88 results on '"Chionh, Yok Hian"'
Search Results
2. Reciprocal regulation of TORC signaling and tRNA modifications by Elongator enforces nutrient-dependent cell fate
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Candiracci, Julie, Migeot, Valerie, Chionh, Yok-Hian, Bauer, Fanelie, Brochier, Thomas, Russell, Brandon, Shiozaki, Kazuhiro, Dedon, Peter, and Hermand, Damien
- Subjects
Biochemistry and Cell Biology ,Biological Sciences ,Underpinning research ,1.1 Normal biological development and functioning ,Cell Differentiation ,Cell Proliferation ,Gene Expression Regulation ,Fungal ,Glycogen Synthase Kinase 3 ,Mechanistic Target of Rapamycin Complex 1 ,Mechanistic Target of Rapamycin Complex 2 ,Mitosis ,Nutrients ,Peptide Chain Elongation ,Translational ,Phosphorylation ,RNA ,Transfer ,Schizosaccharomyces ,Signal Transduction - Abstract
Nutrient availability has a profound impact on cell fate. Upon nitrogen starvation, wild-type fission yeast cells uncouple cell growth from cell division to generate small, round-shaped cells that are competent for sexual differentiation. The TORC1 (TOR complex 1) and TORC2 complexes exert opposite controls on cell growth and cell differentiation, but little is known about how their activity is coordinated. We show that transfer RNA (tRNA) modifications by Elongator are critical for this regulation by promoting the translation of both key components of TORC2 and repressors of TORC1. We further identified the TORC2 pathway as an activator of Elongator by down-regulating a Gsk3 (glycogen synthase kinase 3)-dependent inhibitory phosphorylation of Elongator. Therefore, a feedback control is operating between TOR complex (TORC) signaling and tRNA modification by Elongator to enforce the advancement of mitosis that precedes cell differentiation.
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- 2019
3. Facile metabolic reprogramming distinguishes mycobacterial adaptation to hypoxia and starvation: The role of ketosis in starvation-induced persistence
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Dedon, Peter, primary, Davis, Nick, additional, Chionh, Yok Hian, additional, McBee, Megan, additional, Hia, Fabian, additional, Ma, Duanduan, additional, Liang, Cui, additional, Sharaf, Mariam, additional, Cai, Weiling Maggie, additional, Jumpathong, Watthanachai, additional, Levine, Stuart, additional, and Alonso, Sylvie, additional
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- 2023
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- View/download PDF
4. Phase 1 Trial of a Therapeutic Anti–Yellow Fever Virus Human Antibody
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Singapore-MIT Alliance in Research and Technology (SMART), Massachusetts Institute of Technology. Department of Biological Engineering, Low, Jenny G, Ng, Justin HJ, Ong, Eugenia Z, Kalimuddin, Shirin, Wijaya, Limin, Chan, Yvonne FZ, Ng, Dorothy HL, Tan, Hwee-Cheng, Baglody, Anjali, Chionh, Yok-Hian, Lee, Debbie CP, Budigi, Yadunanda, Sasisekharan, Ram, Ooi, Eng-Eong, Singapore-MIT Alliance in Research and Technology (SMART), Massachusetts Institute of Technology. Department of Biological Engineering, Low, Jenny G, Ng, Justin HJ, Ong, Eugenia Z, Kalimuddin, Shirin, Wijaya, Limin, Chan, Yvonne FZ, Ng, Dorothy HL, Tan, Hwee-Cheng, Baglody, Anjali, Chionh, Yok-Hian, Lee, Debbie CP, Budigi, Yadunanda, Sasisekharan, Ram, and Ooi, Eng-Eong
- Abstract
Copyright © 2020 Massachusetts Medical Society. BACKGROUND Insufficient vaccine doses and the lack of therapeutic agents for yellow fever put global health at risk, should this virus emerge from sub-Saharan Africa and South America. METHODS In phase 1a of this clinical trial, we assessed the safety, side-effect profile, and pharmacokinetics of TY014, a fully human IgG1 anti–yellow fever virus monoclonal antibody. In a double-blind, phase 1b clinical trial, we assessed the efficacy of TY014, as compared with placebo, in abrogating viremia related to the administration of live yellow fever vaccine (YF17D-204; Stamaril). The primary safety outcomes were adverse events reported 1 hour after the infusion and throughout the trial. The primary efficacy outcome was the dose of TY014 at which 100% of the participants tested negative for viremia within 48 hours after infusion. RESULTS A total of 27 healthy participants were enrolled in phase 1a, and 10 participants in phase 1b. During phase 1a, TY014 dose escalation to a maximum of 20 mg per kilogram of body weight occurred in 22 participants. During phases 1a and 1b, adverse events within 1 hour after infusion occurred in 1 of 27 participants who received TY014 and in none of the 10 participants who received placebo. At least one adverse event occurred during the trial in 22 participants who received TY014 and in 8 who received placebo. The mean half-life of TY014 was approximately 12.8 days. At 48 hours after the infusion, none of the 5 participants who received the starting dose of TY014 of 2 mg per kilogram had detectable YF17D-204 viremia; these participants remained aviremic throughout the trial. Viremia was observed at 48 hours after the infusion in 2 of 5 participants who received placebo and at 72 hours in 2 more placebo recipients. Symptoms associated with yellow fever vaccine were less frequent in the TY014 group than in the placebo group. CONCLUSIONS This phase 1 trial of TY014 did not identify worrisome safety si
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- 2022
5. Quantitative analysis of ribonucleoside modifications in tRNA by HPLC-coupled mass spectrometry
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Su, Dan, Chan, Clement T Y, Gu, Chen, Lim, Kok Seong, Chionh, Yok Hian, McBee, Megan E, Russell, Brandon S, Babu, I Ramesh, Begley, Thomas J, and Dedon, Peter C
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- 2014
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6. A Platform for Discovery and Quantification of Modified Ribonucleosides in RNA
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Cai, Weiling Maggie, primary, Chionh, Yok Hian, additional, Hia, Fabian, additional, Gu, Chen, additional, Kellner, Stefanie, additional, McBee, Megan E., additional, Ng, Chee Sheng, additional, Pang, Yan Ling Joy, additional, Prestwich, Erin G., additional, Lim, Kok Seong, additional, Ramesh Babu, I., additional, Begley, Thomas J., additional, and Dedon, Peter C., additional
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- 2015
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7. Mycobacterial RNA isolation optimized for non-coding RNA: high fidelity isolation of 5S rRNA from Mycobacterium bovis BCG reveals novel post-transcriptional processing and a complete spectrum of modified ribonucleosides
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Hia, Fabian, Chionh, Yok Hian, Pang, Yan Ling Joy, DeMott, Michael S., McBee, Megan E., and Dedon, Peter C.
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- 2015
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8. Phase 1 Trial of a Therapeutic Anti–Yellow Fever Virus Human Antibody
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Low, Jenny G, Ng, Justin HJ, Ong, Eugenia Z, Kalimuddin, Shirin, Wijaya, Limin, Chan, Yvonne FZ, Ng, Dorothy HL, Tan, Hwee-Cheng, Baglody, Anjali, Chionh, Yok-Hian, Lee, Debbie CP, Budigi, Yadunanda, Sasisekharan, Ram, Ooi, Eng-Eong, Low, Jenny G, Ng, Justin HJ, Ong, Eugenia Z, Kalimuddin, Shirin, Wijaya, Limin, Chan, Yvonne FZ, Ng, Dorothy HL, Tan, Hwee-Cheng, Baglody, Anjali, Chionh, Yok-Hian, Lee, Debbie CP, Budigi, Yadunanda, Sasisekharan, Ram, and Ooi, Eng-Eong
- Abstract
Copyright © 2020 Massachusetts Medical Society. BACKGROUND Insufficient vaccine doses and the lack of therapeutic agents for yellow fever put global health at risk, should this virus emerge from sub-Saharan Africa and South America. METHODS In phase 1a of this clinical trial, we assessed the safety, side-effect profile, and pharmacokinetics of TY014, a fully human IgG1 anti–yellow fever virus monoclonal antibody. In a double-blind, phase 1b clinical trial, we assessed the efficacy of TY014, as compared with placebo, in abrogating viremia related to the administration of live yellow fever vaccine (YF17D-204; Stamaril). The primary safety outcomes were adverse events reported 1 hour after the infusion and throughout the trial. The primary efficacy outcome was the dose of TY014 at which 100% of the participants tested negative for viremia within 48 hours after infusion. RESULTS A total of 27 healthy participants were enrolled in phase 1a, and 10 participants in phase 1b. During phase 1a, TY014 dose escalation to a maximum of 20 mg per kilogram of body weight occurred in 22 participants. During phases 1a and 1b, adverse events within 1 hour after infusion occurred in 1 of 27 participants who received TY014 and in none of the 10 participants who received placebo. At least one adverse event occurred during the trial in 22 participants who received TY014 and in 8 who received placebo. The mean half-life of TY014 was approximately 12.8 days. At 48 hours after the infusion, none of the 5 participants who received the starting dose of TY014 of 2 mg per kilogram had detectable YF17D-204 viremia; these participants remained aviremic throughout the trial. Viremia was observed at 48 hours after the infusion in 2 of 5 participants who received placebo and at 72 hours in 2 more placebo recipients. Symptoms associated with yellow fever vaccine were less frequent in the TY014 group than in the placebo group. CONCLUSIONS This phase 1 trial of TY014 did not identify worrisome safety si
- Published
- 2021
9. Pyruvate Kinase Regulates the Pentose-Phosphate Pathway in Response to Hypoxia in Mycobacterium tuberculosis
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Massachusetts Institute of Technology. Department of Biological Engineering, Zhong, Wenhe, Guo, Jingjing, Cui, Liang, Chionh, Yok Hian, Li, Kuohan, El Sahili, Abbas, Cai, Qixu, Yuan, Meng, Michels, Paul A.M., Fothergill-Gilmore, Linda A., Walkinshaw, Malcolm D., Mu, Yuguang, Lescar, Julien, Dedon, Peter C, Massachusetts Institute of Technology. Department of Biological Engineering, Zhong, Wenhe, Guo, Jingjing, Cui, Liang, Chionh, Yok Hian, Li, Kuohan, El Sahili, Abbas, Cai, Qixu, Yuan, Meng, Michels, Paul A.M., Fothergill-Gilmore, Linda A., Walkinshaw, Malcolm D., Mu, Yuguang, Lescar, Julien, and Dedon, Peter C
- Abstract
In response to the stress of infection, Mycobacterium tuberculosis (Mtb) reprograms its metabolism to accommodate nutrient and energetic demands in a changing environment. Pyruvate kinase (PYK) is an essential glycolytic enzyme in the phosphoenolpyruvate–pyruvate–oxaloacetate node that is a central switch point for carbon flux distribution. Here we show that the competitive binding of pentose monophosphate inhibitors or the activator glucose 6-phosphate (G6P) to MtbPYK tightly regulates the metabolic flux. Intriguingly, pentose monophosphates were found to share the same binding site with G6P. The determination of a crystal structure of MtbPYK with bound ribose 5-phosphate (R5P), combined with biochemical analyses and molecular dynamic simulations, revealed that the allosteric inhibitor pentose monophosphate increases PYK structural dynamics, weakens the structural network communication, and impairs substrate binding. G6P, on the other hand, primes and activates the tetramer by decreasing protein flexibility and strengthening allosteric coupling. Therefore, we propose that MtbPYK uses these differences in conformational dynamics to up- and down-regulate enzymic activity. Importantly, metabolome profiling in mycobacteria reveals a significant increase in the levels of pentose monophosphate during hypoxia, which provides insights into how PYK uses dynamics of the tetramer as a competitive allosteric mechanism to retard glycolysis and facilitate metabolic reprogramming toward the pentose-phosphate pathway for achieving redox balance and an anticipatory metabolic response in Mtb.
