5 results on '"Sanya Chadha"'
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2. Genetic manipulation of Leishmania donovani threonyl tRNA synthetase facilitates its exploration as a potential therapeutic target.
- Author
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Sanya Chadha, Ramachandran Vijayan, Sakshi Gupta, Manoj Munde, Samudrala Gourinath, and Rentala Madhubala
- Subjects
Arctic medicine. Tropical medicine ,RC955-962 ,Public aspects of medicine ,RA1-1270 - Abstract
Aminoacyl tRNA synthetases are central enzymes required for protein synthesis. These enzymes are the known drug targets in bacteria and fungi. Here, we for the first time report the functional characterization of threonyl tRNA synthetase (LdThrRS) of Leishmania donovani, a protozoan parasite, the primary causative agent of visceral leishmaniasis.Recombinant LdThrRS (rLdThrRS) was expressed in E. coli and purified. The kinetic parameters for rLdThrRS were determined. The subcellular localization of LdThrRS was done by immunofluorescence analysis. Heterozygous mutants of LdThrRS were generated in Leishmania promastigotes. These genetically manipulated parasites were checked for their proliferation, virulence, aminoacylation activity and sensitivity to the known ThrRS inhibitor, borrelidin. An in silico generated structural model of L. donovani ThrRS was compared to that of human.Recombinant LdThrRS displayed aminoacylation activity, and the protein is possibly localized to both the cytosol and mitochondria. The comparison of the 3D-model of LdThrRS to human ThrRS displayed considerable similarity. Heterozygous parasites showed restrictive growth phenotype and had attenuated infectivity. These heterozygous parasites were more susceptible to inhibition by borrelidin. Several attempts to obtain ThrRS homozygous null mutants were not successful, indicating its essentiality for the Leishmania parasite. Borrelidin showed a strong affinity for LdThrRS (KD: 0.04 μM) and was effective in inhibiting the aminoacylation activity of the rLdThrRS (IC50: 0.06 μM). Borrelidin inhibited the promastigotes (IC50: 21 μM) stage of parasites. Our data shows that LdThrRS is essential for L. donovani survival and is likely to bind with small drug-like molecules with strong affinity, thus making it a potential target for drug discovery efforts.
- Published
- 2018
- Full Text
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3. Genetic Validation of Leishmania donovani Lysyl-tRNA Synthetase Shows that It Is Indispensable for Parasite Growth and Infectivity
- Author
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Sanya Chadha, N. Arjunreddy Mallampudi, Debendra K. Mohapatra, and Rentala Madhubala
- Subjects
Leishmania donovani ,lysyl-tRNA synthetase ,drug targets ,genetic validation ,Microbiology ,QR1-502 - Abstract
ABSTRACT Leishmania donovani is a protozoan parasite that causes visceral leishmaniasis. Increasing resistance and severe side effects of existing drugs have led to the need to identify new chemotherapeutic targets. Aminoacyl-tRNA synthetases (aaRSs) are ubiquitous and are required for protein synthesis. aaRSs are known drug targets for bacterial and fungal pathogens. Here, we have characterized and evaluated the essentiality of L. donovani lysyl-tRNA synthetase (LdLysRS). Two different coding sequences for lysyl-tRNA synthetases are annotated in the Leishmania genome database. LdLysRS-1 (LdBPK_150270.1), located on chromosome 15, is closer to apicomplexans and eukaryotes, whereas LdLysRS-2 (LdBPK_300130.1), present on chromosome 30, is closer to bacteria. In the present study, we have characterized LdLysRS-1. Recombinant LdLysRS-1 displayed aminoacylation activity, and the protein localized to the cytosol. The LdLysRS-1 heterozygous mutants had a restrictive growth phenotype and attenuated