Characterizing Protein Protein Interaction Interactions in Malarial Proteases

Abstract

alaria is one of the deadliest diseases affecting humans with ~409000 deaths reported in 2019 alone. The current treatment regimen which includes artemisinins and their combination therapies is being threatened with the rapid emergence of resistance in South-East Asia and their gradual geographical expansion. This necessitates the need for new antimalarial drug targets that are less prone to resistance. Malarial proteases play crucial roles in almost all biological processes and are mostly substrate-specific, making them excellent targets for drug intervention strategies. Cysteine proteases, specifically falcipains, are principally responsible for the degradation of hemoglobin, the principal source of nutrients for blood-stage plasmodium parasites. Our study, through a combination of bioinformatic and mutagenesis analysis, has identified a single amino acid within both falcipains, falcipain-2 (FP2) and falcipain-3 (FP3), responsible for mediating interactions with hemoglobin. This approach is beneficial as the identified residue lies at an exosite protruding away from the active site, thus would likely be less prone to drug pressure. Further, we characterized the interactions between falcipains and their natural macromolecular inhibitor, falstatin which plays crucial roles during the invasion of RBC and hepatocytes. Falstatin was earlier shown to be unique as compared to its homologues and that only a single loop is sufficient for the inhibition of falcipains. Our current study suggests that falstatin interacts with FP2 in a multimeric form with ten units of falstatin interacting with ten units of FP2 in a 1:1 stoichiometry. A model of FP2-falstatin is proposed and key residues that could participate in this oligomerization were identified. The recombinant expression of a member belonging to another important class of proteases, plasmepsin IX (PMIX) has been achieved. With the recent developments surrounding PMIX (and PMX), it is advantageous to standardize recombinant protein expression, which could significantly accelerate PMIX biochemical characterization and compound inhibition efforts. Together, our study aims to comprehensively characterize the functioning of falcipains and their interactions with natural partners such as hemoglobin and falstatin, and attempt to find novel chemotherapeutic targets. Keywords: Malaria, Cysteine proteases, Protein-protein interaction, Falstatin, Aspartic proteases, Hemoglobin hydrolysis, Plasmepsin IX, Host-Parasite interactions v ACKNOWLEDGMENTS I would like to take this opportunity to thank everyone who helped me throughout the journey of this Ph.D. First and foremost, I would like to extend my sincere thanks to my mentor Dr. Kailash C. Pandey for giving me this opportunity to work at Parasite-Host Biology Group (formerly HPIBG) and guiding me throughout this journey. I extend my heartfelt gratitude for his continuous support, guidance, motivation, and encouragement in helping me become a better researcher. Next, I would like to thank my guide, Dr. Sriram Seshadri, for putting faith in me and giving me this chance to work with him. I sincerely thank him for really understanding my situation and reaching out to me and extending to me all the support he could in finishing this arduous task. I would also like to thank both my host institutions without which this work could not be possible. I thank ICMR National Institute of Malaria Research (NIMR), New Delhi, for permitting me to pursue research and utilize its scientific facilities and resources. I am grateful to Nirma University (NU), Ahmedabad for providing the opportunity to pursue my doctoral research. I take this moment to thank all the staff and personnel for their support in all administrative aspects at both these esteemed institutions. Special thanks to the Council of Scientific and Industrial Research (CSIR) and the Indian Council of Medical Research (ICMR) for financial assistance throughout my Ph.D., without which this journey would be impossible. I would also like to thank many esteemed scientists who helped me throughout my research. My heartfelt thanks to Dr. Rajnikant Dixit who motivated me with his cheerful attitude and pushed me to work harder. I thank Dr. Ajay Saxena, Dr. Agam Prasad, Dr. Brijesh Rathi, Dr. Mayur Kajla, and Dr. Soumyananda Chakraborti for their valuable scientific inputs and for permitting me to use their laboratory facilities. Special thanks to Dr. Paco Pino, for his generous gift of synthetic constructs, substrates, and inhibitors. I would like to thank all my past and present lab members Dr. Akansha, Sonia, Dr. Bhumika, Dr. Kapil, Dr. Atul, Dr. Vandana, Preeti and Cherish for their support and motivation throughout my tenure at NIMR. I also thank my fellow members from the former HPIBG group and the institute for their support, pleasurable company, and maintaining a nurturing environment. I thank my friends at NIMR, ISNU, and my former IISER buddies for creating a relaxing and positive atmosphere throughout my Ph.D. tenure. Last but not the least, I owe it all to my family for their unconditional love and support and for providing me the strength to move forward in this journey.