Biochemical Characterization of a Novel Heme-based Adenylate Cyclase from Leishmania major

Through the billion years of evolution of the earth’s biosphere, chemistry of the atmosphere changed from a primitive unbreathable mixture of gas to oxygen-rich air. According to the geologic time scale, in the Neoproterozoic-Cambrian period there was a major rise in atmospheric oxygen, and the start of initial evolution and subsequent ‘explosion’ of complex life. Since then the aerobic life, from tiny bacteria to civilized human, absolutely depends on the oxygen for survival. To harness the atmospheric oxygen in beneficial way, globins are evolved in the living system. Globins are a family of globular heme-containing protein that also shares billion years of evolutionary history. Globins occur in all three kingdoms of life and can be classified into single-domain globins and chimeric globins. The latter comprise the globin-coupled sensors (GCS) that is a chimeric protein, composed of oxygen-sensing/binding globin domain and functional/catalytic domain. Until our work, it was thought that GCSs are present only in bacteria and archaea, and absent in eukaryotes. We have discovered a unique GCS (HemAC-Lm) made of neuroglobin/cytoglobin like domain coupled with adenylate cyclase domain in unicellular eukaryotic organism Leishmania major. Hypoxia is a condition in which the tissue or a region of the body is deprived of adequate oxygen supply. Hypoxia induced cell death has been implicated in a number of pathophysiological conditions like heart disease, cancer, cerebrovascular disease, and chronic lung disease, which are responsible for two-thirds of all deaths annually worldwide. Although transcriptional activator hypoxia-inducible factor 1 (HIF-1) in mammals plays a key role in hypoxia mediated adaptive responses, the exact mechanism of hypoxia induced cell death is obscure till date. Second messenger 3’,5’-cyclic adenosine monophosphate (cAMP) is known to play an important role in a number of metabolic and developmental processes in all living organisms. However the role of cAMP signalling in hypoxia induced cell death is little bit contradictory. Due to the very unique domain architecture, fusion of heme-bound globin and adenylate cyclase domains, HemAC-Lm may hold the answer to actual role of cAMP signalling during hypoxia. Leishmaniasis remains one of the world's most devastating neglected tropical diseases, causing substantial mortality or permanent deformity to nearly 12 million affected individual worldwide. Despite of the prevalence of the disease there is no vaccine or prophylactic drug available to treat the disease. Current treatments for the leishmaniasis are unsatisfactory due to their route of administration, toxicity, expense and the widespread resistance to first-line antimonial drugs. Therefore we are in a desperate search for new drug targets and effective chemotherapeutic agents against leishmaniasis. cAMP signalling in Leishmania is thought to play an important role in its survival and infectivity and like several other diseases as Parkinson’s disease, schizophrenia, asthma, diabetes, this signalling pathway can also be used as potential antileishmanial drug targets. The exclusivity of HemAC-Lm among living being, provide the opportunity to investigate possible mechanisms of inhibition in the framework of novel drug target discovery. In this dissertation an effort is made to unravel the secrets of HemAC-Lm through the biochemical and functional characterization. The findings of our work are discussed in three consecutive chapters. Chapter 1 presents the cloning, expression, biochemical and structural characterization of HemAC-Lm in detail. Chapter 2 deals with physiological significance of the protein under normoxic condition by generating half knockout, conditional knockdown and overexpressing strains corresponding to the HemAC-Lm gene. Chapter 3 deals with essentiality of HemAC-Lm under hypoxic condition and how increase or decrease in its intracellular concentration leads to ROS mediated cell death within L. major

NAD(P)H Cytochrome b5 Oxidoreductase Deficiency in Leishmania major Results in Impaired Linoleate Synthesis Followed by Increased Oxidative Stress and Cell Death

NAD(P)H cytochrome b5 oxidoreductase (Ncb5or) participates in a variety of metabolic conversions. Results: A new Ncb5or acts as a redox partner of �12 fatty acid desaturase. Conclusion: Ncb5or null mutant suffers from impaired linoleate synthesis leading to oxidative stress and cell death. Significance: Our results have exposed a novel fatty acid synthesis pathway in L. major that differs from the mammalian system

Costimulatory CD226 Signaling Regulates Proliferation of Memory-like NK Cells in Healthy Individuals with Latent <i>Mycobacterium tuberculosis</i> Infection

It is now widely accepted that NK cells can acquire memory, and this makes them more effective to protect against some pathogens. Prior reports indicate memory-like NK cells (mlNKs) in murine model of Mycobacterium tuberculosis (Mtb) as well as in healthy individuals with latent TB infection (LTBI). The increased expression of CD226 was evident in mlNKs from LTBI+ people after stimulation with γ-irradiated Mtb (γ-Mtb). We thus evaluated the contribution of costimulatory CD226 signaling in the functionality of mlNKs in LTBI+ people. We found that blockade of CD226 signaling using the antibody- or CRISPR/Cas9-mediated deletion of the CD226 gene in NK cells diminished the proliferation of mlNKs from LTBI+ people. Blocking CD226 signaling also reduced the phosphorylation of FOXO1 and cMyc expression. Additionally, cMyc inhibition using a chemical inhibitor reduced proliferation by mlNKs from LTBI+ people. Moreover, blocking CD226 signaling reduced glycolysis in NK cells, and the inhibition of glycolysis led to reduced effector function of mlNKs from LTBI+ people. Overall, our results provide a role for CD226 signaling in mlNK responses to Mtb