41 results on '"Tripathi, Timir"'
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2. Interactions and interplay of MLOs with classical membrane-bound organelles
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Sasidharan, Santanu, primary, Nag, Niharika, additional, Tripathi, Timir, additional, and Saudagar, Prakash, additional
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- 2023
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3. Preface
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Saudagar, Prakash, primary and Tripathi, Timir, additional
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- 2023
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4. Integration of spectroscopic and computational data to analyze protein structure, function, folding, and dynamics
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Prince, Kavya, primary, Sasidharan, Santanu, additional, Nag, Niharika, additional, Tripathi, Timir, additional, and Saudagar, Prakash, additional
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- 2023
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5. Contributors
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Antifeeva, Iuliia A., primary, Arakawa, Hirofumi, additional, Boccaccio, Graciela Lidia, additional, Boyko, Solomiia, additional, Brocca, Stefania, additional, Darling, April L., additional, Djaja, Nathalie A., additional, Fefilova, Anna S., additional, Fernández-Alvarez, Ana Julia, additional, Ferreira, Luisa A., additional, Fonin, Alexander V., additional, Ganser, Laura R., additional, Giudice, Jimena, additional, Grandori, Rita, additional, Haider, Raza, additional, Hansma, Helen Greenwood, additional, Ilyinsky, Nikolay, additional, Ivanov, Pavel, additional, Janis, Brett, additional, Jiang, Hao, additional, Joshi, Ashish, additional, Kuznetsova, Irina M., additional, Libich, David S., additional, Longhi, Sonia, additional, Menze, Michael A., additional, Mokin, Yakov I., additional, Mukhopadhyay, Samrat, additional, Myong, Sua, additional, Nag, Niharika, additional, Nesterov, Semen, additional, Ng, Woei Shyuan, additional, Noda, Nobuo N., additional, Parra, George L., additional, Pathak, Sunita, additional, Pattanashetty, Swastik G., additional, Pesce, Giulia, additional, Putnam, Andrea, additional, Riggs, Claire L., additional, Sasidharan, Santanu, additional, Saudagar, Prakash, additional, Seydoux, Geraldine, additional, Sielaff, Hendrik, additional, Silonov, Sergey A., additional, Spruijt, Evan, additional, Strader, Lucia C., additional, Surewicz, Witold K., additional, Thomas, María Gabriela, additional, Todd Stukenberg, P., additional, Tripathi, Timir, additional, Turoverov, Konstantin K., additional, Uversky, Vladimir N., additional, Walimbe, Anuja, additional, Zaslavsky, Boris Y., additional, and Zhao, Ziqing Winston, additional
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- 2023
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6. Fundamentals of spectroscopy for biomolecular structure and dynamics
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Nag, Niharika, primary, Sasidharan, Santanu, additional, Saudagar, Prakash, additional, and Tripathi, Timir, additional
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- 2023
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7. Contributors
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Aarthy, Murali, primary, Acharya, Bidisha, additional, Aggarwal, Priyanka, additional, Ahmad, Nabeel, additional, Aiswarya, Natarajan, additional, Alici, Hakan, additional, Al-Khafaji, Khattab, additional, Assaiya, Anshul, additional, Bansal, Saurabh, additional, Bhar, Suparna, additional, Bhardwaj, Aparna, additional, Bhatia, Dhiraj, additional, Bhavesh, Neel Sarovar, additional, Bons, Joanna, additional, Chattopadhyay, Krishnananda, additional, Chetri, Purna Bahadur, additional, Coskuner-Weber, Orkid, additional, De, Soumya, additional, Dodero, Veronica Isabel, additional, Elias, Jack A., additional, Emperador, Agusti, additional, Giambruno, Roberto, additional, Giampà, Marco, additional, Giri, Rajanish, additional, Graether, Steffen P., additional, Gupta, Akshita, additional, Gupta, Smita, additional, Haque, Md Anzarul, additional, Hasekioglu, Orkun, additional, Hassan, Md Imtaiyaz, additional, Hebbar, Prajna N., additional, Hema Naveena, A., additional, Herrera, Maria Georgina, additional, Illig, Alexander-Maurice, additional, Islam, Asimul, additional, Janežič, Matej, additional, Jeyakanthan, Jeyaraman, additional, Kamle, Suchitra, additional, Kasahara, Kota, additional, Kaur, Punit, additional, Khan, Heena, additional, Kumar, Janesh, additional, Kumar, Prateek, additional, Kumar, Ritesh, additional, Kumar, Saravanan, additional, Kumar, Vijay, additional, Kumari, Pooja, additional, Kumari, Varsha, additional, Kundu, Bishwajit, additional, Lee, Chun Geun, additional, Li, Dawei, additional, Lin, Xubo, additional, Maiti, Snigdha, additional, Mandal, Narattam, additional, Mhashal, Anil, additional, Mishra, Parul, additional, Mohan, Ajitha, additional, Mukherjee, Sandip, additional, Mukherjee, Sunandan, additional, Nag, Niharika, additional, Nageswar, Koomity V., additional, Nasim, Fouzia, additional, Nayak, Santoshi, additional, Neog, Siddharth, additional, Nithin, Chandran, additional, O’Broin, Amy, additional, Olubiyi, Olujide O., additional, Orellana, Laura, additional, Padhi, Aditya K., additional, Padhy, Amrita Arpita, additional, Patil, Dipak N., additional, Prajapati, Vijay Kumar, additional, Qureshi, Insaf Ahmed, additional, Rahul, Chandrashekar Narayanan, additional, Rajwar, Anjali, additional, Ramadan, Kristijan, additional, Rashmi, S., additional, Ratnakar, Tadi Sai, additional, Rawat, Arun Kumar, additional, Rose, Jacob, additional, Roy, Anwesha, additional, Rupert, Jakob, additional, Sahoo, Subhashree, additional, Saibo, Nikita V., additional, Samantray, Suman, additional, Samarth, Nikhil H., additional, Sanjeevi, Madhumathi, additional, Sannigrahi, Achinta, additional, Sasidharan, Santanu, additional, Saudagar, Prakash, additional, Schilling, Birgit, additional, Sekar, Kanagaraj, additional, Sharma, Mansi, additional, Sharma, Pradeep, additional, Sharma, Sujata, additional, Sharma, Vinita, additional, Shivani, Kummari, additional, Singh, Abhay Narayan, additional, Singh, Anamika, additional, Singh, Appu K., additional, Singh, Meenakshi, additional, Singh, Sanjeev Kumar, additional, Singh, Satyendra, additional, Singh, Shailza, additional, Singh, Shalini, additional, Singh, Sudhir Kumar, additional, Singh, Tej P., additional, Taskin-Tok, Tugba, additional, Theiss, Arianne L., additional, Tonali, Nicolo, additional, Tripathi, Timir, additional, Trivedi, Vishal, additional, Uversky, Vladimir N., additional, Vaswani, Payal, additional, Venuprasad, K., additional, Zacco, Elsa, additional, and Zhang, Kam Y.J., additional
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- 2022
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8. Preface
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Tripathi, Timir, primary and Dubey, Vikash Kumar, additional
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- 2022
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9. Computational methods to study intrinsically disordered proteins
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Kumar, Prateek, primary, Bhardwaj, Aparna, additional, Uversky, Vladimir N., additional, Tripathi, Timir, additional, and Giri, Rajanish, additional
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- 2022
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10. Strategies to improve the expression and solubility of recombinant proteins in E. coli
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Nag, Niharika, primary, Khan, Heena, additional, and Tripathi, Timir, additional
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- 2022
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11. Methods to determine the oligomeric structure of proteins
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Chetri, Purna Bahadur, primary, Khan, Heena, additional, and Tripathi, Timir, additional
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- 2022
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12. Experimental methods to study intrinsically disordered proteins
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Nag, Niharika, primary, Chetri, Purna Bahadur, additional, Uversky, Vladimir N., additional, Giri, Rajanish, additional, and Tripathi, Timir, additional
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- 2022
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13. Experimental methods to study the thermodynamics of protein–protein interactions
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Sasidharan, Santanu, primary, Nag, Niharika, additional, Tripathi, Timir, additional, and Saudagar, Prakash, additional
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- 2022
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14. Opisthorchis viverrini Proteome and Host–Parasite Interactions
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Suttiprapa, Sutas, primary, Sotillo, Javier, additional, Smout, Michael, additional, Suyapoh, Watcharapol, additional, Chaiyadet, Sujittra, additional, Tripathi, Timir, additional, Laha, Thewarach, additional, and Loukas, Alex, additional
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- 2018
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15. Corrigendum to "Biofunctionalized chrysin-conjugated gold nanoparticles neutralize Leishmania parasites with high efficacy" [Int. J. Biol. Macromol. Volume 205, 30 April 2022, Pages 211-219].