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- 2020
10. A multidimensional platform for the purification of non-coding RNA species
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Chionh, Yok Hian, Ho, Chia-Hua, Pruksakorn, Dumnoensun, Babu, Ramesh I., Ng, Chee Sheng, Hia, Fabian, McBee, Megan E., Su, Dan, Pang, Yan Ling Joy, Gu, Chen, Dong, Hongping, Prestwich, Erin G., Shi, Pei-Yong, Preiser, Peter Rainer, Alonso, Sylvie, and Dedon, Peter C.
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- 2013
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11. Phase 1 Trial of a Therapeutic Anti–Yellow Fever Virus Human Antibody
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Low, Jenny G., primary, Ng, Justin H.J., additional, Ong, Eugenia Z., additional, Kalimuddin, Shirin, additional, Wijaya, Limin, additional, Chan, Yvonne F.Z., additional, Ng, Dorothy H.L., additional, Tan, Hwee-Cheng, additional, Baglody, Anjali, additional, Chionh, Yok-Hian, additional, Lee, Debbie C.P., additional, Budigi, Yadunanda, additional, Sasisekharan, Ram, additional, and Ooi, Eng-Eong, additional
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- 2020
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12. Thienopyrimidinone Derivatives That Inhibit Bacterial tRNA (Guanine37-N¹)-Methyltransferase (TrmD) by Restructuring the Active Site with a Tyrosine-Flipping Mechanism
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Zhong, Wenhe, Pasunooti, Kalyan Kumar, Balamkundu, Seetharamsing, Wong, Yee Hwa, Nah, Qianhui, Gadi, Vinod, Gnanakalai, Shanmugavel, Chionh, Yok Hian, McBee, Megan E., Gopal, Pooja, Lim, Siau Hoi, Olivier, Nelson, Buurman, Ed T., Dick, Thomas, Liu, Chuan Fa, Lescar, Julien, Dedon, Peter C, and Massachusetts Institute of Technology. Department of Biological Engineering
- Abstract
Among the >120 modified ribonucleosides in the prokaryotic epitranscriptome, many tRNA modifications are critical to bacterial survival, which makes their synthetic enzymes ideal targets for antibiotic development. Here we performed a structure-based design of inhibitors of tRNA-(N1G37) methyltransferase, TrmD, which is an essential enzyme in many bacterial pathogens. On the basis of crystal structures of TrmDs from Pseudomonas aeruginosa and Mycobacterium tuberculosis, we synthesized a series of thienopyrimidinone derivatives with nanomolar potency against TrmD in vitro and discovered a novel active site conformational change triggered by inhibitor binding. This tyrosine-flipping mechanism is uniquely found in P. aeruginosa TrmD and renders the enzyme inaccessible to the cofactor S-adenosyl-l-methionine (SAM) and probably to the substrate tRNA. Biophysical and biochemical structure-activity relationship studies provided insights into the mechanisms underlying the potency of thienopyrimidinones as TrmD inhibitors, with several derivatives found to be active against Gram-positive and mycobacterial pathogens. These results lay a foundation for further development of TrmD inhibitors as antimicrobial agents.
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- 2019
13. Thienopyrimidinone Derivatives That Inhibit Bacterial tRNA (Guanine37-N¹)-Methyltransferase (TrmD) by Restructuring the Active Site with a Tyrosine-Flipping Mechanism
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Massachusetts Institute of Technology. Department of Biological Engineering, Zhong, Wenhe, Pasunooti, Kalyan Kumar, Balamkundu, Seetharamsing, Wong, Yee Hwa, Nah, Qianhui, Gadi, Vinod, Gnanakalai, Shanmugavel, Chionh, Yok Hian, McBee, Megan E., Gopal, Pooja, Lim, Siau Hoi, Olivier, Nelson, Buurman, Ed T., Dick, Thomas, Liu, Chuan Fa, Lescar, Julien, Dedon, Peter C, Massachusetts Institute of Technology. Department of Biological Engineering, Zhong, Wenhe, Pasunooti, Kalyan Kumar, Balamkundu, Seetharamsing, Wong, Yee Hwa, Nah, Qianhui, Gadi, Vinod, Gnanakalai, Shanmugavel, Chionh, Yok Hian, McBee, Megan E., Gopal, Pooja, Lim, Siau Hoi, Olivier, Nelson, Buurman, Ed T., Dick, Thomas, Liu, Chuan Fa, Lescar, Julien, and Dedon, Peter C
- Abstract
Among the >120 modified ribonucleosides in the prokaryotic epitranscriptome, many tRNA modifications are critical to bacterial survival, which makes their synthetic enzymes ideal targets for antibiotic development. Here we performed a structure-based design of inhibitors of tRNA-(N1G37) methyltransferase, TrmD, which is an essential enzyme in many bacterial pathogens. On the basis of crystal structures of TrmDs from Pseudomonas aeruginosa and Mycobacterium tuberculosis, we synthesized a series of thienopyrimidinone derivatives with nanomolar potency against TrmD in vitro and discovered a novel active site conformational change triggered by inhibitor binding. This tyrosine-flipping mechanism is uniquely found in P. aeruginosa TrmD and renders the enzyme inaccessible to the cofactor S-adenosyl-l-methionine (SAM) and probably to the substrate tRNA. Biophysical and biochemical structure-activity relationship studies provided insights into the mechanisms underlying the potency of thienopyrimidinones as TrmD inhibitors, with several derivatives found to be active against Gram-positive and mycobacterial pathogens. These results lay a foundation for further development of TrmD inhibitors as antimicrobial agents.
