 123 for alanine. The homodimer is a minor form of PAP, observed only in the cytosol of cells and not in the apoplast. Taken together, these data support a novel mechanism for the limitation of Depurination of autologous ribosomes by molecules of the protein that escape transport to the cell wall by the endomembrane system.
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Depurination within the intergenic region of brome mosaic virus rna3 inhibits viral replication in vitro and in vivo
Nucleic Acids Research, 2008Co-Authors: Rajita A Karran, Katalin A. HudakAbstract:: Pokeweed antiviral protein (PAP) is a glycosidase of plant origin that has been shown to depurinate some viral RNAs in vitro. We have demonstrated previously that treatment of Brome mosaic virus (BMV) RNAs with PAP inhibited their translation in a cell-free system and decreased their accumulation in barley protoplasts. In the current study, we map the Depurination sites on BMV RNA3 and describe the mechanism by which replication of the viral RNA is inhibited by Depurination. Specifically, we demonstrate that the viral replicase exhibited reduced affinity for depurinated positive-strand RNA3 compared with intact RNA3, resulting in less negative-strand product. This decrease was due to Depurination within the intergenic region of RNA3, between ORF3 and 4, and distant from the 3' terminal core promoter required for initiation of negative-strand RNA synthesis. Depurination within the intergenic region alone inhibited the binding of the replicase to full-length RNA3, whereas Depurination outside the intergenic region permitted the replicase to initiate negative-strand synthesis; however, elongation of the RNA product was stalled at the abasic nucleotide. These results support a role of the intergenic region in controlling negative-strand RNA synthesis and contribute new insight into the effect of Depurination by PAP on BMV replication.
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Depurination of brome mosaic virus rna3 in vivo results in translation dependent accelerated degradation of the viral rna
Journal of Biological Chemistry, 2008Co-Authors: Rikesh Gandhi, Mahira Manzoor, Katalin A. HudakAbstract:Abstract Pokeweed antiviral protein (PAP) is a ribosome-inactivating protein isolated from the pokeweed plant (Phytolacca americana) that exhibits antiviral activity against several plant and animal viruses. We have shown previously that PAP depurinates Brome mosaic virus (BMV) RNAs in vitro and that prior incubation of these RNAs with PAP reduced their synthesis in barley protoplasts. To investigate the post-transcriptional effect of PAP on viral RNA in vivo, we transcribed BMV RNA3 and expressed PAP in the yeast, Saccharomyces cerevisiae, which is a surrogate host for BMV. With an inducible transcription system, we show that the half-life of RNA3 in PAP-expressing cells was significantly less than in cells expressing PAPx, its enzymatically inactive form. PAP bound to RNA3 and depurinated the RNA within open reading frames 3 and 4 and within untranslated regions of the RNA. The depurinated RNA was associated with polysomes, caused ribosomes to stall at the point of Depurination, and was targeted for accelerated degradation by components of the No-go decay pathway. As a consequence of translation elongation arrest and increased RNA degradation, expression of PAP in yeast also decreased the level of protein 3a, encoded by the 5′-proximal open reading frame 3 of BMV RNA3. These data provide the first evidence of viral RNA Depurination in vivo by any ribosome-inactivating protein and support our hypothesis that Depurination contributes to the antiviral activity of PAP, by enhancing viral RNA degradation and reducing translation of viral protein product.
Xiaoping Li - One of the best experts on this subject based on the ideXlab platform.