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Raj S, Sasidharan S, Tripathi T, and Saudagar P
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- 2024
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16. Computational resources and chemoinformatics for translational health research.
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Tripathi T, Singh DB, and Tripathi T
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- Precision Medicine, Cheminformatics, Drug Discovery
- Abstract
The integration of computational resources and chemoinformatics has revolutionized translational health research. It has offered a powerful set of tools for accelerating drug discovery. This chapter overviews the computational resources and chemoinformatics methods used in translational health research. The resources and methods can be used to analyze large datasets, identify potential drug candidates, predict drug-target interactions, and optimize treatment regimens. These resources have the potential to transform the drug discovery process and foster personalized medicine research. We discuss insights into their various applications in translational health and emphasize the need for addressing challenges, promoting collaboration, and advancing the field to fully realize the potential of these tools in transforming healthcare., (Copyright © 2024. Published by Elsevier Inc.)
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- 2024
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17. Functional expression, localization, and biochemical characterization of thioredoxin glutathione reductase from air-breathing magur catfish, Clarias magur.
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Koner D, Nag N, Kalita P, Padhi AK, Tripathi T, and Saha N
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- Animals, Phylogeny, Glutathione metabolism, Antioxidants, Thioredoxin-Disulfide Reductase genetics, Thioredoxins genetics, Glutathione Reductase genetics, Catfishes genetics, Catfishes metabolism, Platyhelminths
- Abstract
The glutathione (GSH) and thioredoxin (Trx) systems regulate cellular redox homeostasis and maintain antioxidant defense in most eukaryotes. We earlier reported the absence of gene coding for the glutathione reductase (GR) enzyme of the GSH system in the facultative air-breathing catfish, Clarias magur. Here, we identified three thioredoxin reductase (TrxR) genes, one of which was later confirmed as a thioredoxin glutathione reductase (TGR). We then characterized the novel recombinant TGR enzyme of C. magur (CmTGR). The tissue-specific expression of the txnrd genes and the tissue-specific activity of the TrxR enzyme were analyzed. The recombinant CmTGR is a dimer of ~133 kDa. The protein showed TrxR activity with 5,5'-diothiobis (2-nitrobenzoic acid) reduction assay with a K
m of 304.40 μM and GR activity with a Km of 58.91 μM. Phylogenetic analysis showed that the CmTGR was related to the TrxRs of fishes and distantly related to the TGRs of platyhelminth parasites. The structural analysis revealed the conserved glutaredoxin active site and FAD- and NADPH-binding sites. To our knowledge, this is the first report of the presence of a TGR in any fish. This unusual presence of TGR in C. magur is crucial as it helps maintain redox homeostasis under environmental stressors-induced oxidative stress., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)- Published
- 2023
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18. Hotspot residues and resistance mutations in the nirmatrelvir-binding site of SARS-CoV-2 main protease: Design, identification, and correlation with globally circulating viral genomes.
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Padhi AK and Tripathi T
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- Antiviral Agents chemistry, Binding Sites, Coronavirus 3C Proteases, Cysteine Endopeptidases metabolism, Genome, Viral, Humans, Mutation, Pandemics, Protease Inhibitors chemistry, Viral Nonstructural Proteins chemistry, COVID-19 genetics, SARS-CoV-2 genetics
- Abstract
Shortly after the onset of the COVID-19 pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has acquired numerous variations in its intracellular proteins to adapt quickly, become more infectious, and ultimately develop drug resistance by mutating certain hotspot residues. To keep the emerging variants at bay, including Omicron and subvariants, FDA has approved the antiviral nirmatrelvir for mild-to-moderate and high-risk COVID-19 cases. Like other viruses, SARS-CoV-2 could acquire mutations in its main protease (M
pro ) to adapt and develop resistance against nirmatrelvir. Employing a unique high-throughput protein design technique, the hotspot residues, and signatures of adaptation of Mpro having the highest probability of mutating and rendering nirmatrelvir ineffective were identified. Our results show that ∼40% of the designed mutations in Mpro already exist in the globally circulating SARS-CoV-2 lineages and several predicted mutations. Moreover, several high-frequency, designed mutations were found to be in corroboration with the experimentally reported nirmatrelvir-resistant mutants and are naturally occurring. Our work on the targeted design of the nirmatrelvir-binding site offers a comprehensive picture of potential hotspot sites and resistance mutations in Mpro and is thus crucial in comprehending viral adaptation, robust antiviral design, and surveillance of evolving Mpro variations., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)- Published
- 2022
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19. Antibody-receptor bioengineering and its implications in designing bioelectronic devices.
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Dkhar DS, Kumari R, Mahapatra S, Divya, Kumar R, Tripathi T, and Chandra P
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- Antibodies, Antigens, Bioengineering, Biosensing Techniques methods
- Abstract
Antibodies play a crucial role in the defense mechanism countering pathogens or foreign antigens in eukaryotes. Its potential as an analytical and diagnostic tool has been exploited for over a century. It forms immunocomplexes with a specific antigen, which is the basis of immunoassays and aids in developing potent biosensors. Antibody-based sensors allow for the quick and accurate detection of various analytes. Though classical antibodies have prolonged been used as bioreceptors in biosensors fabrication due to their increased fragility, they have been engineered into more stable fragments with increased exposure of their antigen-binding sites in the recent era. In biosensing, the formats constructed by antibody engineering can enhance the signal since the resistance offered by a conventional antibody is much more than these fragments. Hence, signal amplification can be observed when antibody fragments are utilized as bioreceptors instead of full-length antibodies. We present the first systematic review on engineered antibodies as bioreceptors with the description of their engineering methods. The detection of various target analytes, including small molecules, macromolecules, and cells using antibody-based biosensors, has been discussed. A comparison of the classical polyclonal, monoclonal, and engineered antibodies as bioreceptors to construct highly accurate, sensitive, and specific sensors is also discussed., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)
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- 2022
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20. Biofunctionalized Chrysin-conjugated gold nanoparticles neutralize Leishmania parasites with high efficacy.