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- 2019
14. tRNA epitranscriptomics and biased codon are linked to proteome expression in
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Massachusetts Institute of Technology. Department of Biological Engineering, Ng, Chee Sheng, Sinha, Ameya, Gu, Chen, Babu, Indrakanti Ramesh, Chionh, Yok Hian, Dedon, Peter C, Aniweh, Yaw, Nah, Qianhui, Preiser, Peter R, Massachusetts Institute of Technology. Department of Biological Engineering, Ng, Chee Sheng, Sinha, Ameya, Gu, Chen, Babu, Indrakanti Ramesh, Chionh, Yok Hian, Dedon, Peter C, Aniweh, Yaw, Nah, Qianhui, and Preiser, Peter R
- Abstract
Among components of the translational machinery, ribonucleoside modifications on tRNAs are emerging as critical regulators of cell physiology and stress response. Here, we demonstrate highly coordinated behavior of the repertoire of tRNA modifications of Plasmodium falciparum throughout the intra-erythrocytic developmental cycle (IDC). We observed both a synchronized increase in 22 of 28 modifications from ring to trophozoite stage, consistent with tRNA maturation during translational up-regulation, and asynchronous changes in six modifications. Quantitative analysis of ~2,100 proteins across the IDC revealed that up- and down-regulated proteins in late but not early stages have a marked codon bias that directly correlates with parallel changes in tRNA modifications and enhanced translational efficiency. We thus propose a model in which tRNA modifications modulate the abundance of stage-specific proteins by enhancing translation efficiency of codon-biased transcripts for critical genes. These findings reveal novel epitranscriptomic and translational control mechanisms in the development and pathogenesis of Plasmodium parasites., Singapore. National Research Foundation, Singapore-MIT Alliance (Graduate Fellowship)
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- 2019
15. Pyruvate Kinase Regulates the Pentose-Phosphate Pathway in Response to Hypoxia in Mycobacterium tuberculosis
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Zhong, Wenhe, primary, Guo, Jingjing, additional, Cui, Liang, additional, Chionh, Yok Hian, additional, Li, Kuohan, additional, El Sahili, Abbas, additional, Cai, Qixu, additional, Yuan, Meng, additional, Michels, Paul A.M., additional, Fothergill-Gilmore, Linda A., additional, Walkinshaw, Malcolm D., additional, Mu, Yuguang, additional, Lescar, Julien, additional, and Dedon, Peter C., additional
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- 2019
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16. Thienopyrimidinone Derivatives That Inhibit Bacterial tRNA (Guanine37-N1)-Methyltransferase (TrmD) by Restructuring the Active Site with a Tyrosine-Flipping Mechanism
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Zhong, Wenhe, primary, Pasunooti, Kalyan Kumar, additional, Balamkundu, Seetharamsing, additional, Wong, Yee Hwa, additional, Nah, Qianhui, additional, Gadi, Vinod, additional, Gnanakalai, Shanmugavel, additional, Chionh, Yok Hian, additional, McBee, Megan E., additional, Gopal, Pooja, additional, Lim, Siau Hoi, additional, Olivier, Nelson, additional, Buurman, Ed T., additional, Dick, Thomas, additional, Liu, Chuan Fa, additional, Lescar, Julien, additional, and Dedon, Peter C., additional
- Published
- 2019
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17. Crystal structure and catalytic mechanism of the essential m1G37 tRNA methyltransferase TrmD from Pseudomonas aeruginosa
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Jaroensuk, Juthamas, primary, Wong, Yee Hwa, additional, Zhong, Wenhe, additional, Liew, Chong Wai, additional, Maenpuen, Somchart, additional, Sahili, Abbas E., additional, Atichartpongkul, Sopapan, additional, Chionh, Yok Hian, additional, Nah, Qianhui, additional, Thongdee, Narumon, additional, McBee, Megan E., additional, Prestwich, Erin G., additional, DeMott, Michael S., additional, Chaiyen, Pimchai, additional, Mongkolsuk, Skorn, additional, Dedon, Peter C., additional, Lescar, Julien, additional, and Fuangthong, Mayuree, additional
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- 2019
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18. Targeting the Bacterial Epitranscriptome for Antibiotic Development: Discovery of Novel tRNA-(N1G37) Methyltransferase (TrmD) Inhibitors
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Zhong, Wenhe, primary, Koay, Ann, additional, Ngo, Anna, additional, Li, Yan, additional, Nah, Qianhui, additional, Wong, Yee Hwa, additional, Chionh, Yok Hian, additional, Ng, Hui Qi, additional, Koh-Stenta, Xiaoying, additional, Poulsen, Anders, additional, Foo, Klement, additional, McBee, Megan, additional, Choong, Meng Ling, additional, El Sahili, Abbas, additional, Kang, Congbao, additional, Matter, Alex, additional, Lescar, Julien, additional, Hill, Jeffrey, additional, and Dedon, Peter, additional
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- 2019
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19. Rational Engineering and Characterization of an mAb that Neutralizes Zika Virus by Targeting a Mutationally Constrained Quaternary Epitope
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Massachusetts Institute of Technology. Department of Biological Engineering, Tharakaraman, Kannan, Subramanian, Vidya, Quinlan, Devin Scott, Sasisekharan, Ram, Watanabe, Satoru, Chan, Kuan Rong, Huan, Jia, Chionh, Yok Hian, Raguram, Aditya, McBee, Megan, Ong, Eugenia Z., Gan, Esther S., Tan, Hwee Cheng, Tyagi, Anu, Bhushan, Shashi, Lescar, Julien, Vasudevan, Subhash G., Ooi, Eng Eong, Massachusetts Institute of Technology. Department of Biological Engineering, Tharakaraman, Kannan, Subramanian, Vidya, Quinlan, Devin Scott, Sasisekharan, Ram, Watanabe, Satoru, Chan, Kuan Rong, Huan, Jia, Chionh, Yok Hian, Raguram, Aditya, McBee, Megan, Ong, Eugenia Z., Gan, Esther S., Tan, Hwee Cheng, Tyagi, Anu, Bhushan, Shashi, Lescar, Julien, Vasudevan, Subhash G., and Ooi, Eng Eong
- Abstract
Following the recent emergence of Zika virus (ZIKV), many murine and human neutralizing anti-ZIKV antibodies have been reported. Given the risk of virus escape mutants, engineering antibodies that target mutationally constrained epitopes with therapeutically relevant potencies can be valuable for combating future outbreaks. Here, we applied computational methods to engineer an antibody, ZAb_FLEP, that targets a highly networked and therefore mutationally constrained surface formed by the envelope protein dimer. ZAb_FLEP neutralized a breadth of ZIKV strains and protected mice in distinct in vivo models, including resolving vertical transmission and fetal mortality in infected pregnant mice. Serial passaging of ZIKV in the presence of ZAb_FLEP failed to generate viral escape mutants, suggesting that its epitope is indeed mutationally constrained. A single-particle cryo-EM reconstruction of the Fab-ZIKV complex validated the structural model and revealed insights into ZAb_FLEP's neutralization mechanism. ZAb_FLEP has potential as a therapeutic in future outbreaks. Tharakaraman et al. describe the engineering and validation of a neutralizing anti-Zika antibody (ZAb_FLEP) that targets a mutationally constrained surface epitope formed by the envelope protein. ZAb_FLEP neutralizes ZIKV strains in vitro and protects mice and unborn pups from Zika infection in vivo, indicating its potential as a therapeutic candidate., National Institutes of Health (U.S.) (Award 1R01AI111395), Singapore. National Research Foundation
- Published
- 2018
20. Allosteric pyruvate kinase-based “logic gate” synergistically senses energy and sugar levels in Mycobacterium tuberculosis
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Massachusetts Institute of Technology. Department of Biological Engineering, Dedon, Peter C, Zhong, Wenhe, Cui, Liang, Goh, Boon Chong, Cai, Qixu, Ho, Peiying, Chionh, Yok Hian, Yuan, Meng, Sahili, Abbas El, Fothergill-Gilmore, Linda A., Walkinshaw, Malcolm D., Lescar, Julien, Massachusetts Institute of Technology. Department of Biological Engineering, Dedon, Peter C, Zhong, Wenhe, Cui, Liang, Goh, Boon Chong, Cai, Qixu, Ho, Peiying, Chionh, Yok Hian, Yuan, Meng, Sahili, Abbas El, Fothergill-Gilmore, Linda A., Walkinshaw, Malcolm D., and Lescar, Julien
- Abstract
Pyruvate kinase (PYK) is an essential glycolytic enzyme that controls glycolytic flux and is critical for ATP production in all organisms, with tight regulation by multiple metabolites. Yet the allosteric mechanisms governing PYK activity in bacterial pathogens are poorly understood. Here we report biochemical, structural and metabolomic evidence that Mycobacterium tuberculosis (Mtb) PYK uses AMP and glucose-6-phosphate (G6P) as synergistic allosteric activators that function as a molecular "OR logic gate" to tightly regulate energy and glucose metabolism. G6P was found to bind to a previously unknown site adjacent to the canonical site for AMP. Kinetic data and structural network analysis further show that AMP and G6P work synergistically as allosteric activators. Importantly, metabolome profiling in the Mtb surrogate, Mycobacterium bovis BCG, reveals significant changes in AMP and G6P levels during nutrient deprivation, which provides insights into how a PYK OR gate would function during the stress of Mtb infection.
- Published
- 2018
21. tRNA epitranscriptomics and biased codon are linked to proteome expression in Plasmodium falciparum
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Ng, Chee Sheng, primary, Sinha, Ameya, additional, Aniweh, Yaw, additional, Nah, Qianhui, additional, Babu, Indrakanti Ramesh, additional, Gu, Chen, additional, Chionh, Yok Hian, additional, Dedon, Peter C, additional, and Preiser, Peter R, additional
- Published
- 2018
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22. Rational Engineering and Characterization of an mAb that Neutralizes Zika Virus by Targeting a Mutationally Constrained Quaternary Epitope
- Author
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Tharakaraman, Kannan, primary, Watanabe, Satoru, additional, Chan, Kuan Rong, additional, Huan, Jia, additional, Subramanian, Vidya, additional, Chionh, Yok Hian, additional, Raguram, Aditya, additional, Quinlan, Devin, additional, McBee, Megan, additional, Ong, Eugenia Z., additional, Gan, Esther S., additional, Tan, Hwee Cheng, additional, Tyagi, Anu, additional, Bhushan, Shashi, additional, Lescar, Julien, additional, Vasudevan, Subhash G., additional, Ooi, Eng Eong, additional, and Sasisekharan, Ram, additional
- Published
- 2018
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23. Allosteric pyruvate kinase-based “logic gate” synergistically senses energy and sugar levels in Mycobacterium tuberculosis
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Zhong, Wenhe, primary, Cui, Liang, additional, Goh, Boon Chong, additional, Cai, Qixu, additional, Ho, Peiying, additional, Chionh, Yok Hian, additional, Yuan, Meng, additional, Sahili, Abbas El, additional, Fothergill-Gilmore, Linda A., additional, Walkinshaw, Malcolm D., additional, Lescar, Julien, additional, and Dedon, Peter C., additional
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- 2017
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24. Allosteric Pyruvate Kinase-based 'Logic Gate' Synergistically Senses Energy and Sugar Levels in Mycobacterium Tuberculosis
- Author
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Zhong, Wenhe, Cui, Liang, Goh, Boon Chong, Cai, Qixu, Ho, Peiying, Chionh, Yok Hian, Yuan, Meng, Sahili, A.E., Fothergill-Gilmore, Linda A., Walkinshaw, Malcolm D., Lescar, Julien, Dedon, Peter C., Zhong, Wenhe, Cui, Liang, Goh, Boon Chong, Cai, Qixu, Ho, Peiying, Chionh, Yok Hian, Yuan, Meng, Sahili, A.E., Fothergill-Gilmore, Linda A., Walkinshaw, Malcolm D., Lescar, Julien, and Dedon, Peter C.
