Personal Profile

Research:

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Our lab focusses on understanding the mechanism of GPI anchor biosynthesis in Candida albicans.

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Glycosylphosphatidylinositol (GPI) biosynthesis is a ubiquitous post-translational modification in eukaryotes which attaches a glycolipid anchor to proteins. GPI anchored proteins (GPI-APs) form a major part of the outer surface of C. albicans cell wall. They are required for survival, signalling, cell-wall biogenesis, host-recognition, adhesion, virulence and numerous enzymic activities. They also contribute to cell wall integrity, invasion and protection against immune cells.

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The GPI anchor is first synthesized in the ER via a sequential pathway involving several enzymes and further attached to proteins that carry GPI anchor attachment signal sequence. Later steps of GPI anchor remodelling take place in ER and Golgi after protein transfer followed by presentation of GPI-APs at plasma membrane and/or cell wall. The schematic representation below shows GPI biosynthetic pathway in ER lumen. 

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Figure. GPI biosynthetic pathway occurring in ER lumen in yeast (Komath et al. (2018), International Union of Biochemistry and Molecular Biology, volume 70, number 5, pages 355–383)

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My work focusses on structural, functional and phenotypic glycosylphosphatidylinositol transamidase (GPIT), a key enzyme in GPI biosynthesis pathway that acts as an endopeptidase which removes the signal sequence from the C-terminal end of newly translated protein and transfers the complete precursor glycoprotein in the ER to C-terminal end of newly synthesized protein in ER lumen. In mammals and yeast, GPI transamidase (GPIT) is supposed to be made up of 5 subunits, GAA1(mammals)/Gaa1(yeast), PIGK/Gpi8, PIGT/Gpi16, PIGS/Gpi17 and PIGU/Gab1.

Several structural characterization studies in understanding GPIT in humans and yeast is underway. In S. cerevisiae, a complex constituting Gpi8, Gpi16 and Gaa1 with molecular weight of ~430-650 kDa was isolated on blue native PAGE after solubilization in digitonin. The combined molecular weight of the individual monomers is expected to be ~200 kDa, suggesting that the monomers form a trimer in solution. Based on these data and other information obtained through molecular weight analysis of human (~480-720 kDa) and yeast GPIT, it is expected that the enzyme complex might exists as a dimer of heteropentamer. No other studies regarding the roles or stoichiometry of GPIT complex as whole in C. albicans is available. Understanding the structure and function of GPIT will gives us insight into molecular details of the catalytic mechanism of the enzyme and enable generation of species-specific inhibitors of this crucial biosynthetic step in the long run, which would have significant clinical implications.

Previous analysis of mutants associated with GPIT had proved that they show various phenotypic changes as compared to the wild type. Such phenotypic changes in these mutants (defects in growth, increased cell size and cell wall alterations, hyperfilamentation, sensitivity to tunicamycin and resistance towards towards azoles and amphotericin B, reduced enzymatic activity and GPI-AP expression and other changes) suggests the importance or essentiality of GPIT not only in GPI biosynthetic pathway but also in maintaining the growth and viability of such lower eukaryotes. Therefore, phenotypic characterization of mutants associated with each subunit of GPIT should give much insight into their importance in C. albicans. Furthermore, any regulatory mechanism associated with GPI biosynthetic pathway and other pathway could also be understood through phenotypic characterization.