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Raj S, Sasidharan S, Tripathi T, and Saudagar P
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- Animals, Flavonoids pharmacology, Gold pharmacology, Mammals, Antiprotozoal Agents pharmacology, Leishmania, Metal Nanoparticles, Parasites
- Abstract
Current treatments for leishmaniasis involve various drugs, including miltefosine and amphotericin B, which are associated with several side effects and high costs. Long-term use of these drugs may lead to the development of resistance, thereby reducing their efficiency. Chrysin (CHY) is a well-known, non-toxic flavonoid with antioxidant, antiviral, anti-inflammatory, anti-cancer, hepatoprotective, and neuroprotective properties. Recently we have shown that CHY targets the MAP kinase 3 enzyme of Leishmania and neutralizes the parasite rapidly. However, CHY is associated with low bioavailability, poor absorption, and rapid excretion issues, limiting its usage. In this study, we developed and tested a novel CHY-gold nanoformulation with improved efficacy against the parasites. The reducing power of CHY was utilized to reduce and conjugate with gold nanoparticles. Gold nanoparticles, which are already known for their anti-leishmanial properties, along with conjugated CHY, exhibited a decreased parasite burden in mammalian macrophages. Our findings showed that this biofunctionalized nanoformulation could be used as a potential therapeutic tool against leishmaniasis., (Copyright © 2022. Published by Elsevier B.V.)
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- 2022
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21. Phase separation of FG-nucleoporins in nuclear pore complexes.
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Nag N, Sasidharan S, Uversky VN, Saudagar P, and Tripathi T
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- Active Transport, Cell Nucleus, Amyloid chemistry, Amyloid metabolism, Animals, Biophysical Phenomena, Humans, Molecular Dynamics Simulation, Neurodegenerative Diseases metabolism, Neurodegenerative Diseases pathology, Nuclear Pore Complex Proteins chemistry, Nuclear Envelope metabolism, Nuclear Pore Complex Proteins metabolism
- Abstract
The nuclear envelope (NE) is a bilayer membrane that separates and physically isolates the genetic material from the cytoplasm. Nuclear pore complexes (NPCs) are cylindrical structures embedded in the NE and remain the sole channel of communication between the nucleus and the cytoplasm. The interior of NPCs contains densely packed intrinsically disordered FG-nucleoporins (FG-Nups), consequently forming a permeability barrier. This barrier facilitates the selection and specificity of the cargoes that are imported, exported, or shuttled through the NPCs. Recent studies have revealed that FG-Nups undergo the process of liquid-liquid phase separation into liquid droplets. Moreover, these liquid droplets mimic the permeability barrier observed in the interior of NPCs. This review highlights the phase separation of FG-Nups occurring inside the NPCs rooted in the NE. We discuss the phase separation of FG-Nups and compare the different aspects contributing to their phase separation. Furthermore, several diseases caused by the aberrant phase separation of the proteins are examined with respect to NEs. By understanding the fundamental process of phase separation at the nuclear membrane, the review seeks to explore the parameters influencing this phenomenon as well as its importance, ultimately paving the way for better research on the structure-function relationship of biomolecular condensates., (Copyright © 2022 Elsevier B.V. All rights reserved.)
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- 2022
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22. Targeted design of drug binding sites in the main protease of SARS-CoV-2 reveals potential signatures of adaptation.
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Padhi AK and Tripathi T
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- Amino Acid Sequence, Antiviral Agents pharmacology, Binding Sites drug effects, Binding Sites genetics, Coronavirus 3C Proteases antagonists & inhibitors, Coronavirus 3C Proteases genetics, Coronavirus 3C Proteases metabolism, Genetic Fitness genetics, Hepacivirus drug effects, Hepacivirus enzymology, Ligands, Models, Molecular, Oligopeptides chemistry, Oligopeptides pharmacology, Proline analogs & derivatives, Proline chemistry, Proline pharmacology, Reproducibility of Results, SARS-CoV-2 drug effects, Selection, Genetic genetics, Structure-Activity Relationship, COVID-19 Drug Treatment, Adaptation, Physiological genetics, Antiviral Agents chemistry, Coronavirus 3C Proteases chemistry, Drug Design, Drug Resistance, Viral genetics, Mutation, SARS-CoV-2 enzymology, SARS-CoV-2 genetics
- Abstract
Several existing drugs are currently being tested worldwide to treat COVID-19 patients. Recent data indicate that SARS-CoV-2 is rapidly evolving into more transmissible variants. It is therefore highly possible that SARS-CoV-2 can accumulate adaptive mutations modulating drug susceptibility and hampering viral antigenicity. Thus, it is vital to predict potential non-synonymous mutation sites and predict the evolution of protein structural modifications leading to drug tolerance. As two FDA-approved anti-hepatitis C virus (HCV) drugs, boceprevir, and telaprevir, have been shown to effectively inhibit SARS-CoV-2 by targeting the main protease (M
pro ), here we used a high-throughput interface-based protein design strategy to identify mutational hotspots and potential signatures of adaptation in these drug binding sites of Mpro . Several mutants exhibited reduced binding affinity to these drugs, out of which hotspot residues having a strong tendency to undergo positive selection were identified. The data further indicated that these anti-HCV drugs have larger footprints in the mutational landscape of Mpro and hence encompass the highest potential for positive selection and adaptation. These findings are crucial in understanding the potential structural modifications in the drug binding sites of Mpro and thus its signatures of adaptation. Furthermore, the data could provide systemic strategies for robust antiviral design and discovery against COVID-19 in the future., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)- Published
- 2021
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23. Prevalence and functionality of intrinsic disorder in human FG-nucleoporins.
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Lyngdoh DL, Nag N, Uversky VN, and Tripathi T
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- Active Transport, Cell Nucleus, Cell Nucleus metabolism, Computational Biology methods, Cytoplasm metabolism, Databases, Genetic, Glycine chemistry, Humans, Intrinsically Disordered Proteins chemistry, Models, Molecular, Nuclear Pore chemistry, Nuclear Pore metabolism, Nuclear Pore Complex Proteins physiology, Phenylalanine chemistry, Protein Binding, Protein Folding, Protein Interaction Maps, Intrinsically Disordered Proteins metabolism, Nuclear Pore Complex Proteins chemistry, Nuclear Pore Complex Proteins metabolism
- Abstract
The nuclear-cytoplasmic transport of biomolecules is assisted by the nuclear pores composed of evolutionarily conserved proteins termed nucleoporins (Nups). The central Nups, characterized by multiple FG-repeats, are highly dynamic and contain a high level of intrinsically disordered regions (IDPRs). FG-Nups bind several protein partners and play critical roles in molecular interactions and the regulation of cellular functions through their IDPRs. In the present study, we performed a multiparametric bioinformatics analysis to characterize the prevalence and functionality of IDPRs in human FG-Nups. These analyses revealed that the sequence of all FG-Nups contained >50% IDPRs (except Nup54 and Nup358). Nup98, Nup153, and POM121 were extremely disordered with ~80% IDPRs. The functional disorder-based binding regions in the FG-Nups were identified. The phase separation behavior of FG-Nups indicated that all FG-Nups have the potential to undergo liquid-to-liquid phase separation that could stabilize their liquid state. The inherent structural flexibility in FG-Nups is mechanistically and functionally advantageous. Since certain FG-Nups interact with disease-relevant protein aggregates, their complexes can be exploited for drug design. Furthermore, consideration of the FG-Nups from the intrinsic disorder perspective provides critical information that can guide future experimental studies to uncover novel pathways associated with diseases linked with protein misfolding and aggregation., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2021 Elsevier B.V. All rights reserved.)