- Abstract
Pyruvate kinase (PYK) is an essential glycolytic enzyme that controls glycolytic flux and is critical for ATP production in all organisms, with tight regulation by multiple metabolites. Yet the allosteric mechanisms governing PYK activity in bacterial pathogens are poorly understood. Here we report biochemical, structural and metabolomic evidence that Mycobacterium tuberculosis (Mtb) PYK uses AMP and glucose-6-phosphate (G6P) as synergistic allosteric activators that function as a molecular "OR logic gate" to tightly regulate energy and glucose metabolism. G6P was found to bind to a previously unknown site adjacent to the canonical site for AMP. Kinetic data and structural network analysis further show that AMP and G6P work synergistically as allosteric activators. Importantly, metabolome profiling in the Mtb surrogate, Mycobacterium bovis BCG, reveals significant changes in AMP and G6P levels during nutrient deprivation, which provides insights into how a PYK OR gate would function during the stress of Mtb infection. © 2017 The Author(s).
- Published
- 2017
25. The role of sequence context, nucleotide pool balance and stress in 2′-deoxynucleotide misincorporation in viral, bacterial and mammalian RNA
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Massachusetts Institute of Technology. Center for Environmental Health Sciences, Massachusetts Institute of Technology. Department of Biological Engineering, Dedon, Peter C, Wang, Jin, Dong, Hongping, Chionh, Yok Hian, McBee, Megan E., Sirirungruang, Sasilada, Cunningham, Richard P., Shi, Pei-Yong, Massachusetts Institute of Technology. Center for Environmental Health Sciences, Massachusetts Institute of Technology. Department of Biological Engineering, Dedon, Peter C, Wang, Jin, Dong, Hongping, Chionh, Yok Hian, McBee, Megan E., Sirirungruang, Sasilada, Cunningham, Richard P., and Shi, Pei-Yong
- Abstract
The misincorporation of 2′-deoxyribonucleotides (dNs) into RNA has important implications for the function of non-coding RNAs, the translational fidelity of coding RNAs and the mutagenic evolution of viral RNA genomes. However, quantitative appreciation for the degree to which dN misincorporation occurs is limited by the lack of analytical tools. Here, we report a method to hydrolyze RNA to release 2′-deoxyribonucleotide-ribonucleotide pairs (dNrN) that are then quantified by chromatography-coupled mass spectrometry (LC-MS). Using this platform, we found misincorporated dNs occurring at 1 per 10[superscript 3] to 10[superscript 5] ribonucleotide (nt) in mRNA, rRNAs and tRNA in human cells, Escherichia coli, Saccharomyces cerevisiae and, most abundantly, in the RNA genome of dengue virus. The frequency of dNs varied widely among organisms and sequence contexts, and partly reflected the in vitro discrimination efficiencies of different RNA polymerases against 2′-deoxyribonucleoside 5′-triphosphates (dNTPs). Further, we demonstrate a strong link between dN frequencies in RNA and the balance of dNTPs and ribonucleoside 5′-triphosphates (rNTPs) in the cellular pool, with significant stress-induced variation of dN incorporation. Potential implications of dNs in RNA are discussed, including the possibilities of dN incorporation in RNA as a contributing factor in viral evolution and human disease, and as a host immune defense mechanism against viral infections., National Institutes of Health (U.S.) (Grant ES022858), Singapore. National Research Foundation, Singapore-MIT Alliance for Research and Technology
- Published
- 2017
26. Methylation at position 32 of tRNA catalyzed by TrmJ alters oxidative stress response in Pseudomonas aeruginosa
- Author
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Massachusetts Institute of Technology. Department of Biological Engineering, Chionh, Yok Hian, DeMott, Michael S, Dedon, Peter C, Jaroensuk, Juthamas, Atichartpongkul, Sopapan, Wong, Yee Hwa, Liew, Chong Wai, McBee, Megan E., Thongdee, Narumon, Prestwich, Erin G., Mongkolsuk, Skorn, Lescar, Julien, Fuangthong, Mayuree, Massachusetts Institute of Technology. Department of Biological Engineering, Chionh, Yok Hian, DeMott, Michael S, Dedon, Peter C, Jaroensuk, Juthamas, Atichartpongkul, Sopapan, Wong, Yee Hwa, Liew, Chong Wai, McBee, Megan E., Thongdee, Narumon, Prestwich, Erin G., Mongkolsuk, Skorn, Lescar, Julien, and Fuangthong, Mayuree
- Abstract
Bacteria respond to environmental stresses using a variety of signaling and gene expression pathways, with translational mechanisms being the least well understood. Here, we identified a tRNA methyltransferase in Pseudomonas aeruginosa PA14, trmJ, which confers resistance to oxidative stress. Analysis of tRNA from a trmJ mutant revealed that TrmJ catalyzes formation of Cm, Um, and, unexpectedly, Am. Defined in vitro analyses revealed that tRNA[superscript Met(CAU)] and tRNA[superscript Trp(CCA)] are substrates for Cm formation, tRNA[superscript Gln(UUG)], tRNA[superscript Pro(UGG)], tRNA[superscript Pro(CGG)] and tRNA[superscript His(GUG)] for Um, and tRNA[superscript Pro(GGG)] for Am. tRNA[superscript Ser(UGA)], previously observed as a TrmJ substrate in Escherichia coli, was not modified by PA14 TrmJ. Position 32 was confirmed as the TrmJ target for Am in tRNA[superscriptPro(GGG)] and Um in tRNA[superscript Gln(UUG)] by mass spectrometric analysis. Crystal structures of the free catalytic N-terminal domain of TrmJ show a 2-fold symmetrical dimer with an active site located at the interface between the monomers and a flexible basic loop positioned to bind tRNA, with conformational changes upon binding of the SAM-analog sinefungin. The loss of TrmJ rendered PA14 sensitive to H2O2 exposure, with reduced expression of oxyR-recG, katB-ankB, and katE. These results reveal that TrmJ is a tRNA:Cm32/Um32/Am32 methyltransferase involved in translational fidelity and the oxidative stress response., National Science Foundation (U.S.) (CHE-1308839), Agilent Technologies, Singapore-MIT Alliance for Research and Technology (SMART)
- Published
- 2017
27. Three distinct 3-methylcytidine (m3C) methyltransferases modify tRNA and mRNA in mice and humans
- Author
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Xu, Luang, primary, Liu, Xinyu, additional, Sheng, Na, additional, Oo, Kyaw Soe, additional, Liang, Junxin, additional, Chionh, Yok Hian, additional, Xu, Juan, additional, Ye, Fuzhou, additional, Gao, Yong-Gui, additional, Dedon, Peter C., additional, and Fu, Xin-Yuan, additional
- Published
- 2017
- Full Text
- View/download PDF
28. tRNA-mediated codon-biased translation in mycobacterial hypoxic persistence
- Author
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Chionh, Yok Hian, primary, McBee, Megan, additional, Babu, I. Ramesh, additional, Hia, Fabian, additional, Lin, Wenwei, additional, Zhao, Wei, additional, Cao, Jianshu, additional, Dziergowska, Agnieszka, additional, Malkiewicz, Andrzej, additional, Begley, Thomas J., additional, Alonso, Sylvie, additional, and Dedon, Peter C., additional
- Published
- 2016
- Full Text
- View/download PDF
29. Thienopyrimidinone Derivatives That Inhibit Bacterial tRNA (Guanine37‑N1)‑Methyltransferase (TrmD) by Restructuring the Active Site with a Tyrosine-Flipping Mechanism.
- Author
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Zhong, Wenhe, Pasunooti, Kalyan Kumar, Balamkundu, Seetharamsing, Wong, Yee Hwa, Nah, Qianhui, Gadi, Vinod, Gnanakalai, Shanmugavel, Chionh, Yok Hian, McBee, Megan E., Gopal, Pooja, Lim, Siau Hoi, Olivier, Nelson, Buurman, Ed T., Dick, Thomas, Liu, Chuan Fa, Lescar, Julien, and Dedon, Peter C.
- Published
- 2019
- Full Text
- View/download PDF
30. Thienopyrimidinone Derivatives That Inhibit Bacterial tRNA (Guanine37-N1)-Methyltransferase (TrmD) by Restructuring the Active Site with a Tyrosine-Flipping Mechanism
- Author
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Zhong, Wenhe, Pasunooti, Kalyan Kumar, Balamkundu, Seetharamsing, Wong, Yee Hwa, Nah, Qianhui, Gadi, Vinod, Gnanakalai, Shanmugavel, Chionh, Yok Hian, McBee, Megan E., Gopal, Pooja, Lim, Siau Hoi, Olivier, Nelson, Buurman, Ed T., Dick, Thomas, Liu, Chuan Fa, Lescar, Julien, and Dedon, Peter C.
- Abstract
Among the >120 modified ribonucleosides in the prokaryotic epitranscriptome, many tRNA modifications are critical to bacterial survival, which makes their synthetic enzymes ideal targets for antibiotic development. Here we performed a structure-based design of inhibitors of tRNA-(N1G37) methyltransferase, TrmD, which is an essential enzyme in many bacterial pathogens. On the basis of crystal structures of TrmDs from Pseudomonas aeruginosaand Mycobacterium tuberculosis, we synthesized a series of thienopyrimidinone derivatives with nanomolar potency against TrmD in vitro and discovered a novel active site conformational change triggered by inhibitor binding. This tyrosine-flipping mechanism is uniquely found in P. aeruginosaTrmD and renders the enzyme inaccessible to the cofactor S-adenosyl-l-methionine (SAM) and probably to the substrate tRNA. Biophysical and biochemical structure–activity relationship studies provided insights into the mechanisms underlying the potency of thienopyrimidinones as TrmD inhibitors, with several derivatives found to be active against Gram-positive and mycobacterial pathogens. These results lay a foundation for further development of TrmD inhibitors as antimicrobial agents.