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wild type rta and less toxic variants have distinct requirements for png1 for their Depurination activity and toxicity in saccharomyces cerevisiae
PLOS ONE, 2014Co-Authors: Xiaoping Li, Nilgun E. TumerAbstract:: Ricin A chain (RTA) undergoes retrograde trafficking and is postulated to use components of the endoplasmic reticulum (ER) associated degradation (ERAD) pathway to enter the cytosol to depurinate ribosomes. However, it is not known how RTA evades degradation by the proteasome after entry into the cytosol. We observed two distinct trafficking patterns among the precursor forms of wild type RTA and nontoxic variants tagged with enhanced green fluorescent protein (EGFP) at their C-termini in yeast. One group, which included wild type RTA, underwent ER-to-vacuole transport, while another group, which included the G83D variant, formed aggregates in the ER and was not transported to the vacuole. Peptide: N-glycanase (Png1), which catalyzes degradation of unfolded glycoproteins in the ERAD pathway affected Depurination activity and toxicity of wild type RTA and G83D variant differently. PreG83D variant was deglycosylated by Png1 on the ER membrane, which reduced its Depurination activity and toxicity by promoting its degradation. In contrast, wild type preRTA was deglycosylated by the free pool of Png1 in the cytosol, which increased its Depurination activity, possibly by preventing its degradation. These results indicate that wild type RTA has a distinct requirement for Png1 compared to the G83D variant and is deglycosylated by Png1 in the cytosol as a possible strategy to avoid degradation by the ERAD pathway to reach the ribosome.
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arginine residues on the opposite side of the active site stimulate the catalysis of ribosome Depurination by ricin a chain by interacting with the p protein stalk
Journal of Biological Chemistry, 2013Co-Authors: Xiaoping Li, Jennifer Nielsen Kahn, Peter C Kahn, Przemyslaw Grela, Nilgun E. TumerAbstract:Ricin inhibits protein synthesis by depurinating the α-sarcin/ricin loop (SRL). Ricin holotoxin does not inhibit translation unless the disulfide bond between the A (RTA) and B (RTB) subunits is reduced. Ricin holotoxin did not bind ribosomes or depurinate them but could depurinate free RNA. When RTA is separated from RTB, arginine residues located at the interface are exposed to the solvent. Because this positively charged region, but not the active site, is blocked by RTB, we mutated arginine residues at or near the interface of RTB to determine if they are critical for ribosome binding. These variants were structurally similar to wild type RTA but could not bind ribosomes. Their Km values and catalytic rates (kcat) for an SRL mimic RNA were similar to those of wild type, indicating that their activity was not altered. However, they showed an up to 5-fold increase in Km and up to 38-fold decrease in kcat toward ribosomes. These results suggest that the stalk binding stimulates the catalysis of ribosome Depurination by RTA. The mutated arginines have side chains behind the active site cleft, indicating that the ribosome binding surface of RTA is on the opposite side of the surface that interacts with the SRL. We propose that stalk binding stimulates the catalysis of ribosome Depurination by orienting the active site of RTA toward the SRL and thereby allows docking of the target adenine into the active site. This model may apply to the translation factors that interact with the stalk.
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a relatively low level of ribosome Depurination by mutant forms of ricin toxin a chain can trigger protein synthesis inhibition cell signaling and apoptosis in mammalian cells
The International Journal of Biochemistry & Cell Biology, 2012Co-Authors: Amanda E Jetzt, Nilgun E. Tumer, Jushun Cheng, Xiaoping Li, Wendie S CohickAbstract:The A chain of the plant toxin ricin (RTA) is an N-glycosidase that inhibits protein synthesis by removing a specific adenine from the 28S rRNA. RTA also induces ribotoxic stress, which activates stress-induced cell signaling cascades and apoptosis. However, the mechanistic relationship between Depurination, protein synthesis inhibition and apoptosis remains an open question. We previously identified two RTA mutants that suggested partial independence of these processes in a yeast model. The goals of this study were to establish an endogenous RTA expression system in mammalian cells and utilize RTA mutants to examine the relationship between Depurination, protein synthesis inhibition, cell signaling and apoptosis in mammalian cells. The non-transformed epithelial cell line MAC-T was transiently transfected with plasmid vectors encoding precursor (pre) or mature forms of wild-type (WT) RTA or mutants. PreRTA was glycosylated indicating that the native signal peptide targeted RTA to the ER in mammalian cells. Mature RTA was not glycosylated and thus served as a control to detect changes in catalytic activity. Both pre- and mature WT RTA induced ribosome Depurination, protein synthesis inhibition, activation of cell signaling and apoptosis. Analysis of RTA mutants showed for the first time that Depurination can be reduced by 40% in mammalian cells with minimal effects on inhibition of protein synthesis, activation of cell signaling and apoptosis. We further show that protein synthesis inhibition by RTA correlates more linearly with apoptosis than ribosome Depurination.