- Published
- 2021
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24. One year update on the COVID-19 pandemic: Where are we now?
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Mishra SK and Tripathi T
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- Adenosine Monophosphate analogs & derivatives, Adenosine Monophosphate therapeutic use, Alanine analogs & derivatives, Alanine therapeutic use, Amides therapeutic use, Animals, Antiviral Agents therapeutic use, COVID-19 immunology, COVID-19 transmission, COVID-19 Vaccines, Chloroquine therapeutic use, Clinical Trials as Topic, Coronavirus genetics, Coronavirus Infections transmission, Drug Combinations, Drug Repositioning, Glucocorticoids therapeutic use, Humans, Hydroxychloroquine therapeutic use, Indoles therapeutic use, Ivermectin therapeutic use, Lopinavir therapeutic use, Mutation, Pandemics, Phytotherapy, Plant Extracts therapeutic use, Pyrazines therapeutic use, Ritonavir therapeutic use, SARS-CoV-2 genetics, SARS-CoV-2 immunology, SARS-CoV-2 pathogenicity, Spike Glycoprotein, Coronavirus, Tinospora, Viral Zoonoses, COVID-19 therapy
- Abstract
We are living through an unprecedented crisis with the rapid spread of the new coronavirus disease (COVID-19) worldwide within a short time. The timely availability of thousands of SARS-CoV-2 genomes has enabled the scientific community to study the origin, structures, and pathogenesis of the virus. The pandemic has spurred research publication and resulted in an unprecedented number of therapeutic proposals. Because the development of new drugs is time consuming, several strategies, including drug repurposing and repositioning, are being tested to treat patients with COVID-19. Researchers have developed several potential vaccine candidates that have shown promise in phase II and III trials. As of 12 November 2020, 164 candidate vaccines are in preclinical evaluation, and 48 vaccines are in clinical evaluation, of which four have cleared phase III trials (Pfizer/BioNTech's BNT162b2, Moderna's mRNA-1273, University of Oxford & AstraZeneca's AZD1222, and Gamaleya's Sputnik V vaccine). Despite the acquisition of a vast body of scientific information, treatment depends only on the clinical management of the disease through supportive care. At the pandemic's 1-year mark, we summarize current information on SARS-CoV-2 origin and biology, and advances in the development of therapeutics. The updated information presented here provides a comprehensive report on the scientific progress made in the past year in understanding of SARS-CoV-2 biology and therapeutics., (Copyright © 2020 Elsevier B.V. All rights reserved.)
- Published
- 2021
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25. Engineering glutathione S-transferase with a point mutation at conserved F136 residue increases the xenobiotic-metabolizing activity.
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Kalita J, Shukla H, and Tripathi T
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- Amino Acid Sequence, Animals, Binding Sites genetics, Catalysis, Fasciola genetics, Glutathione genetics, Kinetics, Molecular Docking Simulation methods, Sequence Alignment, Glutathione Transferase genetics, Inactivation, Metabolic genetics, Point Mutation genetics, Xenobiotics metabolism
- Abstract
Glutathione S-transferases (GSTs) are multifunctional enzymes that play major roles in a wide range of biological processes, including cellular detoxification, biosynthesis, metabolism, and transport. The dynamic structural scaffold and diverse functional roles of GSTs make them important for enzyme engineering and for exploring novel biotechnological applications. The present study reported a significant gain-of-function activity in GST caused by a point mutation at the conserved F136 residue. The fluorescence quenching and kinetic data suggested that both binding affinity and catalytic efficiency of the mutant enzyme to the substrates 1-chloro-2,4-dinitrobenzene (CDNB), as well as the glutathione (GSH), is increased. Molecular docking showed that the mutation improves the binding interactions of the GSH with several binding-site residues. The simulation of molecular dynamics revealed that the mutant enzyme gained increased structural rigidity than the wild-type enzyme. The mutation also altered the residue interaction network (RIN) of the GSH-binding residues. These phenomena suggested that mutations led to conformational alterations and dominant differential motions in the enzyme that lead to increased rigidity and modifications in RIN. Collectively, engineering GST with a single point mutation at conserved F136 can significantly increase its xenobiotic activity by increasing the catalytic efficiency that may be exploited for biotechnological applications., Competing Interests: Declaration of competing interest The authors declare no conflict of interest exists., (Copyright © 2020 Elsevier B.V. All rights reserved.)
- Published
- 2020
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26. Conserved Arg451 residue is critical for maintaining the stability and activity of thioredoxin glutathione reductase.
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Kalita P, Shukla H, Das KC, and Tripathi T
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- Animals, Catalytic Domain, Fasciola enzymology, Helminth Proteins genetics, Helminth Proteins metabolism, Molecular Dynamics Simulation, Multienzyme Complexes genetics, Multienzyme Complexes metabolism, NADH, NADPH Oxidoreductases genetics, NADH, NADPH Oxidoreductases metabolism, Point Mutation, Principal Component Analysis, Protein Stability, Protein Structure, Secondary genetics, Protein Unfolding, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Thermodynamics, Arginine chemistry, Helminth Proteins chemistry, Multienzyme Complexes chemistry, NADH, NADPH Oxidoreductases chemistry
- Abstract
Thioredoxin glutathione reductase (TGR), a potential anthelminthic drug target causes NADPH-dependent transfer of electrons to both thioredoxins and glutathione systems. In the present study, we showed that a single point mutation conserved at Arg451 position is critical for maintaining the structure-function of FgTGR. The current biochemical results showed that R451A mutation significantly decreases both oxidoreductase activities (glutathione reductase and thioredoxin reductase) of the enzyme. Computational analyses using molecular dynamics simulation provided an in-depth insight into the structural alterations caused as a result of the mutation. Furthermore, the different regions of the mutant FgTGR structure were found to be altered in flexibility/rigidity as a result of the mutation. This led to mutant-specific conformational alterations and dominant differential motions that contributed to the abrogated function of mutant FgTGR. These results were confirmed using GdnHCl-induced denaturation-based stability studies. Moreover, mutation reduced the free energy of stabilization of the protein, thereby destabilizing the mutant protein structure. Therefore, these findings displayed differential dynamics in the FgTGR structure and highlighted the relevance of residue-level interactions in the protein. Thus, the current study provided a basis for exploiting regions other than the active site of TGR for inhibitory effect and development of novel antihelminthics., (Copyright © 2019 Elsevier Inc. All rights reserved.)
- Published
- 2019
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27. Development of multi-epitope driven subunit vaccine against Fasciola gigantica using immunoinformatics approach.