- Published
- 2024
- Full Text
- View/download PDF
31. tRNA-mediated codon-biased translation in mycobacterial hypoxic persistence
- Author
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Massachusetts Institute of Technology. Center for Environmental Health Sciences, Massachusetts Institute of Technology. Department of Biological Engineering, Massachusetts Institute of Technology. Department of Chemistry, Singapore-MIT Alliance in Research and Technology (SMART), Chionh, Yok Hian, McBee, Megan E, Babu, I. Ramesh, Dedon, Peter C, Cao, Jianshu, McBee, Megan, Hia, Fabian, Lin, Wenwei, Zhao, Wei, Dziergowska, Agnieszka, Malkiewicz, Andrzej, Begley, Thomas J., Alonso, Sylvie, Dedon, Peter C., Massachusetts Institute of Technology. Center for Environmental Health Sciences, Massachusetts Institute of Technology. Department of Biological Engineering, Massachusetts Institute of Technology. Department of Chemistry, Singapore-MIT Alliance in Research and Technology (SMART), Chionh, Yok Hian, McBee, Megan E, Babu, I. Ramesh, Dedon, Peter C, Cao, Jianshu, McBee, Megan, Hia, Fabian, Lin, Wenwei, Zhao, Wei, Dziergowska, Agnieszka, Malkiewicz, Andrzej, Begley, Thomas J., Alonso, Sylvie, and Dedon, Peter C.
- Abstract
Microbial pathogens adapt to the stress of infection by regulating transcription, translation and protein modification. We report that changes in gene expression in hypoxia-induced non-replicating persistence in mycobacteria—which models tuberculous granulomas—are partly determined by a mechanism of tRNA reprogramming and codon-biased translation. Mycobacterium bovis BCG responded to each stage of hypoxia and aerobic resuscitation by uniquely reprogramming 40 modified ribonucleosides in tRNA, which correlate with selective translation of mRNAs from families of codon-biased persistence genes. For example, early hypoxia increases wobble cmo[superscript 5]U in tRNA[superscript Thr(UGU)], which parallels translation of transcripts enriched in its cognate codon, ACG, including the DosR master regulator of hypoxic bacteriostasis. Codon re-engineering of dosR exaggerates hypoxia-induced changes in codon-biased DosR translation, with altered dosR expression revealing unanticipated effects on bacterial survival during hypoxia. These results reveal a coordinated system of tRNA modifications and translation of codon-biased transcripts that enhance expression of stress response proteins in mycobacteria., Singapore-MIT Alliance for Research and Technology (SMART), National Institute of Environmental Health Sciences (grants ES017010 and ES002109), National Science Foundation (U.S.) (grant CHE-1308839), Singapore-MIT Alliance for Research and Technology (SMART) (SMA3 Graduate Fellowship)
- Published
- 2016
32. Methylation at position 32 of tRNA catalyzed by TrmJ alters oxidative stress response inPseudomonas aeruginosa
- Author
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Jaroensuk, Juthamas, primary, Atichartpongkul, Sopapan, additional, Chionh, Yok Hian, additional, Wong, Yee Hwa, additional, Liew, Chong Wai, additional, McBee, Megan E., additional, Thongdee, Narumon, additional, Prestwich, Erin G., additional, DeMott, Michael S., additional, Mongkolsuk, Skorn, additional, Dedon, Peter C., additional, Lescar, Julien, additional, and Fuangthong, Mayuree, additional
- Published
- 2016
- Full Text
- View/download PDF
33. The role of sequence context, nucleotide pool balance and stress in 2′-deoxynucleotide misincorporation in viral, bacterial and mammalian RNA
- Author
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Wang, Jin, primary, Dong, Hongping, additional, Chionh, Yok Hian, additional, McBee, Megan E., additional, Sirirungruang, Sasilada, additional, Cunningham, Richard P., additional, Shi, Pei-Yong, additional, and Dedon, Peter C., additional
- Published
- 2016
- Full Text
- View/download PDF
34. Quantitative analysis of tRNA modifications by HPLC-coupled mass spectrometry
- Author
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Massachusetts Institute of Technology. Center for Environmental Health Sciences, Massachusetts Institute of Technology. Department of Biological Engineering, Su, Dan, Chan, Clement T. Y., Gu, Chen, Lim, Kok Seong, Russell, Brandon S., Ramesh Babu, I., Dedon, Peter C., Chionh, Yok Hian, McBee, Megan E., Begley, Thomas J., Massachusetts Institute of Technology. Center for Environmental Health Sciences, Massachusetts Institute of Technology. Department of Biological Engineering, Su, Dan, Chan, Clement T. Y., Gu, Chen, Lim, Kok Seong, Russell, Brandon S., Ramesh Babu, I., Dedon, Peter C., Chionh, Yok Hian, McBee, Megan E., and Begley, Thomas J.
- Abstract
Post-transcriptional modification of RNA is an important determinant of RNA quality control, translational efficiency, RNA-protein interactions and stress response. This is illustrated by the observation of toxicant-specific changes in the spectrum of tRNA modifications in a stress-response mechanism involving selective translation of codon-biased mRNA for crucial proteins. To facilitate systems-level studies of RNA modifications, we developed a liquid chromatography–mass spectrometry (LC-MS) technique for the quantitative analysis of modified ribonucleosides in tRNA. The protocol includes tRNA purification by HPLC, enzymatic hydrolysis, reversed-phase HPLC resolution of the ribonucleosides, and identification and quantification of individual ribonucleosides by LC-MS via dynamic multiple reaction monitoring (DMRM). In this approach, the relative proportions of modified ribonucleosides are quantified in several micrograms of tRNA in a 15-min LC-MS run. This protocol can be modified to analyze other types of RNA by modifying the steps for RNA purification as appropriate. By comparison, traditional methods for detecting modified ribonucleosides are labor- and time-intensive, they require larger RNA quantities, they are modification-specific or require radioactive labeling., National Institute of Environmental Health Sciences (ES002109), National Institute of Environmental Health Sciences (ES015037), National Institute of Environmental Health Sciences (ES017010), Westaway Research Fund, Massachusetts Institute of Technology (Merck Fellowship), David H. Koch Institute for Integrative Cancer Research at MIT (Graduate Fellowship), Howard Hughes Medical Institute (International Student Research Fellowship), Singapore-MIT Alliance for Research and Technology
- Published
- 2015
35. Mycobacterial RNA isolation optimized for non-coding RNA: high fidelity isolation of 5S rRNA from Mycobacterium bovis BCG reveals novel post-transcriptional processing and a complete spectrum of modified ribonucleosides
- Author
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Massachusetts Institute of Technology. Center for Environmental Health Sciences, Massachusetts Institute of Technology. Department of Biological Engineering, Chionh, Yok Hian, Pang, Yan Ling Joy, DeMott, Michael S., McBee, Megan E., Dedon, Peter C., Hia, Fabian, Massachusetts Institute of Technology. Center for Environmental Health Sciences, Massachusetts Institute of Technology. Department of Biological Engineering, Chionh, Yok Hian, Pang, Yan Ling Joy, DeMott, Michael S., McBee, Megan E., Dedon, Peter C., and Hia, Fabian
- Abstract
A major challenge in the study of mycobacterial RNA biology is the lack of a comprehensive RNA isolation method that overcomes the unusual cell wall to faithfully yield the full spectrum of non-coding RNA (ncRNA) species. Here, we describe a simple and robust procedure optimized for the isolation of total ncRNA, including 5S, 16S and 23S ribosomal RNA (rRNA) and tRNA, from mycobacteria, using Mycobacterium bovis BCG to illustrate the method. Based on a combination of mechanical disruption and liquid and solid-phase technologies, the method produces all major species of ncRNA in high yield and with high integrity, enabling direct chemical and sequence analysis of the ncRNA species. The reproducibility of the method with BCG was evident in bioanalyzer electrophoretic analysis of isolated RNA, which revealed quantitatively significant differences in the ncRNA profiles of exponentially growing and non-replicating hypoxic bacilli. The method also overcame an historical inconsistency in 5S rRNA isolation, with direct sequencing revealing a novel post-transcriptional processing of 5S rRNA to its functional form and with chemical analysis revealing seven post-transcriptional ribonucleoside modifications in the 5S rRNA. This optimized RNA isolation procedure thus provides a means to more rigorously explore the biology of ncRNA species in mycobacteria., Singapore-MIT Alliance for Research and Technology, National Institute of Environmental Health Sciences (ES017010), National Institute of Environmental Health Sciences (ES002109), Singapore-MIT Alliance (Graduate Fellowship), Singapore. National Research Foundation
- Published
- 2015
36. 2′-O Methylation of Internal Adenosine by Flavivirus NS[subscript 5] Methyltransferase
- Author
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Dong, Hongping, Chang, David C., Ho, Chia-Hua, Lim, Siew Pheng, Chionh, Yok Hian, Hia, Fabian, Lee, Yie Hou, Kukkaro, Petra, Lok, Shee-Mei, Dedon, Peter C., Shi, Pei-Yong, Massachusetts Institute of Technology. Department of Biological Engineering, and Dedon, Peter C.
