Cholesterol-induced Conformational Plasticity and Oligomerization of GPCRs: Novel Insights in Health and Disease
G protein-coupled receptors (GPCRs) are the largest class of molecules involved in signal transduction across membranes, and represent major drug targets in all clinical areas. The serotonin1A receptor is an important neurotransmitter receptor of the GPCR superfamily and is implicated in the generation and modulation of various cognitive, behavioral and developmental functions. We previously demonstrated that membrane cholesterol is necessary for ligand binding, and G-protein coupling of serotonin 1A receptors. Interestingly, recently reported crystal structures of GPCRs have shown structural evidence of cholesterol binding site(s). In this context, we reported the presence of cholesterol recognition/interaction amino acid consensus (CRAC) motifs in the serotonin1A receptor. We also showed that the receptor is more stable and compact in the presence of membrane cholesterol. Our recent results utilizing coarse-grain molecular dynamics simulations to analyze the molecular nature of receptor-cholesterol interaction offer interesting insight in cholesterol binding site(s) in the receptor and oligomerization of the receptor. We showed utilizing homo-FRET that the serotonin1A receptor is constitutively oligomerized in live cells, with the possibility of higher order oligomers of the receptor. Progress in deciphering molecular details of the nature of GPCR-cholesterol interaction in the membrane would lead to better insight into our overall understanding of GPCR function in health and disease.
February 12, 2018
Prof. Souvik Maiti, CSIR-IGIB
Venue & Time: C V Raman Hall at 3:30 PM
January 08, 2018
Prof. Hemalatha Balaram
Venue & Time: C V Raman Hall at
January 04, 2018
Prof. Anindya Dutta, University of Virginia School of Medicine
Noncoding RNAs in regulation of differentiation and cancer
I will use examples from skeletal muscle differentiation and prostate and brain cancers to illustrate the importance of (a) short noncoding RNAs like microRNAs and (b) long noncoding RNAs (lncRNAs). We will begin our discussion of lncRNA with H19, which promotes muscle differentiation by producing a microRNA and MUNC lncRNA which is an eRNA,but also has an independent role in promoting the expression of several myogenic RNAs. We will then move to cancers to discuss the discovery of DRAIC and its importance in regulation of cancer progression (with significant contribution from an IACS alumnus, Dr. Shekhar Saha. Finally, if time permits, we will address the identification of tens of lncRNAs that predict outcome in gliomas and glioblastomas. Overall this talk will introduce the students to lncRNAs, but more important, will encourage them to ask questions during the seminar itself that will guide the direction in which the seminar will proceed.
Glutathione, an unusual tripeptide, is the most abundant small molecular weight thiol compound of living cells. Discovered more than 125 years ago, it plays many vital roles that includes its function as the cells redox buffer. The degradation of glutathione was first described in 1952 with the discovery of the g-glutamyl- transpeptidase enzyme. For more than 5 decades, this enzyme, which acts on non-cytosolic pools of glutathione was thought to be the only enzyme involved in glutathione degradation in living cells. Recent studies from our lab have thrown fresh light on glutathione degradation with the discovery of alternate enzymes for glutathione degradation. This has also led to a better understanding on the roles of glutathione degradation. One role that will be discussed in detail here is the role in calcium signalling. Yeasts have two channels that pump calcium into the cytoplasm, Cch1p and Yvc1p. Yvc1p is a yeast vacuolar TRP channel, while Cch1p is a plasma membrane L-type channel. Yvc1p and Cch1p werefound to be activated by redox through glutathionylation. The uncovering of the mechanisms by which these signalling pathways are regulated by redox has led to new insights on the role of glutathione degradation in cell signalling.