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Kalita P, Lyngdoh DL, Padhi AK, Shukla H, and Tripathi T
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- Amino Acid Sequence, Animals, Helminth Proteins chemistry, Helminth Proteins immunology, Molecular Docking Simulation, Molecular Dynamics Simulation, Protein Structure, Tertiary, Thermodynamics, Vaccines, Subunit chemistry, Computational Biology, Epitopes immunology, Fasciola immunology, Vaccines, Subunit immunology
- Abstract
Fascioliasis, a serious helminth disease of the livestock population, results from infection with the parasite Fasciola. Despite the alarming increase in drug resistance, a safe and fully effective vaccine for fascioliasis is still not available. In the present study, we employed high-throughput immunoinformatics approaches to design a multi-epitope based subunit vaccine using seven important F. gigantica proteins (cathepsin B, cathepsin L, leucyl aminopeptidase, thioredoxin glutathione reductase, fatty acid binding protein-1, saposin-like protein-2, and 14-3-3 protein epsilon). The CTL, HTL, and B-cell epitopes were selected for designing the vaccine on the basis of their immunogenic behavior and binding affinity. The engineered vaccine showed potential immunogenic efficacy by elaborating the IFN-γ and humoral response. The modeled structure of the vaccine was docked with the toll-like receptor-2 immune receptor, and the molecular dynamics simulation was performed to understand the stability, interaction, and dynamics of the complex. Finally, in silico cloning of the resulting vaccine was performed to create the plasmid construct of vaccine for expression in an appropriate biological system. Experimental evaluation of the designed vaccine construct in an animal model may result in a novel and immunogenic vaccine that may confer protection against F. gigantica infection., (Copyright © 2019 Elsevier B.V. All rights reserved.)
- Published
- 2019
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28. Unfolding of Acinetobacter baumannii MurA proceeds through a metastable intermediate: A combined spectroscopic and computational investigation.
- Author
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Sonkar A, Shukla H, Shukla R, Kalita J, and Tripathi T
- Subjects
- Acrylamide chemistry, Bacterial Proteins isolation & purification, Fluorescence, Hydrogen Bonding, Molecular Dynamics Simulation, Principal Component Analysis, Protein Structure, Secondary, Recombinant Proteins isolation & purification, Solvents chemistry, Thermodynamics, Time Factors, Urea pharmacology, Acinetobacter baumannii metabolism, Bacterial Proteins chemistry, Bacterial Proteins metabolism, Protein Unfolding drug effects, Spectrum Analysis
- Abstract
Peptidoglycan (PG) is the main constituent of the bacterial cell wall. The enzyme UDP‑N‑acetylglucosamine enolpyruvyl transferase (MurA) catalyzes the transfer of enolpyruvate from phosphoenolpyruvate to uridinediphospho‑N‑acetylglucosamine, which is the first committed step of PG biosynthesis. In this study, we have systematically examined the urea-induced unfolding of Acinetobacter baumannii MurA (AbMurA) using various optical spectroscopic techniques and molecular dynamics (MD) simulations. The urea-induced unfolding of AbMurA was a three-state process, where a metastable intermediate conformation state is populated between 3.0 and 4.0 M. Above 6.0 M urea, AbMurA gets completely unfolded. The transition from the native structure to the partially unfolded metastable state involves ~30% loss of native contacts but little change in the radius of gyration or core hydration properties. The intermediate-to-unfolded state transition was characterized by a large increase in the radius of gyration. MD trajectories simulated in different unfolding conditions suggest that urea destabilizes AbMurA structure weakening hydrophobic interactions and the hydrogen bond network. We observed a clear correlation between both in vitro and in silico studies. To our knowledge, this is also the first report on unfolding/stability analysis of any MurA enzyme., (Copyright © 2018 Elsevier B.V. All rights reserved.)
- Published
- 2019
- Full Text
- View/download PDF
29. Point mutation A394E in the central intrinsic disordered region of Rna14 leads to chromosomal instability in fission yeast.
- Author
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Sonkar A, Lyngdoh DL, Shukla R, Shukla H, Tripathi T, and Ahmed S
- Subjects
- Alleles, Chromosome Aberrations, Genotype, RNA Splicing, Transcription Termination, Genetic, Amino Acid Substitution, Chromosomal Instability, Intrinsically Disordered Proteins genetics, Point Mutation, Schizosaccharomyces genetics, Schizosaccharomyces pombe Proteins genetics, mRNA Cleavage and Polyadenylation Factors genetics
- Abstract
Accurate chromosomal segregation is crucial for the maintenance of genomic integrity. Rna14 is a major component of the yeast pre-mRNA 3'-end processing factor, the cleavage factor IA complex, and is involved in cleavage and polyadenylation of mRNA in the nucleus. Rna14 is also essential for the maintenance of genomic integrity in fission yeast Schizosaccharomyces pombe. In the present study, we report that a non-homologous mutation, A394E that is present in the central intrinsic disordered region of Rna14 leads to chromosomal instability in fission yeast. This mutation was shown to disrupt chromosome segregation and 3'-end maturation, and also affects the pre-mRNA splicing in vivo at non-permissive temperatures. We observed that a significant part of Rna14 is intrinsically disordered, that includes the N- and C-terminal of Rna14, as well as the central region containing the HAT repeats and the mutation within amino acid residues 372-435. These regions are crucial for the function of Rna14 as they are involved in the interaction of Rna14 with other proteins., (Copyright © 2018 Elsevier B.V. All rights reserved.)
- Published
- 2018
- Full Text
- View/download PDF
30. Role of the glutaredoxin domain and FAD in the stabilization of thioredoxin glutathione reductase.
- Author
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Kalita P, Shukla H, Gadhave K, Giri R, and Tripathi T
- Subjects
- Animals, Catalysis, Dithionitrobenzoic Acid metabolism, Fasciola enzymology, Flavin-Adenine Dinucleotide metabolism, Glutaredoxins genetics, Glutaredoxins isolation & purification, Glutaredoxins metabolism, Helminth Proteins genetics, Helminth Proteins isolation & purification, Helminth Proteins metabolism, Multienzyme Complexes genetics, Multienzyme Complexes isolation & purification, Multienzyme Complexes metabolism, Mutation, NADH, NADPH Oxidoreductases genetics, NADH, NADPH Oxidoreductases isolation & purification, NADH, NADPH Oxidoreductases metabolism, NADP metabolism, Protein Binding, Protein Conformation drug effects, Protein Stability, Protein Unfolding drug effects, Thioredoxins chemistry, Thioredoxins genetics, Thioredoxins isolation & purification, Thioredoxins metabolism, Tryptophan chemistry, Urea chemistry, Flavin-Adenine Dinucleotide chemistry, Glutaredoxins chemistry, Helminth Proteins chemistry, Multienzyme Complexes chemistry, NADH, NADPH Oxidoreductases chemistry, Protein Domains
- Abstract
Thioredoxin glutathione reductase (TGRsec) is a multi-domain flavoprotein that plays a principal role in redox homeostasis maintenance. We have previously demonstrated the role of selenocysteine in maintaining TGRsec structure-function, but the role of the glutaredoxin (Grx) domain and FAD is still unclear. In the present study, the urea-induced unfolding of recombinant Fasciola gigantica TGRsec (FgTGRsec) and its N-terminal truncated variant (ΔNTD-FgTGRsec) were examined to understand the role of the Grx domain and FAD in the stabilization of FgTGRsec and ΔNTD-FgTGRsec. Our results showed that both proteins underwent unfolding in a three state manner. First, the protein undergoes a conformational transition rendering a near-native state with no FAD bound, and then full unfolding of the apo-dimer occurs without dissociation. The Grx domain stabilized the global FgTGRsec structure and positively regulated FgTGRsec activity, and alteration in the FAD microenvironment was directly proportional to the loss of thioredoxin reductase (TrxR) and glutathione reductase activities. Based on these results, we concluded that the Grx domain stabilizes the full-length FgTGRsec protein for efficient catalysis. Thus, we suggest that in platyhelminth parasites, during evolution, the Grx domain merged with the TrxR domain to confer higher catalytic activity and provide additional structural stability to the full-length TGR., (Copyright © 2018 Elsevier Inc. All rights reserved.)