infectivity. LdLysRS-1 appears to be an essential gene, as a chromosomal knockout of LdLysRS-1 could be generated when the gene was provided on a rescuing plasmid. Cladosporin, a fungal secondary metabolite and a known inhibitor of LysRS, was more potent against promastigotes (50% inhibitory concentration [IC50], 4.19 µM) and intracellular amastigotes (IC50, 1.09 µM) than were isomers of cladosporin (3-epi-isocladosporin and isocladosporin). These compounds exhibited low toxicity to mammalian cells. The specificity of inhibition of parasite growth caused by these inhibitors was further assessed using LdLysRS-1 heterozygous mutant strains and rescue mutant promastigotes. These inhibitors inhibited the aminoacylation activity of recombinant LdLysRS. Our data provide a framework for the development of a new class of drugs against this parasite. IMPORTANCE Aminoacyl-tRNA synthetases are housekeeping enzymes essential for protein translation, providing charged tRNAs for the proper construction of peptide chains. These enzymes provide raw materials for protein translation and also ensure fidelity of translation. L. donovani is a protozoan parasite that causes visceral leishmaniasis. It is a continuously proliferating parasite that depends heavily on efficient protein translation. Lysyl-tRNA synthetase is one of the aaRSs which charges lysine to its cognate tRNA. Two different coding sequences for lysyl-tRNA synthetases (LdLysRS) are present in this parasite. LdLysRS-1 is closer to apicomplexans and eukaryotes, whereas LdLysRS-2 is closer to bacteria. Here, we have characterized LdLysRS-1 of L. donovani. LdLysRS-1 appears to be an essential gene, as the chromosomal null mutants did not survive. The heterozygous mutants showed slower growth kinetics and exhibited attenuated virulence. This study also provides a platform to explore LdLysRS-1 as a potential drug target.
- Published
- 2017
- Full Text
- View/download PDF
4. RUFY3 links Arl8b and JIP4-Dynein complex to regulate lysosome size and positioning
- Author
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Mahak Sharma, Sanya Chadha, Prateek Chawla, Sheetal Sharma, Gaurav Kumar, Kanupriya Sethi, and Amit Tuli
- Subjects
Dynein complex ,medicine.anatomical_structure ,Lysosome ,medicine ,Biology ,Cell biology - Abstract
The whole-cell scale spatial organization of lysosomes is regulated by their bidirectional motility on microtubule tracks. Small GTP-binding (G) protein, Arl8b, stimulates the anterograde transport of lysosomes by recruiting adaptor protein SKIP (also known as PLEKHM2), which in turn couples the microtubule motor kinesin-1. Here, we have identified an Arl8b effector, RUN and FYVE domain-containing protein family member 3, RUFY3, which drives the retrograde transport of lysosomes. Artificial targeting of RUFY3 to the surface of mitochondria was sufficient to drive their perinuclear positioning. We find that RUFY3 interacts with the JIP4-Dynein-Dynactin complex and mediates Arl8b association with the retrograde motor complex. The mobile fraction of the total lysosomes per cell was significantly enhanced upon RUFY3 depletion, suggesting that RUFY3 maintains the lysosomes clustering within the perinuclear cloud. Expectedly, RUFY3 knockdown disrupted the perinuclear positioning of lysosomes upon nutrient starvation and/or serum depletion, although lysosome continued to undergo fusion with autophagosomes. Interestingly, lysosome fission events were more frequent in RUFY3-depleted cells and accordingly, there was a striking reduction in lysosome size, an effect that was also observed in dynein and JIP4 depleted cells. These findings indicate that the dynein-dependent “perinuclear cloud” arrangement of lysosomes also regulates the size of these proteolytic compartments and, likely, their cellular roles.