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shiga toxin 1 is more dependent on the p proteins of the ribosomal stalk for Depurination activity than shiga toxin 2
The International Journal of Biochemistry & Cell Biology, 2011Co-Authors: Jiachi Chiou, Xiaoping Li, Miguel Remacha, Juan P G Ballesta, Nilgun E. TumerAbstract:Abstract Shiga toxins produced by Escherichia coli O157:H7 are responsible for food poisoning and hemolytic uremic syndrome (HUS). The A subunits of Shiga toxins (Stx1A and Stx2A) inhibit translation by depurinating a specific adenine in the large rRNA. To determine if Stx1A and Stx2A require the ribosomal stalk for Depurination, their activity and cytotoxicity were examined in the yeast P protein deletion mutants. Stx1A and Stx2A were less toxic and depurinated ribosomes less in a strain lacking P1/P2 on the ribosome and in the cytosol (ΔP2) than in a strain lacking P1/P2 on the ribosome, but containing free P2 in the cytosol (ΔP1). To determine if cytoplasmic P proteins facilitated Depurination, Stx1A and Stx2A were expressed in the P0ΔAB mutant, in which the binding sites for P1/P2 were deleted on the ribosome, and P1/P2 accumulated in the cytosol. Stx1A was less toxic and depurinated ribosomes less in P0ΔAB, suggesting that intact binding sites for P1/P2 were critical. In contrast, Stx2A was toxic and depurinated ribosomes in P0ΔAB as in wild type, suggesting that it did not require the P1/P2 binding sites. Depurination of ΔP1, but not P0ΔAB ribosomes increased upon addition of purified P1α/P2β in vitro, and the increase was greater for Stx1 than for Stx2. We conclude that cytoplasmic P proteins stimulate Depurination by Stx1 by facilitating the access of the toxin to the ribosome. Although ribosomal stalk is important for Stx1 and Stx2 to depurinate the ribosome, Stx2 is less dependent on the stalk proteins for activity than Stx1 and can depurinate ribosomes with an incomplete stalk better than Stx1.
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pentameric organization of the ribosomal stalk accelerates recruitment of ricin a chain to the ribosome for Depurination
Journal of Biological Chemistry, 2010Co-Authors: Xiaoping Li, Marek Tchorzewski, Przemyslaw Grela, Dawid Krokowski, Nilgun E. TumerAbstract:Ribosome inactivating proteins (RIPs) depurinate a universally conserved adenine in the α-sarcin/ricin loop (SRL) and inhibit protein synthesis at the translation elongation step. We previously showed that ribosomal stalk is required for Depurination of the SRL by ricin toxin A chain (RTA). The interaction between RTA and ribosomes was characterized by a two-step binding model, where the stalk structure could be considered as an important interacting element. Here, using purified yeast ribosomal stalk complexes assembled in vivo, we show a direct interaction between RTA and the isolated stalk complex. Detailed kinetic analysis of these interactions in real time using surface plasmon resonance (SPR) indicated that there is only one type of interaction between RTA and the ribosomal stalk, which represents one of the two binding steps of the interaction with ribosomes. Interactions of RTA with the isolated stalk were relatively insensitive to salt, indicating that nonelectrostatic interactions were dominant. We compared the interaction of RTA with the full pentameric stalk complex containing two pairs of P1/P2 proteins with its interaction with the trimeric stalk complexes containing only one pair of P1/P2 and found that the rate of association of RTA with the pentamer was higher than with either trimer. These results demonstrate that the stalk is the main landing platform for RTA on the ribosome and that pentameric organization of the stalk accelerates recruitment of RTA to the ribosome for Depurination. Our results suggest that multiple copies of the stalk proteins might also increase the scavenging ability of the ribosome for the translational GTPases.