- Subjects
viruses ,education ,virus diseases - Abstract
RNA modification plays an important role in modulating host-pathogen interaction. Flavivirus NS5 protein encodes N-7 and 2′-O methyltransferase activities that are required for the formation of 5′ type I cap (m[superscript 7]GpppAm) of viral RNA genome. Here we reported, for the first time, that flavivirus NS5 has a novel internal RNA methylation activity. Recombinant NS5 proteins of West Nile virus and Dengue virus (serotype 4; DENV-4) specifically methylates polyA, but not polyG, polyC, or polyU, indicating that the methylation occurs at adenosine residue. RNAs with internal adenosines substituted with 2′-O-methyladenosines are not active substrates for internal methylation, whereas RNAs with adenosines substituted with N6-methyladenosines can be efficiently methylated, suggesting that the internal methylation occurs at the 2′-OH position of adenosine. Mass spectroscopic analysis further demonstrated that the internal methylation product is 2′-O-methyladenosine. Importantly, genomic RNA purified from DENV virion contains 2′-O-methyladenosine. The 2′-O methylation of internal adenosine does not require specific RNA sequence since recombinant methyltransferase of DENV-4 can efficiently methylate RNAs spanning different regions of viral genome, host ribosomal RNAs, and polyA. Structure-based mutagenesis results indicate that K61-D146-K181-E217 tetrad of DENV-4 methyltransferase forms the active site of internal methylation activity; in addition, distinct residues within the methyl donor (S-adenosyl-L-methionine) pocket, GTP pocket, and RNA-binding site are critical for the internal methylation activity. Functional analysis using flavivirus replicon and genome-length RNAs showed that internal methylation attenuated viral RNA translation and replication. Polymerase assay revealed that internal 2′-O-methyladenosine reduces the efficiency of RNA elongation. Collectively, our results demonstrate that flavivirus NS5 performs 2′-O methylation of internal adenosine of viral RNA in vivo and host ribosomal RNAs in vitro.
- Published
- 2011
37. Identification of N6,N6-Dimethyladenosine in Transfer RNA from Mycobacterium bovis Bacille Calmette-Guérin
- Author
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Chan, Tsz Yan Clement, Chionh, Yok Hian, Ho, Chia-Hua, Lim, Kok Seong, Babu, I. Ramesh, Ang, Emily, Wenwei, Lin, Alonso, Sylvie, Dedon, Peter C., Massachusetts Institute of Technology. Department of Biological Engineering, Massachusetts Institute of Technology. Department of Chemistry, Chan, Tsz Yan Clement, Lim, Kok Seong, Babu, I. Ramesh, and Dedon, Peter C.
- Abstract
There are more than 100 different ribonucleoside structures incorporated as post-transcriptional modifications, mainly in tRNA and rRNA of both prokaryotes and eukaryotes, and emerging evidence suggests that these modifications function as a system in the translational control of cellular responses. However, our understanding of this system is hampered by the paucity of information about the complete set of RNA modifications present in individual organisms. To this end, we have employed a chromatography-coupled mass spectrometric approach to define the spectrum of modified ribonucleosides in microbial species, starting with Mycobacterium bovis BCG. This approach revealed a variety of ribonucleoside candidates in tRNA from BCG, of which 12 were definitively identified based on comparisons to synthetic standards and 5 were tentatively identified by exact mass comparisons to RNA modification databases. Among the ribonucleosides observed in BCG tRNA was one not previously described in tRNA, which we have now characterized as N6,N6-dimethyladenosine., National Institute of Environmental Health Sciences (ES017010), Singapore-MIT Alliance for Research and Technology, Massachusetts Institute of Technology (Westaway Fund), Merck Research Laboratories (Merck/CSBi Fellowship)
- Published
- 2011
38. Identification of N[superscript 6],N[superscript 6]-Dimethyladenosine in Transfer RNA from Mycobacterium bovis Bacille Calmette-Guérin
- Author
-
Massachusetts Institute of Technology. Department of Biological Engineering, Massachusetts Institute of Technology. Department of Chemistry, Chan, Tsz Yan Clement, Lim, Kok Seong, Babu, I. Ramesh, Dedon, Peter C., Chionh, Yok Hian, Ho, Chia-Hua, Ang, Emily, Wenwei, Lin, Alonso, Sylvie, Massachusetts Institute of Technology. Department of Biological Engineering, Massachusetts Institute of Technology. Department of Chemistry, Chan, Tsz Yan Clement, Lim, Kok Seong, Babu, I. Ramesh, Dedon, Peter C., Chionh, Yok Hian, Ho, Chia-Hua, Ang, Emily, Wenwei, Lin, and Alonso, Sylvie
- Abstract
There are more than 100 different ribonucleoside structures incorporated as post-transcriptional modifications, mainly in tRNA and rRNA of both prokaryotes and eukaryotes, and emerging evidence suggests that these modifications function as a system in the translational control of cellular responses. However, our understanding of this system is hampered by the paucity of information about the complete set of RNA modifications present in individual organisms. To this end, we have employed a chromatography-coupled mass spectrometric approach to define the spectrum of modified ribonucleosides in microbial species, starting with Mycobacterium bovis BCG. This approach revealed a variety of ribonucleoside candidates in tRNA from BCG, of which 12 were definitively identified based on comparisons to synthetic standards and 5 were tentatively identified by exact mass comparisons to RNA modification databases. Among the ribonucleosides observed in BCG tRNA was one not previously described in tRNA, which we have now characterized as N6,N6-dimethyladenosine., National Institute of Environmental Health Sciences (ES017010), Singapore-MIT Alliance for Research and Technology, Massachusetts Institute of Technology (Westaway Fund), Merck Research Laboratories (Merck/CSBi Fellowship)
- Published
- 2014
39. Mycobacterial RNA isolation optimized for non-coding RNA: high fidelity isolation of 5S rRNA from Mycobacterium bovis BCG reveals novel post-transcriptional processing and a complete spectrum of modified ribonucleosides
- Author
-
Hia, Fabian, primary, Chionh, Yok Hian, additional, Pang, Yan Ling Joy, additional, DeMott, Michael S., additional, McBee, Megan E., additional, and Dedon, Peter C., additional
- Published
- 2014
- Full Text
- View/download PDF
40. A multidimensional platform for the purification of non-coding RNA species
- Author
-
Massachusetts Institute of Technology. Center for Environmental Health Sciences, Massachusetts Institute of Technology. Department of Biological Engineering, McBee, Megan E., Su, Dan, Pang, Yan Ling Joy, Gu, Chen, Prestwich, Erin, Dedon, Peter C., Indrakanti, Ramesh Babu, Chionh, Yok Hian, Ho, Chia-Hua, Pruksakorn, Dumnoensun, Ng, Chee Sheng, Hia, Fabian, Dong, Hongping, Shi, Pei-Yong, Preiser, Peter Rainer, Alonso, Sylvie, Massachusetts Institute of Technology. Center for Environmental Health Sciences, Massachusetts Institute of Technology. Department of Biological Engineering, McBee, Megan E., Su, Dan, Pang, Yan Ling Joy, Gu, Chen, Prestwich, Erin, Dedon, Peter C., Indrakanti, Ramesh Babu, Chionh, Yok Hian, Ho, Chia-Hua, Pruksakorn, Dumnoensun, Ng, Chee Sheng, Hia, Fabian, Dong, Hongping, Shi, Pei-Yong, Preiser, Peter Rainer, and Alonso, Sylvie
- Abstract
A renewed interest in non-coding RNA (ncRNA) has led to the discovery of novel RNA species and post-transcriptional ribonucleoside modifications, and an emerging appreciation for the role of ncRNA in RNA epigenetics. Although much can be learned by amplification-based analysis of ncRNA sequence and quantity, there is a significant need for direct analysis of RNA, which has led to numerous methods for purification of specific ncRNA molecules. However, no single method allows purification of the full range of cellular ncRNA species. To this end, we developed a multidimensional chromatographic platform to resolve, isolate and quantify all canonical ncRNAs in a single sample of cells or tissue, as well as novel ncRNA species. The applicability of the platform is demonstrated in analyses of ncRNA from bacteria, human cells and plasmodium-infected reticulocytes, as well as a viral RNA genome. Among the many potential applications of this platform are a system-level analysis of the dozens of modified ribonucleosides in ncRNA, characterization of novel long ncRNA species, enhanced detection of rare transcript variants and analysis of viral genomes., Singapore-MIT Alliance for Research and Technology, National Institute of Environmental Health Sciences (ES017010), National Institute of Environmental Health Sciences (ES002109)
- Published
- 2013
41. 2′-O Methylation of Internal Adenosine by Flavivirus NS[subscript 5] Methyltransferase
- Author
-
Massachusetts Institute of Technology. Department of Biological Engineering, Dedon, Peter C., Dong, Hongping, Chang, David C., Ho, Chia-Hua, Lim, Siew Pheng, Chionh, Yok Hian, Hia, Fabian, Lee, Yie Hou, Kukkaro, Petra, Lok, Shee-Mei, Shi, Pei-Yong, Massachusetts Institute of Technology. Department of Biological Engineering, Dedon, Peter C., Dong, Hongping, Chang, David C., Ho, Chia-Hua, Lim, Siew Pheng, Chionh, Yok Hian, Hia, Fabian, Lee, Yie Hou, Kukkaro, Petra, Lok, Shee-Mei, and Shi, Pei-Yong
- Abstract
