Signal Transduction Laboratory Bhupat and Jyoti Mehta School of Biosciences Indian Institute of Technology Madras, Chennai
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Research

Our work covers the area of signal transduction using two model systems: G protein coupled receptors (GPCRs) and Calcium binding proteins (CaBPs). In both these systems we strive to be able to understand ligand-mediated switching mechanism, form off-state to on-state of this class of molecules. Our ultimate goal is to understand the structure-function relationship of these molecules and their biochemical and medical ramifications.

 

We investigate the process of transduction of signals through a combination of biochemistry and cell biology based studies. We regularly use molecules from a variety of organisms to show, both the universality of the phenomena and its complex nature. We hope that the knowledge generated enables us to ask newer questions in the field of translational research and better understanding of the associated human diseases.

Peptide Chemistry
A novel strategy in peptide purification has been developed using recombinant DNA technology. Harnessing the possibilities in protein purification and peptide chemistry, we have developed a easy method to synthesize peptides of various lengths by expressing the peptides as fusion proteins with GST tag, followed by affinity purification, tag cleavage and tag removal with subsequent lyophilization to obtain pure peptides. Kumar, P. and Aradhyam, G.K. Easy and efficient protocol for purification of recombinant peptides. Protein Expression and Purification. 95:129-135 (2014).
GPCR Mechanism APJ Receptor (APJR), is a cardiac GPCR activated by its endogenous ligand- Apelin. We are interested in analyzing the ligand binding and activation mechanism of GPCRs using APJ receptor as a model. APJR is involved in Gai, Gas and Gaq mediated signaling. Apart from classical signaling pathways, APJR also exert its physiological effects via b-arrestin. Understanding the receptor - ligand interaction and mapping ligand binding sites and establishing signaling bias of APJR are the prime areas of our GPCR research.

 

Fig. (Top) Fluorescence Microscopic Image showing the membrane expression of APJR and its internalization upon ligand activation

 

Left, Schematic describing the pathways activated upon ligand binding to GPCR

Calcium Binding Proteins

Calcium-binding proteins are highly essential in regulating secondary messengers in a cell, specifically altering the calcium homeostasis and its related cellular functions. In our lab, we work on biophysical and biochemical characterization of two calcium-binding proteins, Calnuc and Calumenin.

A multi-domain, multi-functional, calcium- and DNA-binding protein has been reported in numerous cellular events. We have identified two new high affinity zinc-binding sites in the N-terminal region of the protein and also serine protease catalytic triad and explained its activity. Further studies are aimed at studying the biophysical characterization of Calnuc using spectroscopic and thermodynamic techniques. Fig (left), Zinc-binding studies in human Calnuc using ITC studeis. Kanuru, M., Raman, R., and Aradhyam, G. K. Serine protease activity of calnuc: regulation by Zn2+ and G proteins. J Biol Chem. 288(3):1762-73 (2013) i. Calnuc

Calumenin is a ER resident with multiple calcium-binding sites, involved in calcium homeostasis. Calumenin is an intrinsically disordered protein which acquires a well defined structure upon binding to calcium.

 

We are interested in looking for the nucleation site of folding and studying the ion-binding properties using fragment and mutational studies and aimed in identifying the functional role of calumenin in human.

Fig. Changes in secondary structure of Calumenin upon binding to calcium

ii. Calumenin
Protein Regulation i. EGF-CFC Proteins

Cryptic, a cellular protein, is involved in developmental process in regulating right-left axis symmetry. We have identified the transcriptional regulatory mechanism of Cryptic expression by transcriptional factor Snail. Also we have have established the functional role of Snail in maintaining the 'Epithelial-Mesenchymal Transition (EMT)' by regulating the expression of Cripto-1. Both Cripto-1 and Cryptic belongs to EGF CFC family of proteins.

Fig.(above) Snail mediated L-R axis specification through Cryptic repression. Gupta, K., Pilli, V. S. S., Aradhyam, G. K. Left-right axis asymmetry determining human Cryptic gene is transcriptionally repressed by Snail. BMC Dev Biol. 16(1): 1-10 (2016)

Mitofusins

Mitofusins are mitochondrial proteins involved in mitochondrial fusion. Mitofusin-1 has GTPase domain, heptad repeats and transmembrane domain. We are interested in analyzing the evolutionary relationship of mitofusins and their impact in structure-function relationship.

 

Fig.(above) Schematic Representation of domain arrangement in Mitofusins.