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Summer Research Fellowship Programme of India's Science Academies

Molecular characterization of Otubain: A probable drug target of Leishmania donovani

Clayton Fernando Rencilin

Kalasalingam Academy of Research and Education (KARE), Virudhunagar, Tamilnadu 626 126.

Dr. Nahid Ali

Division of Infectious Diseases and Immunology, Indian Institute of Chemical Biology (IICB), Kolkata, West Bengal - 700 032.

ABSTRACT

Leishmaniasis is one of the protozoan parasite diseases caused by the genus of leishmania. It affects over 150 million people all over the world. The transmission is done by the sandfly, an insect. It has two forms, one is cutaneous leishmaniasis that occurs in skin and mucosal layer and the other one is visceral leishmaniasis that involves internal organs of humans. My work is on visceral leishmaniasis (VL) which is caused by the species of Leishmania donovani. This species has a promastigote form when it is in sandfly but in humans it has amastigote form.

Our work is on host pathogen interaction i.e about the genes which are responsible for causing virulence, in particular those involved in its survival inside macrophages. The role of Ubiquitin in transcriptional regulation is important for parasite. But enzymes involved in ubiquitination/deubiquitination of these organism have not been well characterized. Otubain is a protease family member related to OTU (Ovarian Tumour Proteases). Otubain is the second largest DUB (Deubiquitinase) family in mammals and also it plays a role of immune regulation, tumour regulation, promote T cell anergy, etc.

In our effort to characterize one of the deubiquitinase enzymes from L.donovani, we isolated and cloned Otubain gene from the parasite, followed by over-expresseion in Rosetta (strain of E.coli). The protein was purified by Ni-NTA chromatography (Affinity Chromatography) and Size Exclusion Chromatography. The identity of the purified protein of interest (OTU) was confirmed by SDS-PAGE. Enzyme activity test was performed by studying interaction between the Otubain protein and substrate and also Otubain protein and inhibitor. By these, we found that our protein has deubiquitinase activity and it was inhibited by inhibitors of E-64 (transepoxysuccinyl-L-leucylamodo (4-guanido) butane) and leupeptin.

To characterize the gene OTU by biophysical tools like dynamic light scattering, circular dichorism, MALDI-TOF-MS/MS, western blotting, etc. Based on this work, identification and characterization of otubain in L. donovani could be a promising alternative way to search for new therapeutic targets for leishmaniasis.

Keywords: Leishmania, Otubain

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