- Published
- 2018
- Full Text
- View/download PDF
31. Biochemical and thermodynamic comparison of the selenocysteine containing and non-containing thioredoxin glutathione reductase of Fasciola gigantica.
- Author
-
Kalita P, Shukla H, Shukla R, and Tripathi T
- Subjects
- Animals, Binding Sites, Cattle, Glutathione metabolism, Liver enzymology, Models, Molecular, Molecular Docking Simulation, Multienzyme Complexes isolation & purification, NADH, NADPH Oxidoreductases isolation & purification, Oxidation-Reduction, Protein Binding, Thermodynamics, Fasciola enzymology, Liver parasitology, Multienzyme Complexes chemistry, Multienzyme Complexes metabolism, NADH, NADPH Oxidoreductases chemistry, NADH, NADPH Oxidoreductases metabolism, Selenocysteine chemistry, Selenocysteine metabolism
- Abstract
The thiol-disulfide redox metabolism in platyhelminth parasites depends entirely on a single selenocysteine (Sec) containing flavoenzyme, thioredoxin glutathione reductase (TGR) that links the classical thioredoxin (Trx) and glutathione (GSH) systems. In the present study, we investigated the catalytic and structural properties of different variants of Fasciola gigantica TGR to understand the role of Sec. The recombinant full-length Sec containing TGR (FgTGRsec), TGR without Sec (FgTGR) and TGRsec without the N-terminal glutaredoxin (Grx) domain (∆NTD-FgTGRsec) were purified to homogeneity. Biochemical studies revealed that Sec597 is responsible for higher thioredoxin reductase (TrxR) and glutathione reductase (GR) activity of FgTGRsec. The N-terminal Grx domain was found to positively regulate the DTNB-based TrxR activity of FgTGRsec. The FgTGRsec was highly sensitive to inhibition by auranofin (AF). The structure of FgTGR was modeled, and the inhibitor AF was docked, and binding sites were identified. Unfolding studies suggest that all three proteins are highly cooperative molecules since during GdnHCl-induced denaturation, a monophasic unfolding of the proteins without stabilization of any intermediate is observed. The Cm for GdnHCl induced unfolding of FgTGR was higher than FgTGRsec and ∆NTD-FgTGRsec suggesting that FgTGR without Sec was more stable in solution than the other protein variants. The free energy of stabilization for the proteins was also determined. To our knowledge, this is also the first report on unfolding and stability analysis of any TGR., (Copyright © 2018 Elsevier B.V. All rights reserved.)
- Published
- 2018
- Full Text
- View/download PDF
32. Aminoacyl-tRNA synthetases: Structure, function, and drug discovery.
- Author
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Rajendran V, Kalita P, Shukla H, Kumar A, and Tripathi T
- Subjects
- Amino Acids genetics, Amino Acyl-tRNA Synthetases antagonists & inhibitors, Amino Acyl-tRNA Synthetases genetics, Amino Acyl-tRNA Synthetases metabolism, Autoantibodies chemistry, Autoantibodies immunology, Enzyme Inhibitors chemistry, Humans, Mutation, RNA, Transfer chemistry, Signal Transduction genetics, Amino Acids chemistry, Amino Acyl-tRNA Synthetases chemistry, Drug Discovery, Structure-Activity Relationship
- Abstract
Aminoacyl-tRNA synthetases (AARSs) are the enzymes that catalyze the aminoacylation reaction by covalently linking an amino acid to its cognate tRNA in the first step of protein translation. Beyond this classical function, these enzymes are also known to have a role in several metabolic and signaling pathways that are important for cell viability. Study of these enzymes is of great interest to the researchers due to its pivotal role in the growth and survival of an organism. Further, unfolding the interesting structural and functional aspects of these enzymes in the last few years has qualified them as a potential drug target against various diseases. Here we review the classification, function, and the conserved as well the appended structural architecture of these enzymes in detail, including its association with multi-synthetase complexes. We also considered their role in human diseases in terms of mutations and autoantibodies against AARSs. Finally, we have discussed the available inhibitors against AARSs. This review offers comprehensive information on AARSs under a single canopy that would be a good inventory for researchers working in this area., (Copyright © 2018 Elsevier B.V. All rights reserved.)
- Published
- 2018
- Full Text
- View/download PDF
33. Salt-regulated reversible fibrillation of Mycobacterium tuberculosis isocitrate lyase: Concurrent restoration of structure and activity.
- Author
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Shukla H, Kumar R, Sonkar A, Mitra K, Akhtar MS, and Tripathi T
- Subjects
- Kinetics, Protein Structure, Quaternary, Isocitrate Lyase chemistry, Isocitrate Lyase metabolism, Mycobacterium tuberculosis enzymology, Protein Multimerization drug effects, Salts pharmacology
- Abstract
Protein fibrillation is associated with a number of neurodegenerative diseases. Nevertheless, several proteins not related to disease can also form fibrils in vitro under specific conditions. In the present study, we demonstrate the reversible fibrillation of a globular protein that is modulated by salt under physiological pH. Mycobacterium tuberculosis Isocitrate lyase (MtbICL) is a crucial enzyme involved in the glyoxylate shunt and a potential drug target against M. tuberculosis infection. Under physiological pH, the enzyme self-assembles into a fibrillar structure in the absence of salt in vitro. The mature fibrillar structure of MtbICL is dynamic and restores its tetrameric structure as well as activity with the addition of salt. The kinetics of fibril formation was investigated spectroscopically using 8-Anilinonaphthalene-1-sulfonic acid (ANS). Further, Transmission electron microscopy (TEM) and Atomic force microscopy (AFM) imaging also confirmed the formation of elongated fibrils in the absence of salt. The results indicate the balance between stabilizing forces and the localized electrostatic repulsions destabilizing the tetrameric MtbICL is adjusted via ion shielding. Our result is in congruence of the hypothesis that amyloid formation is an intrinsic property of most, if not all natural proteins under an appropriate set of conditions., (Copyright © 2017 Elsevier B.V. All rights reserved.)
- Published
- 2017
- Full Text
- View/download PDF
34. Alterations in conformational topology and interaction dynamics caused by L418A mutation leads to activity loss of Mycobacterium tuberculosis isocitrate lyase.
- Author
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Shukla H, Shukla R, Sonkar A, and Tripathi T
- Subjects
- Mycobacterium tuberculosis metabolism, Protein Conformation, Alanine genetics, Isocitrate Lyase metabolism, Lysine genetics, Molecular Dynamics Simulation, Mycobacterium tuberculosis enzymology, Point Mutation
- Abstract
Mycobacterium tuberculosis isocitrate lyase (MtbICL) is a key enzyme of the glyoxylate cycle that catalyzes the cleavage of isocitrate to succinate and glyoxylate and is a potential antituberculosis drug target. The aim of this research was to explore the structural alterations induced by L418A point mutation that caused the loss of enzyme activity. In-depth structural analyses were carried out for understanding the influence of L418A mutation using techniques, viz. molecular dynamics, principal component analysis, time-dependent secondary structure, residue interaction network and molecular docking. Since L418A mutation site is structurally far from the active site, it cannot influence the binding of the substrate directly. Our results showed that collective motions, residual mobility, and flexibility of the enzyme increased upon mutation. The mutated residue changed the global conformational dynamics of the system along with the residue-residue interaction network, leading to a loss of the enzyme activity. The docking results suggest that L418A mutation influenced the binding interactions of the substrate with several residues in the active site of MtbICL. This study provides information on the structural dynamics of MtbICL and highlights the importance of residue level interactions in the protein. Thus, our results may provide significant guidance to the scientific community engaged in designing potent inhibitors targeting MtbICL., (Copyright © 2017 Elsevier Inc. All rights reserved.)