Rajita A Karran - One of the best experts on this subject based on the ideXlab platform.
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Depurination of brome mosaic virus rna3 inhibits its packaging into virus particles
Nucleic Acids Research, 2011Co-Authors: Rajita A Karran, Katalin A. HudakAbstract:Packaging of the segmented RNA genome of Brome mosaic virus (BMV) into discrete particles is an essential step in the virus life cycle; however, questions remain regarding the mechanism of RNA packaging and the degree to which the viral coat protein controls the process. In this study, we used a plant-derived glycosidase, Pokeweed antiviral protein, to remove 14 specific bases from BMV RNA3 to examine the effect of Depurination on virus assembly. Depurination of A771 within ORF3 and A1006 in the intergenic region inhibited coat protein binding and prevented RNA3 incorporation into particles. The disruption of interaction was not based on sequence identity, as mutation of these two purines to pyrimidines did not decrease coat protein-binding affinity. Rather, we suggest that base removal results in decreased thermodynamic stability of local RNA structures required for packaging, and that this instability is detected by coat protein. These results describe a new level of discrimination by coat protein, whereby it recognizes damage to specific viral RNA elements in the form of base removal and selects against incorporating the RNA into particles.
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homodimerization of pokeweed antiviral protein as a mechanism to limit Depurination of pokeweed ribosomes
Molecular Plant Pathology, 2010Co-Authors: Marina E Tourlakis, Rajita A Karran, Leroi V Desouza, Katalin A. HudakAbstract:SUMMARY Ribosome inactivating proteins are glycosidases synthesized by many plants and have been hypothesized to serve in defence against pathogens. These enzymes catalytically remove a conserved purine from the sarcin/ricin loop of the large ribosomal RNA, which has been shown in vitro to limit protein synthesis. The resulting toxicity suggests that plants may possess a mechanism to protect their ribosomes from Depurination during the synthesis of these enzymes. For example, pokeweed antiviral protein (PAP) is cotranslationally inserted into the lumen of the endoplasmic reticulum and travels via the endomembrane system to be stored in the cell wall. However, some PAP may retrotranslocate across the endoplasmic reticulum membrane to be released back into the cytosol, thereby exposing ribosomes to Depurination. In this work, we isolated and characterized a complexed form of the enzyme that exhibits substantially reduced activity. We showed that this complex is a homodimer of PAP and that dimerization involves a peptide that contains a conserved aromatic amino acid, tyrosine 123, located in the active site of the enzyme. Bimolecular fluorescence complementation demonstrated that the homodimer may form in vivo and that dimerization is prevented by the substitution of tyrosine 123 for alanine. The homodimer is a minor form of PAP, observed only in the cytosol of cells and not in the apoplast. Taken together, these data support a novel mechanism for the limitation of Depurination of autologous ribosomes by molecules of the protein that escape transport to the cell wall by the endomembrane system.
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Depurination within the intergenic region of brome mosaic virus rna3 inhibits viral replication in vitro and in vivo
Nucleic Acids Research, 2008Co-Authors: Rajita A Karran, Katalin A. HudakAbstract:: Pokeweed antiviral protein (PAP) is a glycosidase of plant origin that has been shown to depurinate some viral RNAs in vitro. We have demonstrated previously that treatment of Brome mosaic virus (BMV) RNAs with PAP inhibited their translation in a cell-free system and decreased their accumulation in barley protoplasts. In the current study, we map the Depurination sites on BMV RNA3 and describe the mechanism by which replication of the viral RNA is inhibited by Depurination. Specifically, we demonstrate that the viral replicase exhibited reduced affinity for depurinated positive-strand RNA3 compared with intact RNA3, resulting in less negative-strand product. This decrease was due to Depurination within the intergenic region of RNA3, between ORF3 and 4, and distant from the 3' terminal core promoter required for initiation of negative-strand RNA synthesis. Depurination within the intergenic region alone inhibited the binding of the replicase to full-length RNA3, whereas Depurination outside the intergenic region permitted the replicase to initiate negative-strand synthesis; however, elongation of the RNA product was stalled at the abasic nucleotide. These results support a role of the intergenic region in controlling negative-strand RNA synthesis and contribute new insight into the effect of Depurination by PAP on BMV replication.