- Published
- 2021
5. Genetic manipulation of Leishmania donovani threonyl tRNA synthetase facilitates its exploration as a potential therapeutic target
- Author
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Ramachandran Vijayan, Manoj Munde, Sakshi Gupta, Rentala Madhubala, Samudrala Gourinath, and Sanya Chadha
- Subjects
0301 basic medicine ,Leishmania Donovani ,Life Cycles ,Acylation ,Protozoan Proteins ,Gene Expression ,Protozoology ,Pathology and Laboratory Medicine ,Biochemistry ,Parasitic Cell Cycles ,law.invention ,chemistry.chemical_compound ,Drug Delivery Systems ,law ,Drug Discovery ,Protein biosynthesis ,Medicine and Health Sciences ,Aminoacylation ,Post-Translational Modification ,Phylogeny ,Sequence Deletion ,chemistry.chemical_classification ,Protozoans ,Leishmania ,biology ,lcsh:Public aspects of medicine ,Eukaryota ,Recombinant Proteins ,Bacterial Pathogens ,Protein Transport ,Infectious Diseases ,Medical Microbiology ,Recombinant DNA ,Leishmaniasis, Visceral ,Protozoan Life Cycles ,Fatty Alcohols ,Pathogens ,Research Article ,Amastigotes ,lcsh:Arctic medicine. Tropical medicine ,Drug Research and Development ,lcsh:RC955-962 ,Parasitic Life Cycles ,030106 microbiology ,Leishmania donovani ,Microbiology ,03 medical and health sciences ,Protein Domains ,Escherichia coli ,Threonine-tRNA Ligase ,Parasitic Diseases ,Humans ,Amastigote ,Microbial Pathogens ,Pharmacology ,Organisms, Genetically Modified ,Bacteria ,Aminoacyl tRNA synthetase ,Borrelia ,Promastigotes ,Public Health, Environmental and Occupational Health ,Organisms ,Biology and Life Sciences ,Proteins ,lcsh:RA1-1270 ,biology.organism_classification ,Parasitic Protozoans ,030104 developmental biology ,Enzyme ,chemistry ,Parasitology ,Developmental Biology - Abstract
Background Aminoacyl tRNA synthetases are central enzymes required for protein synthesis. These enzymes are the known drug targets in bacteria and fungi. Here, we for the first time report the functional characterization of threonyl tRNA synthetase (LdThrRS) of Leishmania donovani, a protozoan parasite, the primary causative agent of visceral leishmaniasis. Methodology Recombinant LdThrRS (rLdThrRS) was expressed in E. coli and purified. The kinetic parameters for rLdThrRS were determined. The subcellular localization of LdThrRS was done by immunofluorescence analysis. Heterozygous mutants of LdThrRS were generated in Leishmania promastigotes. These genetically manipulated parasites were checked for their proliferation, virulence, aminoacylation activity and sensitivity to the known ThrRS inhibitor, borrelidin. An in silico generated structural model of L. donovani ThrRS was compared to that of human. Conclusions Recombinant LdThrRS displayed aminoacylation activity, and the protein is possibly localized to both the cytosol and mitochondria. The comparison of the 3D-model of LdThrRS to human ThrRS displayed considerable similarity. Heterozygous parasites showed restrictive growth phenotype and had attenuated infectivity. These heterozygous parasites were more susceptible to inhibition by borrelidin. Several attempts to obtain ThrRS homozygous null mutants were not successful, indicating its essentiality for the Leishmania parasite. Borrelidin showed a strong affinity for LdThrRS (KD: 0.04 μM) and was effective in inhibiting the aminoacylation activity of the rLdThrRS (IC50: 0.06 μM). Borrelidin inhibited the promastigotes (IC50: 21 μM) stage of parasites. Our data shows that LdThrRS is essential for L. donovani survival and is likely to bind with small drug-like molecules with strong affinity, thus making it a potential target for drug discovery efforts., Author summary Aminoacyl tRNA synthetases (aaRSs) are ubiquitous enzymes required for protein translation. They play a vital role in helping an organism's survival. Therefore, they have been suggested as favourable targets for the development of antileishmanial drugs. Leishmania, a protozoan parasite that causes leishmaniasis is known to encode 26 aaRSs. In the present study, we have worked on the functional characterization of L. donovani threonyl tRNA synthetase (LdThrRS) protein. We report that the L. donovani encodes a functional copy of ThrRS. The protein is localized in the cytosol and possibly also in mitochondria. The LdThrRS seems to be an essential gene for the parasite since null mutants did not survive. The deletion of one allele of the gene caused reduced growth and attenuated virulence in the heterozygous parasites. These parasites showed increased sensitivity to the known ThrRS inhibitor, borrelidin. Furthermore, borrelidin was found to inhibit the aminoacylation activity of LdThrRS thus, indicating that parasitic ThrRS can be exploited as a drug target for antileishmanial chemotherapy.
- Published
- 2018
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