RNA modification plays an important role in modulating host-pathogen interaction. Flavivirus NS5 protein encodes N-7 and 2′-O methyltransferase activities that are required for the formation of 5′ type I cap (m[superscript 7]GpppAm) of viral RNA genome. Here we reported, for the first time, that flavivirus NS5 has a novel internal RNA methylation activity. Recombinant NS5 proteins of West Nile virus and Dengue virus (serotype 4; DENV-4) specifically methylates polyA, but not polyG, polyC, or polyU, indicating that the methylation occurs at adenosine residue. RNAs with internal adenosines substituted with 2′-O-methyladenosines are not active substrates for internal methylation, whereas RNAs with adenosines substituted with N6-methyladenosines can be efficiently methylated, suggesting that the internal methylation occurs at the 2′-OH position of adenosine. Mass spectroscopic analysis further demonstrated that the internal methylation product is 2′-O-methyladenosine. Importantly, genomic RNA purified from DENV virion contains 2′-O-methyladenosine. The 2′-O methylation of internal adenosine does not require specific RNA sequence since recombinant methyltransferase of DENV-4 can efficiently methylate RNAs spanning different regions of viral genome, host ribosomal RNAs, and polyA. Structure-based mutagenesis results indicate that K61-D146-K181-E217 tetrad of DENV-4 methyltransferase forms the active site of internal methylation activity; in addition, distinct residues within the methyl donor (S-adenosyl-L-methionine) pocket, GTP pocket, and RNA-binding site are critical for the internal methylation activity. Functional analysis using flavivirus replicon and genome-length RNAs showed that internal methylation attenuated viral RNA translation and replication. Polymerase assay revealed that internal 2′-O-methyladenosine reduces the efficiency of RNA elongation. Collectively, our results demonstrate that flavivirus NS5 performs 2′-O methylation of internal adenosine of viral
- Published
- 2012
42. Induction of Functional Human Macrophages from Bone Marrow Promonocytes by M-CSF in Humanized Mice
- Author
-
Li, Yan, primary, Chen, Qingfeng, additional, Zheng, Dahai, additional, Yin, Lu, additional, Chionh, Yok Hian, additional, Wong, Lan Hiong, additional, Tan, Shu Qi, additional, Tan, Thiam Chye, additional, Chan, Jerry K. Y., additional, Alonso, Sylvie, additional, Dedon, Peter C., additional, Lim, Bing, additional, and Chen, Jianzhu, additional
- Published
- 2013
- Full Text
- View/download PDF
43. 2′-O Methylation of Internal Adenosine by Flavivirus NS5 Methyltransferase
- Author
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Dong, Hongping, primary, Chang, David C., additional, Hua, Maggie Ho Chia, additional, Lim, Siew Pheng, additional, Chionh, Yok Hian, additional, Hia, Fabian, additional, Lee, Yie Hou, additional, Kukkaro, Petra, additional, Lok, Shee-Mei, additional, Dedon, Peter C., additional, and Shi, Pei-Yong, additional
- Published
- 2012
- Full Text
- View/download PDF
44. Identification of N6,N6-Dimethyladenosine in Transfer RNA from Mycobacterium bovis Bacille Calmette-Guérin
- Author
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Chan, Clement, primary, Chionh, Yok Hian, additional, Ho, Chia-Hua, additional, Lim, Kok Seong, additional, Babu, I. Ramesh, additional, Ang, Emily, additional, Wenwei, Lin, additional, Alonso, Sylvie, additional, and Dedon, Peter, additional
- Published
- 2011
- Full Text
- View/download PDF
45. Methylation at position 32 of tRNA catalyzed by TrmJ alters oxidative stress response inPseudomonas aeruginosa
- Author
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Narumon Thongdee, Julien Lescar, Juthamas Jaroensuk, Michael S. DeMott, Chong Wai Liew, Mayuree Fuangthong, Yee Hwa Wong, Yok Hian Chionh, Sopapan Atichartpongkul, Skorn Mongkolsuk, Peter C. Dedon, Megan E. McBee, Erin G. Prestwich, Chulabhorn Research Institute, Singapore-MIT Alliance for Research and Technology (SMART), Massachusetts Institute of Technology (MIT), Nanyang Technological University [Singapour], Mahidol University [Bangkok], Center of excellence on environmental health and toxicology, Bangkok, Centre d'Immunologie et de Maladies Infectieuses (CIMI), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Massachusetts Institute of Technology. Department of Biological Engineering, Chionh, Yok Hian, DeMott, Michael S, Dedon, Peter C, and HAL UPMC, Gestionnaire
- Subjects
Models, Molecular ,0301 basic medicine ,[SDV]Life Sciences [q-bio] ,Mutant ,Crystallography, X-Ray ,medicine.disease_cause ,Methylation ,03 medical and health sciences ,Sinefungin ,Bacterial Proteins ,RNA, Transfer ,Catalytic Domain ,Gene expression ,Genetics ,medicine ,Amino Acid Sequence ,Escherichia coli ,tRNA Methyltransferases ,Base Sequence ,030102 biochemistry & molecular biology ,biology ,Nucleic Acid Enzymes ,Active site ,Hydrogen Peroxide ,Molecular biology ,[SDV] Life Sciences [q-bio] ,Oxidative Stress ,RNA, Bacterial ,030104 developmental biology ,Biochemistry ,Pseudomonas aeruginosa ,Transfer RNA ,biology.protein ,T arm - Abstract
Bacteria respond to environmental stresses using a variety of signaling and gene expression pathways, with translational mechanisms being the least well understood. Here, we identified a tRNA methyltransferase in Pseudomonas aeruginosa PA14, trmJ, which confers resistance to oxidative stress. Analysis of tRNA from a trmJ mutant revealed that TrmJ catalyzes formation of Cm, Um, and, unexpectedly, Am. Defined in vitro analyses revealed that tRNA[superscript Met(CAU)] and tRNA[superscript Trp(CCA)] are substrates for Cm formation, tRNA[superscript Gln(UUG)], tRNA[superscript Pro(UGG)], tRNA[superscript Pro(CGG)] and tRNA[superscript His(GUG)] for Um, and tRNA[superscript Pro(GGG)] for Am. tRNA[superscript Ser(UGA)], previously observed as a TrmJ substrate in Escherichia coli, was not modified by PA14 TrmJ. Position 32 was confirmed as the TrmJ target for Am in tRNA[superscriptPro(GGG)] and Um in tRNA[superscript Gln(UUG)] by mass spectrometric analysis. Crystal structures of the free catalytic N-terminal domain of TrmJ show a 2-fold symmetrical dimer with an active site located at the interface between the monomers and a flexible basic loop positioned to bind tRNA, with conformational changes upon binding of the SAM-analog sinefungin. The loss of TrmJ rendered PA14 sensitive to H2O2 exposure, with reduced expression of oxyR-recG, katB-ankB, and katE. These results reveal that TrmJ is a tRNA:Cm32/Um32/Am32 methyltransferase involved in translational fidelity and the oxidative stress response., National Science Foundation (U.S.) (CHE-1308839), Agilent Technologies, Singapore-MIT Alliance for Research and Technology (SMART)
- Published
- 2016
46. tRNA-mediated codon-biased translation in mycobacterial hypoxic persistence
- Author
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Fabian Hia, Sylvie Alonso, Jianshu Cao, Agnieszka Dziergowska, Megan E. McBee, Peter C. Dedon, I. Ramesh Babu, Andrzej Malkiewicz, Yok Hian Chionh, Thomas J. Begley, Wei Zhao, Wenwei Lin, Massachusetts Institute of Technology. Center for Environmental Health Sciences, Massachusetts Institute of Technology. Department of Biological Engineering, Massachusetts Institute of Technology. Department of Chemistry, Singapore-MIT Alliance in Research and Technology (SMART), Chionh, Yok Hian, McBee, Megan E, Babu, I. Ramesh, Dedon, Peter C, and Cao, Jianshu
- Subjects
0301 basic medicine ,Science ,General Physics and Astronomy ,Biology ,Article ,General Biochemistry, Genetics and Molecular Biology ,Microbiology ,Transcriptome ,03 medical and health sciences ,Oxygen Consumption ,Bacterial Proteins ,RNA, Transfer ,Transcription (biology) ,Gene expression ,Protein biosynthesis ,RNA, Messenger ,Codon ,Gene ,Regulation of gene expression ,Genetics ,Multidisciplinary ,fungi ,Translation (biology) ,Gene Expression Regulation, Bacterial ,General Chemistry ,Mycobacterium bovis ,3. Good health ,RNA, Bacterial ,030104 developmental biology ,Protein Biosynthesis ,Transfer RNA ,Protein Processing, Post-Translational - Abstract
Microbial pathogens adapt to the stress of infection by regulating transcription, translation and protein modification. We report that changes in gene expression in hypoxia-induced non-replicating persistence in mycobacteria—which models tuberculous granulomas—are partly determined by a mechanism of tRNA reprogramming and codon-biased translation. Mycobacterium bovis BCG responded to each stage of hypoxia and aerobic resuscitation by uniquely reprogramming 40 modified ribonucleosides in tRNA, which correlate with selective translation of mRNAs from families of codon-biased persistence genes. For example, early hypoxia increases wobble cmo[superscript 5]U in tRNA[superscript Thr(UGU)], which parallels translation of transcripts enriched in its cognate codon, ACG, including the DosR master regulator of hypoxic bacteriostasis. Codon re-engineering of dosR exaggerates hypoxia-induced changes in codon-biased DosR translation, with altered dosR expression revealing unanticipated effects on bacterial survival during hypoxia. These results reveal a coordinated system of tRNA modifications and translation of codon-biased transcripts that enhance expression of stress response proteins in mycobacteria., Singapore-MIT Alliance for Research and Technology (SMART), National Institute of Environmental Health Sciences (grants ES017010 and ES002109), National Science Foundation (U.S.) (grant CHE-1308839), Singapore-MIT Alliance for Research and Technology (SMART) (SMA3 Graduate Fellowship)
- Published
- 2016
47. Mycobacterial RNA isolation optimized for non-coding RNA: high fidelity isolation of 5S rRNA from Mycobacterium bovis BCG reveals novel post-transcriptional processing and a complete spectrum of modified ribonucleosides
- Author
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Megan E. McBee, Yok Hian Chionh, Fabian Hia, Yan Ling Joy Pang, Peter C. Dedon, Michael S. DeMott, Massachusetts Institute of Technology. Center for Environmental Health Sciences, Massachusetts Institute of Technology. Department of Biological Engineering, Chionh, Yok Hian, Pang, Yan Ling Joy, DeMott, Michael S., McBee, Megan E., and Dedon, Peter C.