Feb 20, 2017
Prof. Dipankar Chatterjee, MBU, IISc., Bangalore
Venue & Time: C V Raman Hall at 3-30 PM to 5-00 PM
Second Messengers (p)ppGppand Cyclic di-GMP mediated regulation of Cell Shape, Cell Division and Antibiotic Sensitivity in Mycobacterium smegmatis
Quorum Sensing, a cell to cell communication phenomenon, is involved in modulating the social behavior of bacteria. Nucleotide based second messengers like (p)ppGpp and c-di-GMP are known to regulate such communication in mycobacteria. (p)ppGpp is synthesized by bacteria to face any kind of stress; while the signalling nucleotide c-di-GMP is synthesized principally to switch from motile (planktonic) to sessile (biofilm) life style. We investigated the effect of disrupting (p)ppGpp and c-di-GMP signalling on the antibiotic sensitivity in M. smegmatis. Using Phenotype Microarray (PM) technology, the growth of Δreland ΔdcpAknock out strains was compared to those of the wild-type and respective complemented strains in 240 different antimicrobials. It was found that the knockout mutants displayed enhanced survival in the presence of multiple antibiotics. The PM data was corroborated by the independent determination of minimum inhibitory concentrations of seven different antibiotics. Microscopy analyses revealed that the Δrel and ΔdcpA strains are elongated, multinucleate and multiseptate in M. smegmatis. The higher levels of (p)ppGpp and c-di-GMP caused M. Smegmatisassume coccoid morphology. The overproduction of (p)ppGpp and c-di-GMP, achieved through overexpression of Rel and DcpA proteins, encased the overexpression strains relOE and dcpAOE in a biofilm like matrix.
Molecular Insights into Meiotic Chromosome Synapsis from Single Molecule Analysis
In humans, defects in meiotic chromosome synapsis and segregation results in aneuploidy leading to miscarriages and genetic disorders. Aneuploidy occurs with much higher probability for the female counterpart, and increases dramatically with age. An evolutionarily conserved meiosis-specific proteinaceous structure, the synaptonemal complex (SC), is required for normal synapsis of meiotic chromosomes. However, very little is known about the biochemical properties of SC components or the mechanisms underlying their roles in meiotic chromosome synapsis and recombination. Structural and functional analysis of Saccharomyces cerevisiae Hop1, a key structural component of SC, has begun to reveal important insights into its function in the synapsis of meiotic chromosomes. Toward this end, we showed that Hop1 is a structure-specific DNA-binding protein, displays higher binding affinity for G-quadruplex DNA and the Holliday junction, and causes structural distortion of the latter at the core of the junction. Using atomic force microscopy and magnetic tweezers techniques, we discovered that Hop1 exhibits the ability to bridge non-contiguous DNA segments into intramolecular stem-loop structures in which the DNA segments are fully synapsed within the filamentous protein stems. Additional evidence suggested that Hop1 folds DNA into rigid protein-DNA filaments and higher-order nucleoprotein structures. Importantly, Hop1 promotes robust intra- and intermolecular synapsis between double-stranded DNA molecules, suggesting that juxtaposition of DNA sequences may assist in strand exchange between homologs by recombination-associated proteins. Furthermore, evidence from in vitro and in vivo studies disclosed the existence of G-quadruplex and i-motif structures at meiosis-specific recombination hot spots. Taken together, these studies support the notion that understanding of meiotic chromosome synapsis and recombination must consider protein binding interactions in conjunction with DNA structural motifs at meiosis-specific recombination hotspots.
Feb 6, 2017
Prof. B J Rao, TIFR, Mumbai
Venue & Time: C V Raman Hall, 6th Feb, 2017 at 4:00 PM
Local biochemical problems and non-local solutions in biological designs: some of our recent take-homes
It is a recurring theme that local biochemical reactions often require large-scale systemic non-local changes in cells and tissues, the design of which is not easily intuitive. We explore this theme using DNA Damage Response (DDR) paradigm in normal human cells and Drosophila. We uncover that damaged chromosomes traverse large distances reversibly to repair damaged DNA. Damaged replication forks collapse if the repair remains persistently active where the repair has inbuilt non-local design to attenuate itself. Interestingly, un-repaired dying cells seem to trigger changes across large distances in the tissue-field to provoke additional cellular proliferation, “compensating for death and thereby renewing life”. I illustrate these with examples and argue that most of these biological regulations are based on the “system as a whole” rather than “the parts thereof” based regulation.