The work is focused on the following two major themes.
1. Investigation of the role of human ribonucleases, particularly eosinophil ribonucleases, eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN) in host defense. Human ribonucleases, and naturally occurring protein toxins are being explored to design knowledge-based recombinant toxins.
2. Investigation of crucial housekeeping proteins of M. tuberculosis for their role in survival and virulence of the pathogen. The functioning of caseinolytic protease (Clp) machinery, and RNase P mediated tRNA maturation is being investigated in M. tuberculosis.
Summary of Research:
We been investigating the role of human ribonucleases, including human pancreatic ribonuclease (HPR), EDN and ECP in host defense. HPR has a unique property of having a strong double stranded RNA cleavage activity. We have demonstrated that for the double stranded RNA cleavage activity of HPR glycine 38 is very important, and the basicity of the protein also plays a crucial role. In a very significant study we have shown that the interaction between HPR and ribonuclease inhibitor (RI) can be disrupted by mutating residues involved in HPR-RI binding. The inhibitor-resistant HPR mutants have been shown to manifest cytotoxic activity towards tumor cells. Further, using an inhibitor-resistant HPR mutant a recombinant dimeric HPR variant has been generated which shows specific cytotoxicity to tumor cells. The catalytic mechanism of eosinophil ribonuclease, EDN has also been delineated by our group to understand its role in eosinophil biology. We have also delineated the mechanism of antibacterial activity of ECP, and antiviral activity of EDN.
To understand the mechanism of action of a potent ribonucleolytic protein toxin, restrictocin from Aspergillus, we have identified a crucial histidine residue responsible for the target recognition, delineated the role of various cysteines and prolines in restrictocin catalysis, and also defined the functional boundaries in the toxin molecule. We have also solved the mechanism of specific target recognition and RNA hydrolysis by ribonucleolytic toxin restrictocin by an extensive mutational analysis and molecular modeling.
The knowledge generated by the structure-function investigations was used to develop and engineer restrictocin based recombinant immunotoxins for targeted therapy of cancer. A strategy was developed to enhance the intracellular processing of restrictocin containing immunotoxins by incorporating a proteolytically cleavable spacer in these molecules. It is now possible to use ribonucleolytic toxin restrictocin to construct immunotoxins universally with any potential targeting ligand. We have also shown restrictocin to have anti-HIV activity which is dependent on its specific ribonucleolytic activity.
Saporin, another protein toxin extensively studied in my laboratory, belongs to the family of ribosome-inactivating proteins that potently inhibit protein synthesis in eukaryotic cells, by specifically depurinating rRNA, which results in cell death. We have demonstrated that saporin possesses two catalytic activities, namely RNA N-glycosidase and genomic DNA fragmentation activity, and for its complete cytotoxic activity both these activities are absolutely critical. We have further elucidated the mechanism of binding and internalization of saporin in eukaryotic cells. My group has identified crucial determinants for the potent cytotoxic activity of saporin, and also established its mechanism of apoptosis induction and anti-HIV activity.
Caseinolytic proteases (Clps), members of the heat shock protein family, in many pathogenic bacteria have been shown to be involved in their virulence, survival in the host and dormancy. A similar mechanism may be operative in M. tuberculosis also and the members of the Clp protease family will be attractive drug targets. Our current studies are aimed at understanding the functioning of Clp protease machinery in M. tuberculosis by in vitro biochemical characterizations and by inactivating these proteins in vivo. As a first study in this field, our group has characterized M. tuberculosis ClpC1and established the role of N-terminal domain in its function. Also, we are investigating the regulation of heat shock proteins, particularly the role of transcriptional repressors, HspR and HrcA in M. tuberculosis. We have delineated the mechanism of repression by HspR of M. tuberculosis.
We have reconstituted the RNase P of M. tuberculosis in vitro, and delineated the role of a histidine in the RNR motif in its catalysis. Our studies show that the histidine in the RNR motif of M. tuberculosis is able to substitute optimally for asparagine found in the majority of the protein components of other bacterial RNase P enzymes. Further, we have analyzed the residues in RNase P protein of M. tuberculosis that differ from the residues generally conserved in other well-studied bacterial RNase Ps. We have demonstrated that Phe23, Val27 and Ala70 are involved in substrate binding, while Arg72 and Arg93 are involved in interactions with other residues within the protein to provide the protein a conformation conducive for activity.
Ayush Attery, Prajna Tripathi, Virendra K Patel, Owais Rashid Hakiem, Manish Gupta, Lalit Singh, Kevalanad, Jagdish, Yam Bahadur
• Elected Fellow, The National Academy of Sciences, India
• Elected Fellow, The Indian Academy of Sciences, India
• Sreenivasaya Memorial Award of Society of Biological Chemists (India) for outstanding contributions to Biological Sciences.