Jacques R Fresco - One of the best experts on this subject based on the ideXlab platform.
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A Role for the Mutagenic DNA Self-Catalyzed Depurination Mechanism in the Evolution of 7SL-Derived RNAs
Journal of Molecular Evolution, 2017Co-Authors: Maxwell P. Gold, Jacques R FrescoAbstract:The Alu element, the most prevalent SINE (short interspersed element) in the human genome, is one of the many RNA-encoding genes that evolved from the 7SL RNA gene. During analysis of the evolution of 7SL-derived RNAs, two distinct evolutionary intermediates capable of self-catalyzed DNA Depurination (SDP) were identified. These SDP sequences spontaneously create apurinic sites that can result in increased mutagenesis due to their error-prone repair. This DNA self-Depurination mechanism has been shown both in vitro and in vivo to lead to substitution and short frameshift mutations at a frequency that far exceeds their occurrence due to random errors in DNA replication. In both evolutionary intermediates, the same self-Depurination sequence overlaps motifs necessary for successful transcription and SRP9/14 (signal recognition particle) binding; hence, mutations in this region could disrupt RNA activity. Yet, the 7SL-derived RNAs that arose from the elements capable of SDP show significant diversity in this region, and every new sequence retains the transcription and SRP9/14-binding motifs, even as it has lost the SDP sequence. While some (but not all) of the mutagenesis can be alternatively attributed to CpG decay, the very fact that the self-depurinating sequences are selectively discarded in all cases suggests that this was evolutionarily motivated to prevent further destructive mutagenesis by the SDP mechanism.
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site specific self catalyzed dna Depurination a biological mechanism that leads to mutations and creates sequence diversity
Annual Review of Biochemistry, 2017Co-Authors: Jacques R Fresco, Olga AmosovaAbstract:Self-catalyzed DNA Depurination is a sequence-specific physiological mechanism mediated by spontaneous extrusion of a stem-loop catalytic intermediate. Hydrolysis of the 5′G residue of the 5′GA/TGG loop and of the first 5′A residue of the 5′GAGA loop, together with particular first stem base pairs, specifies their hydrolysis without involving protein, cofactor, or cation. As such, this mechanism is the only known DNA catalytic activity exploited by nature. The consensus sequences for self-Depurination of such G- and A-loop residues occur in all genomes examined across the phyla, averaging one site every 2,000–4,000 base pairs. Because apurinic sites are subject to error-prone repair, leading to substitution and short frameshift mutations, they are both a source of genome damage and a means for creating sequence diversity. Their marked overrepresentation in genomes, and largely unchanging density from the lowest to the highest organisms, indicate their selection over the course of evolution. The mutagenici...