- Published
- 2017
- Full Text
- View/download PDF
35. Unusual thiol-based redox metabolism of parasitic flukes.
- Author
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Tripathi T, Suttiprapa S, and Sripa B
- Subjects
- Animals, Oxidation-Reduction, Antioxidants metabolism, Clonorchis sinensis metabolism, Opisthorchis metabolism, Sulfhydryl Compounds metabolism
- Abstract
Parasitic flukes are exposed to free radicals and, to a greater extent, reactive oxygen species (ROS) during their life cycle. Despite being relentlessly exposed to ROS released by activated immune cells, these parasites can survive for many years in the host. Cellular thiol-based redox metabolism plays a crucial role in parasite survival within their hosts. Evidence shows that oxidative stress and redox homeostasis maintenance are important clinical and pathobiochemical as well as effective therapeutic principles in various diseases. The characterization of redox and antioxidant enzymes is likely to yield good target candidates for novel drugs and vaccines. The absence of active catalase in fluke parasites offers great potential for the development of chemotherapeutic agents that act by perturbing the redox equilibrium of the cell. One of the redox-sensitive enzymes, thioredoxin glutathione reductase (TGR), has been accepted as a drug target against blood fluke infections, and related clinical trials are in progress. TGR is the sole enzyme responsible for Trx and GSH reduction in parasitic flukes. The availability of helminth genomes has accelerated the research on redox metabolism of flukes; however, significant achievements have yet to be attained. The present review summarizes current knowledge on the redox and antioxidant system of the parasitic flukes., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)
- Published
- 2017
- Full Text
- View/download PDF
36. Purification and characterization of two-domain glutaredoxin in the parasitic helminth Fasciola gigantica.
- Author
-
Gupta A, Sripa B, and Tripathi T
- Subjects
- Amino Acid Sequence, Animals, Escherichia coli genetics, Fasciola genetics, Gene Expression, Glutaredoxins chemistry, Glutaredoxins metabolism, Helminth Proteins chemistry, Helminth Proteins metabolism, Organisms, Genetically Modified, Recombinant Proteins genetics, Recombinant Proteins metabolism, Sequence Alignment, Fasciola physiology, Glutaredoxins genetics, Helminth Proteins genetics
- Abstract
Glutaredoxins (Grxs) are small thiol-dependent proteins and key elements of redox signaling as they regulate the redox state of important cellular proteins. In the present study, the complete sequence of a glutaredoxin protein, obtained from the liver fluke Fasciola gigantica, was PCR-amplified and cloned. The 690-bp open reading frame (ORF) encodes a 230-amino acid protein with two conserved domains (FgGrxD1 and FgGrxD2) and has similarities with two monothiol Grxs of Saccharomyces cerevisiae, i.e., ScGrx3 and ScGrx4. The full-length FgGrx along with its two constituent domains were overexpressed in Escherichia coli as hexahistidyl-tagged proteins. The affinity chromatography resulted in almost pure and soluble proteins. The full-length FgGrx and the FgGrxD2 showed reddish-brown color, indicating the presence of bound iron in the second domain. In the insulin based reduction assay, both FgGrx and FgGrxD2 containing the active site motif CGFS exhibited a weak reducing activity, whereas FgGrxD1 was inactive. Additionally, FgGrx did not show any GSH-disulfide transhydrogenase activity when 2-hydroxyethyl disulfide (HED) or de-hydroascorbate (DHA) were taken as substrates. These results indicated the probable role of FgGrx in cellular iron-sulfur homeostasis. FgGrx was found to be reversibly S-glutathionylated, suggesting a potential redox regulation that is likely to take place at the active site Cys158. Since there is only one Cys in FgGrxD2, the Cys158 might be involved in FeS binding. This study is the first report on the presence of Grx in platyhelminthic parasites and provides a starting point for further characterization of the redox network in liver flukes., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)
- Published
- 2017
- Full Text
- View/download PDF
37. UDP-N-Acetylglucosamine enolpyruvyl transferase (MurA) of Acinetobacter baumannii (AbMurA): Structural and functional properties.
- Author
-
Sonkar A, Shukla H, Shukla R, Kalita J, Pandey T, and Tripathi T
- Subjects
- Alkyl and Aryl Transferases antagonists & inhibitors, Amino Acid Sequence, Catalytic Domain, Enzyme Inhibitors metabolism, Enzyme Inhibitors pharmacology, Fosfomycin metabolism, Fosfomycin pharmacology, Hydrogen-Ion Concentration, Kinetics, Molecular Docking Simulation, Phylogeny, Sequence Alignment, Sequence Homology, Amino Acid, Temperature, Acinetobacter baumannii enzymology, Alkyl and Aryl Transferases chemistry, Alkyl and Aryl Transferases metabolism
- Abstract
Peptidoglycan (PG) is the key component of the bacterial cell wall. The enzyme UDP-N-Acetylglucosamine Enolpyruvyl Transferase (MurA) catalyzes the transfer of enolpyruvate from phosphoenolpyruvate (PEP) to uridinediphospho-N-acetylglucosamine (UNAG), which is the first committed step of PG biosynthesis. Here, we present the biochemical and structural features of the MurA enzyme of the opportunistic pathogen Acinetobacter baumannii (AbMurA). The recombinant AbMurA exists as a monomer in solution and shows optimal activity at pH 7.5 and 37°C. The Km for UDP-N-acetylglucosamine was 1.062±0.09mM and for PEP was 1.806±0.23mM. The relative enzymatic activity was inhibited ∼3 fold in the presence of 50mM fosfomycin (FFQ). Superimposition of the AbMurA model with E. coli demonstrated key structural similarity in the FFQ-binding site. AbMurA also has a surface loop that contains the active site Cys116 that interact with FFQ. Sequence analysis indicates the presence of the five conserved amino acids, i.e., K22, C116, D306, D370 and L371, required for the functional activity like other MurA enzymes from different bacteria. MurA enzymes are indispensable for cell integrity and their lack of counterparts in eukaryotes suggests them to be a promising drug target., (Copyright © 2017 Elsevier B.V. All rights reserved.)
- Published
- 2017
- Full Text
- View/download PDF
38. A combined biochemical and computational studies of the rho-class glutathione s-transferase sll1545 of Synechocystis PCC 6803.