- Subjects
RNA, Untranslated ,5.8S ribosomal RNA ,RNA-dependent RNA polymerase ,RNA integrity number ,Computational biology ,Biology ,RNA, Transfer ,23S ribosomal RNA ,RNA, Ribosomal, 16S ,Genetics ,RNA Processing, Post-Transcriptional ,Chromatography, High Pressure Liquid ,RNA, Ribosomal, 5S ,Reproducibility of Results ,RNA ,Ribosomal RNA ,Non-coding RNA ,Mycobacterium bovis ,3. Good health ,RNA, Bacterial ,RNA, Ribosomal, 23S ,RNA editing ,Chromatography, Gel ,Methods Online ,Ribonucleosides - Abstract
A major challenge in the study of mycobacterial RNA biology is the lack of a comprehensive RNA isolation method that overcomes the unusual cell wall to faithfully yield the full spectrum of non-coding RNA (ncRNA) species. Here, we describe a simple and robust procedure optimized for the isolation of total ncRNA, including 5S, 16S and 23S ribosomal RNA (rRNA) and tRNA, from mycobacteria, using Mycobacterium bovis BCG to illustrate the method. Based on a combination of mechanical disruption and liquid and solid-phase technologies, the method produces all major species of ncRNA in high yield and with high integrity, enabling direct chemical and sequence analysis of the ncRNA species. The reproducibility of the method with BCG was evident in bioanalyzer electrophoretic analysis of isolated RNA, which revealed quantitatively significant differences in the ncRNA profiles of exponentially growing and non-replicating hypoxic bacilli. The method also overcame an historical inconsistency in 5S rRNA isolation, with direct sequencing revealing a novel post-transcriptional processing of 5S rRNA to its functional form and with chemical analysis revealing seven post-transcriptional ribonucleoside modifications in the 5S rRNA. This optimized RNA isolation procedure thus provides a means to more rigorously explore the biology of ncRNA species in mycobacteria., Singapore-MIT Alliance for Research and Technology, National Institute of Environmental Health Sciences (ES017010), National Institute of Environmental Health Sciences (ES002109), Singapore-MIT Alliance (Graduate Fellowship), Singapore. National Research Foundation
- Published
- 2014
48. Crystal structure and catalytic mechanism of the essential m 1 G37 tRNA methyltransferase TrmD from Pseudomonas aeruginosa .
- Author
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Jaroensuk J, Wong YH, Zhong W, Liew CW, Maenpuen S, Sahili AE, Atichartpongkul S, Chionh YH, Nah Q, Thongdee N, McBee ME, Prestwich EG, DeMott MS, Chaiyen P, Mongkolsuk S, Dedon PC, Lescar J, and Fuangthong M
- Subjects
- Catalysis, Crystallography, X-Ray, Kinetics, Protein Binding, Protein Conformation, RNA, Transfer metabolism, S-Adenosylmethionine metabolism, Substrate Specificity, tRNA Methyltransferases chemistry, tRNA Methyltransferases isolation & purification, Pseudomonas aeruginosa enzymology, tRNA Methyltransferases metabolism
- Abstract
The tRNA (m
1 G37) methyltransferase TrmD catalyzes m1 G formation at position 37 in many tRNA isoacceptors and is essential in most bacteria, which positions it as a target for antibiotic development. In spite of its crucial role, little is known about TrmD in Pseudomonas aeruginosa ( Pa TrmD), an important human pathogen. Here we present detailed structural, substrate, and kinetic properties of Pa TrmD. The mass spectrometric analysis confirmed the G36G37-containing tRNAs Leu(GAG), Leu(CAG), Leu(UAG), Pro(GGG), Pro(UGG), Pro(CGG), and His(GUG) as Pa TrmD substrates. Analysis of steady-state kinetics with S -adenosyl-l-methionine (SAM) and tRNALeu(GAG) showed that Pa TrmD catalyzes the two-substrate reaction by way of a ternary complex, while isothermal titration calorimetry revealed that SAM and tRNALeu(GAG) bind to Pa TrmD independently, each with a dissociation constant of 14 ± 3 µM. Inhibition by the SAM analog sinefungin was competitive with respect to SAM ( Ki = 0.41 ± 0.07 µM) and uncompetitive for tRNA ( Ki = 6.4 ± 0.8 µM). A set of crystal structures of the homodimeric Pa TrmD protein bound to SAM and sinefungin provide the molecular basis for enzyme competitive inhibition and identify the location of the bound divalent ion. These results provide insights into Pa TrmD as a potential target for the development of antibiotics., (© 2019 Jaroensuk et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)- Published
- 2019
- Full Text
- View/download PDF
49. Thienopyrimidinone Derivatives That Inhibit Bacterial tRNA (Guanine37- N 1 )-Methyltransferase (TrmD) by Restructuring the Active Site with a Tyrosine-Flipping Mechanism.
- Author
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Zhong W, Pasunooti KK, Balamkundu S, Wong YH, Nah Q, Gadi V, Gnanakalai S, Chionh YH, McBee ME, Gopal P, Lim SH, Olivier N, Buurman ET, Dick T, Liu CF, Lescar J, and Dedon PC
- Subjects
- Binding Sites drug effects, Dose-Response Relationship, Drug, Drug Design, Enzyme Inhibitors chemical synthesis, Enzyme Inhibitors chemistry, Models, Molecular, Molecular Structure, Pseudomonas aeruginosa enzymology, Pyrimidines chemical synthesis, Pyrimidines chemistry, Structure-Activity Relationship, Tyrosine chemistry, tRNA Methyltransferases metabolism, Enzyme Inhibitors pharmacology, Pyrimidines pharmacology, Tyrosine pharmacology, tRNA Methyltransferases antagonists & inhibitors
- Abstract
Among the >120 modified ribonucleosides in the prokaryotic epitranscriptome, many tRNA modifications are critical to bacterial survival, which makes their synthetic enzymes ideal targets for antibiotic development. Here we performed a structure-based design of inhibitors of tRNA-(N
1 G37) methyltransferase, TrmD, which is an essential enzyme in many bacterial pathogens. On the basis of crystal structures of TrmDs from Pseudomonas aeruginosa and Mycobacterium tuberculosis , we synthesized a series of thienopyrimidinone derivatives with nanomolar potency against TrmD in vitro and discovered a novel active site conformational change triggered by inhibitor binding. This tyrosine-flipping mechanism is uniquely found in P. aeruginosa TrmD and renders the enzyme inaccessible to the cofactor S -adenosyl-l-methionine (SAM) and probably to the substrate tRNA. Biophysical and biochemical structure-activity relationship studies provided insights into the mechanisms underlying the potency of thienopyrimidinones as TrmD inhibitors, with several derivatives found to be active against Gram-positive and mycobacterial pathogens. These results lay a foundation for further development of TrmD inhibitors as antimicrobial agents.- Published
- 2019
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50. Targeting the Bacterial Epitranscriptome for Antibiotic Development: Discovery of Novel tRNA-(N 1 G37) Methyltransferase (TrmD) Inhibitors.
- Author
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Zhong W, Koay A, Ngo A, Li Y, Nah Q, Wong YH, Chionh YH, Ng HQ, Koh-Stenta X, Poulsen A, Foo K, McBee M, Choong ML, El Sahili A, Kang C, Matter A, Lescar J, Hill J, and Dedon P
- Subjects
- Anti-Bacterial Agents chemistry, Bacterial Proteins chemistry, Bacterial Proteins genetics, Bacterial Proteins metabolism, Drug Discovery, Enzyme Inhibitors chemistry, Kinetics, Methyltransferases chemistry, Methyltransferases genetics, Methyltransferases metabolism, Pseudomonas aeruginosa genetics, Substrate Specificity, Anti-Bacterial Agents pharmacology, Bacterial Proteins antagonists & inhibitors, Drug Evaluation, Preclinical methods, Enzyme Inhibitors pharmacology, Methyltransferases antagonists & inhibitors, Pseudomonas aeruginosa drug effects, Pseudomonas aeruginosa enzymology, RNA, Transfer metabolism
- Abstract
Bacterial tRNA modification synthesis pathways are critical to cell survival under stress and thus represent ideal mechanism-based targets for antibiotic development. One such target is the tRNA-(N
1 G37) methyltransferase (TrmD), which is conserved and essential in many bacterial pathogens. Here we developed and applied a widely applicable, radioactivity-free, bioluminescence-based high-throughput screen (HTS) against 116350 compounds from structurally diverse small-molecule libraries to identify inhibitors of Pseudomonas aeruginosa TrmD ( PaTrmD). Of 285 compounds passing primary and secondary screens, a total of 61 TrmD inhibitors comprised of more than 12 different chemical scaffolds were identified, all showing submicromolar to low micromolar enzyme inhibitor constants, with binding affinity confirmed by thermal stability and surface plasmon resonance. S-Adenosyl-l-methionine (SAM) competition assays suggested that compounds in the pyridine-pyrazole-piperidine scaffold were substrate SAM-competitive inhibitors. This was confirmed in structural studies, with nuclear magnetic resonance analysis and crystal structures of PaTrmD showing pyridine-pyrazole-piperidine compounds bound in the SAM-binding pocket. Five hits showed cellular activities against Gram-positive bacteria, including mycobacteria, while one compound, a SAM-noncompetitive inhibitor, exhibited broad-spectrum antibacterial activity. The results of this HTS expand the repertoire of TrmD-inhibiting molecular scaffolds that show promise for antibiotic development.- Published
- 2019
- Full Text
- View/download PDF
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