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why the dna self Depurination mechanism operates in hb β but not in β globin paralogs hb δ hb ɛ1 hb γ1 and hb γ2
Mutation Research, 2015Co-Authors: Olga Amosova, Juan R Alvarezdominguez, Jacques R FrescoAbstract:Abstract The human β-globin, δ-globin and ɛ-globin genes contain almost identical coding strand sequences centered about codon 6 having potential to form a stem-loop with a 5′GAGG loop. Provided with a sufficiently stable stem, such a structure can self-catalyze Depurination of the loop 5′G residue, leading to a potential mutation hotspot. Previously, we showed that such a hotspot exists about codon 6 of β-globin, with by far the highest incidence of mutations across the gene, including those responsible for 6 anemias (notably Sickle Cell Anemia) and β-thalassemias. In contrast, we show here that despite identical loop sequences, there is no mutational hotspot in the δ- or ɛ1-globin potential self-Depurination sites, which differ by only one or two base pairs in the stem region from that of the β-globin gene. These differences result in either one or two additional mismatches in the potential 7-base pair-forming stem region, thereby weakening its stability, so that either DNA cruciform extrusion from the duplex is rendered ineffective or the lifetime of the stem-loop becomes too short to permit self-catalysis to occur. Having that same loop sequence, paralogs HB-γ1 and HB-γ2 totally lack stem-forming potential. Hence the absence in δ- and ɛ1-globin genes of a mutational hotspot in what must now be viewed as non-functional homologs of the self-Depurination site in β-globin. Such stem-destabilizing variants appeared early among vertebrates and remained conserved among mammals and primates. Thus, this study has revealed conserved sequence determinants of self-catalytic DNA Depurination associated with variability of mutation incidence among human β-globin paralogs.
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self catalytic dna Depurination underlies human β globin gene mutations at codon 6 that cause anemias and thalassemias
Journal of Biological Chemistry, 2013Co-Authors: Juan R Alvarezdominguez, Olga Amosova, Jacques R FrescoAbstract:The human β-globin gene contains an 18-nucleotide coding strand sequence centered at codon 6 and capable of forming a stem-loop structure that can self-catalyze Depurination of the 5′G residue of that codon. The resultant apurinic lesion is subject to error-prone repair, consistent with the occurrence about this codon of mutations responsible for 6 anemias and β-thalassemias and additional substitutions without clinical consequences. The 4-residue loop of this stem-loop-forming sequence shows the highest incidence of mutation across the gene. The loop and first stem base pair-forming residues appeared early in the mammalian clade. The other stem-forming segments evolved more recently among primates, thereby conferring self-Depurination capacity at codon 6. These observations indicate a conserved molecular mechanism leading to β-globin variants underlying phenotypic diversity and disease.
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self catalyzed site specific Depurination of g residues mediated by cruciform extrusion in closed circular dna plasmids
Journal of Biological Chemistry, 2011Co-Authors: Olga Amosova, Veena Kumar, Aaron Deutsch, Jacques R FrescoAbstract:A major variety of “spontaneous” genomic damage is endogenous generation of apurinic sites. Depurination rates vary widely across genomes, occurring with higher frequency at “Depurination hot spots.” Recently, we discovered a site-specific self-catalyzed depurinating activity in short (14–18 nucleotides) DNA stem-loop-forming sequences with a 5′-G(T/A)GG-3′ loop and T·A or G·C as the first base pair at the base of the loop; the 5′-G residue of the loop self-depurinates at least 105-fold faster than random “spontaneous” Depurination at pH 5. Formation of the catalytic intermediate for self-Depurination in double-stranded DNA requires a stem-loop to extrude as part of a cruciform. In this study, evidence is presented for self-catalyzed Depurination mediated by cruciform formation in plasmid DNA in vitro. Cruciform extrusion was confirmed, and its extent was quantitated by digestion of the plasmid with single strand-specific mung bean endonuclease, followed by restriction digestion and sequencing of resulting mung bean-generated fragments. Appearance of the apurinic site in the self-depurinating stem-loop was confirmed by digestion of plasmid DNA with apurinic endonuclease IV, followed by primer extension and/or PCR amplification to detect the endonuclease-generated strand break and identify its location. Self-catalyzed Depurination was contingent on the plasmid being supercoiled and was not observed in linearized plasmids, consistent with the presence of the extruded cruciform in the supercoiled plasmid and not in the linear one. These results indicate that self-catalyzed Depurination is not unique to single-stranded DNA; rather, it can occur in stem-loop structures extruding from double-stranded DNA and therefore could, in principle, occur in vivo.