- Author
-
Pandey T, Shukla R, Shukla H, Sonkar A, Tripathi T, and Singh AK
- Subjects
- Amino Acid Sequence, Benzene Derivatives chemistry, Catalytic Domain, Conserved Sequence, Dichloroacetic Acid chemistry, Hydrogen Bonding, Kinetics, Molecular Dynamics Simulation, Oxidation-Reduction, Phylogeny, Protein Structure, Secondary, Structural Homology, Protein, Substrate Specificity, Bacterial Proteins chemistry, Glutathione Transferase chemistry, Peroxidase chemistry, Synechocystis enzymology
- Abstract
Peroxides are one of the most important radicals that cause oxidative stress. Certain Glutathione S-transferases (GSTs) have been reported to show peroxidase activity. We report a novel peroxidase activity of Synechocystis GST- sll1545. The recombinant protein was purified to homogeneity and characterized. Low Km (0.109μM) and high Vmax (0.663μmolmin
-1 ) values suggest a high preference of sll1545 for cumenehydroperoxide. Disc inhibition assay confirmed the ability of the enzyme to protect cells against peroxide-induced damage. sll1545 has very low sequence and structural similarity with theta and alpha class GSTs that exhibit glutathione-dependent peroxidase activity. Recent data from our laboratory shows that sll1545 is also strongly active against dichloroacetate (DCA), which is a characteristic of zeta class GST. Interestingly, sll1545 shows less than 20% sequence identity with zeta class GST. Molecular dynamic simulation results show that sll1545 was much more structurally different from alpha/theta classes. Our results suggest that sll1545 shows structural variation from zeta, theta/alpha classes of GSTs but have related enzymatic activity. Phylogenetic analysis reveal that sll1545 is evolutionally very distinct from the known GSTs. Overall, the data suggest that Synechocystis sll1545 does not belong to any known GST class and represent a novel GST class, which we have named rho., (Copyright © 2016 Elsevier B.V. All rights reserved.)- Published
- 2017
- Full Text
- View/download PDF
39. Fasciola gigantica thioredoxin glutathione reductase: Biochemical properties and structural modeling.
- Author
-
Gupta A, Kesherwani M, Velmurugan D, and Tripathi T
- Subjects
- Amino Acid Sequence, Animals, Biocatalysis, Chromatography, Gel, Coenzymes metabolism, Computer Simulation, Disulfides metabolism, Glutathione Disulfide chemistry, Insulin metabolism, Kinetics, Molecular Docking Simulation, Multienzyme Complexes isolation & purification, NADH, NADPH Oxidoreductases isolation & purification, Oxidation-Reduction, Protein Binding, Protein Multimerization, Recombinant Proteins chemistry, Recombinant Proteins isolation & purification, Sequence Alignment, Substrate Specificity, Thioredoxins metabolism, Fasciola enzymology, Models, Molecular, Multienzyme Complexes chemistry, Multienzyme Complexes metabolism, NADH, NADPH Oxidoreductases chemistry, NADH, NADPH Oxidoreductases metabolism
- Abstract
Platyhelminth thioredoxin glutathione reductase (TGR) is a multifunctional enzyme that crosstalk between the conventional thioredoxin (Trx) and glutathione (GSH) system. It has been validated as a potential drug target in blood flukes. In the present study, we have performed a biochemical study on Fasciola gigantica TGR with substrates DTNB and GSSG. The Michaelis constant (Km) with DTNB was found to be 4.34±0.12μM while it was 61.15±1.50μM with GSSG. The kinetic results were compared with the TGR activities of other helminths. FgTGR showed typical hysteretic behavior with GSSG as other TGRs. We also described a homology-based structure of FgTGR. The cofactors (NADPH and FAD) and substrates (GSSG and DTNB) were docked, and two possible binding sites for substrates were identified in a single chain. The substrates were found to bind more favorably in the second site of TrxR domains. We also presented the first report on binding interaction of DTNB with a TGR. DTNB forms H-bond with His204 and Arg450 of chain A, Sec597, and Gly598 from chain B, salt-bridge with Lys124, and numerous other hydrophobic interactions. Helminth TGR represents an important enzyme in the redox and antioxidant system; hence, its inhibition can be used as an effective strategy against liver flukes., (Copyright © 2016 Elsevier B.V. All rights reserved.)
- Published
- 2016
- Full Text
- View/download PDF
40. Preferential regeneration of thioredoxin from parasitic flatworm Fasciola gigantica using glutathione system.
- Author
-
Gupta A, Pandey T, Kumar B, and Tripathi T
- Subjects
- Animals, Antioxidants pharmacology, Biocatalysis drug effects, Biological Assay, Computer Simulation, Insulin metabolism, Protein Multimerization drug effects, Recombinant Proteins metabolism, Fasciola metabolism, Glutathione metabolism, Parasites metabolism, Thioredoxins metabolism
- Abstract
The maintenance of cellular redox homeostasis is a crucial adaptive problem faced by parasites, and its disruption can shift the biochemical balance toward the host. The thioredoxin (Trx) system plays a key role in redox metabolism and defense against oxidative stress. In this study, biochemical experiments were performed on Fasciola gigantica Thioredoxin1 (FgTrx1). The recombinant FgTrx1 exists as a monomer and catalyzes the reduction of insulin. FgTrx1 is preferentially regenerated by the glutathione (GSH) system using glutathione reductase (GR). The regeneration of FgTrx1 by the conventional Trx system is much less as compared to the GSH system, suggesting that FgTrx1 could be acting as glutaredoxin (Grx). DNA nicking and hydroperoxide assay suggests that it protects the DNA from radical-induced oxidative damage. Thus, FgTrx1 might play a role in parasite survival as it can regenerate itself even in the absence of the canonical Trx system and also protect the cells from ROS induced damage. Further, we propose that the GR activity of FgTrx1 is not restricted to -CXXC- motif but is regulated by residues present in close proximity to the -CXXC- motif, through manipulation of the redox potential or the pKa of the active site Cys residues., (Copyright © 2015 Elsevier B.V. All rights reserved.)
- Published
- 2015
- Full Text
- View/download PDF
41. Structural, functional and unfolding characteristics of glutathione S-transferase of Plasmodium vivax.
- Author
-
Tripathi T, Na BK, Sohn WM, Becker K, and Bhakuni V
- Subjects
- Animals, Enzyme Stability drug effects, Glutathione metabolism, Guanidine pharmacology, Models, Molecular, Protein Multimerization drug effects, Protein Structure, Quaternary, Salts pharmacology, Solvents pharmacology, Spectrum Analysis, Urea pharmacology, Glutathione Transferase chemistry, Glutathione Transferase metabolism, Plasmodium vivax enzymology, Protein Denaturation drug effects
- Abstract
Glutathione S-transferases (GSTs) of Plasmodium parasites are potential targets for antimalarial drug and vaccine development. We investigated the equilibrium unfolding, functional activity regulation and stability characteristics of the unique GST of Plasmodium vivax (PvGST). Despite high sequence, structural, functional, and evolutionary similarity, the unfolding behavior of PvGST was significantly different from Plasmodium falciparum GST (PfGST). The unfolding pathway of PvGST was non-cooperative with stabilization of an inactive dimeric intermediate. The absence of any compact, folded monomeric intermediate during the unfolding transition suggests that inter-subunit interactions play an important role in stabilizing the protein. Presence of salts effectively inhibited PvGST enzymatic activity by quenching the nucleophilicity of the thiolate anion of GSH. Based on the present findings, together with our previous studies on PfGST, we propose that the regulation of GST enzymatic activity through a dimer-tetramer transition via GSH binding is an exclusive feature of Plasmodium.
- Published
- 2009
- Full Text
- View/download PDF
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