Sunanda Bhattacharya's Lab
Hsp90 Chaperone system
Hsp90 chaperone system influences the DNA damage response and diverse DNA repair pathways by providing client-ship to the multiple components of DNA repair machinery. Hsp90 chaperone system not only provides stability to the individual proteins but also manifest non-canonical function being present in the nucleus. Hsp90 function is regulated by a cohort of cochaperones, which influence Hsp90 activity. It was observed that some Hsp90 cochaperones display plasticity towards specific client folding. Thus, understanding the requirement of cochaperones for specific client can be employed to target the stability of specific clients without altering the overall cellular homeostasis. Similarly, post translational modification of Hsp90, also known as chaperone code, regulates the chaperone function and displays impact on client protein folding and activity. It remains largely unknown, how specific chaperone code regulates the interaction between Hsp90 and cochaperones as well as that with its client. We are currently working to decipher the cochaperone dependence for maintaining homeostasis of Rad51 and Rad52.
Our current research shows that acetylation at K-27th position of Hsp90 negatively impact its association with its cochaperone Aha1 as well as with the recombinase Rad51. K27-acetylated Hsp90 is the substrate for the histone deacetylase. We observe that hda1 deleted strain is epistatic to hsp82K27Q mutant strain and in such background, Hsp90 association with its cochaperone Aha1 is substantially reduced. We also observed that Aha1 is a mediator of the interaction between Rad51 and Hsp90 hence, reduced Hsp90-Aha1 interaction in K27Qhsp82 mutant decreases Hsp90-Rad51 association leading to proteasomal degradation of Rad51, subsequently causing poor gene conversion in that strain background.
Khushboo Rani et al., JBC, 2024
Our lab shows that in yeast, Hsp90 is redistributed to the nucleus upon DNA damage and it is recruited to the damaged chromatin. Using various mutants of Hsp90, that shows loss of nuclear translocation upon DNA damage, we show that those strains display extreme DNA damage sensitivity. We identified that Aha1 promotes the nuclear import of yHSP90α and the cochaperone also shows increased accumulation in the nucleus during DNA damage. Also, the nuclear import of yHSP90α is severely affected in ∆aha1 strain.
Nupur Fangaria et al., MBoC, 2022
We demonstrate that elevated levels of Hsp90 attenuate efficient DNA damage signalling and dictate preferential use of the potentially mutagenic double-strand break repair pathway. We show that under normal physiological conditions, Hsp90 negatively regulates RAD53 transcription to suppress DNA damage checkpoint activation. However, under DNA damaging conditions, RAD53 is derepressed, and the increased level of Rad53p triggers an efficient DNA damage response. A higher abundance of Hsp90 causes increased transcriptional repression on RAD53 in a dose-dependent manner, which could not be fully derepressed even in the presence of DNA damage. Accordingly, cells behave like a rad53 loss-of-function mutant with a drastic failure to up-regulate RAD51 expression and manifest faster accumulation of CLN1 and CLN2 in DNA-damaged G1 arrested cells leading to premature release from checkpoint arrest.
Nidhi Khurana et al., Molecular Biology of the Cell, 2016
We have established that Hsp90 over expression, which is a natural outcome of heat-stressed condition, drives downregulation of SIR2 at the transcription level. Such reduced abundance of SIR2 transcript is maintained through several generations before it gradually returns to its normal level. The reduced pool of Sir2 was functionally active, but its limiting quantity was insufficient to establish silencing across all 32 telomeres. However, it was adequate to silence hidden mating type loci. Using a series of genetic experiments, we have established that heat shock or yHsp90α overexpression causes upregulation of CUP9 that, in turn, represses SIR2 transcription by binding to its upstream activator sequence. Our study shows that the deletion of cup9 causes reversal of the Hsp82 overexpression phenotype and upregulation of SIR2 expression in heat-induced or Hsp82 overexpressing cells. On the other hand, we found that Cup9 overexpression represses SIR2 transcription under normal condition and leads to a failure in the establishment of heterochromatin. The results of our study highlight the mechanism by which environmental factors amend the epigenetic configuration of chromatin.
Shyamasree Laskar et al., Molecular and Cell Biology, 2014
Replication of Plasmodium falciparum during asexual development
Plasmodium falciparum undergoes endoreduplication during schizont stage of development, in which its nuclear, mitochondrial and apicoplast genome are replicated multiple times without cytokinesis. This mode of replication ensures the increase in number of the parasites in a geometric progression, which is crucial for its infectivity. Our laboratory is working on major replication proteins of the parasite deciphering their essential functions in parasite biology.
We have identified one novel topoisomerase from malaria parasite. It is a Type IIB topoisomerase having two subunits PfTopoVIB and PfSpo11, of which PfTopoVIB is unique to the parasite and it has 10% sequence identity with mouse/human TopoVIBL (TopoVIB like) protein. Predicted structures of these two subunits show that the ATP binding pocket of PfTopoVIB, namely, the Bergerat fold, does not superimpose with the similar fold present in human TopoVIBL protein. We establish a likely role of topoisomerase VI in the mitochondrial genome segregation of Plasmodium falciparum during endoreduplication. We show that PfTopoVIB and PfSpo11 remain associated and form the functional holoenzyme within the parasite. The spatiotemporal expression of both subunits of PfTopoVI correlates well with their recruitment to the mitochondrial DNA at the late schizont stage of the parasite. Additionally, the synergistic interaction between PfTopoVI inhibitor and the disruptor of mitochondrial membrane potential, atovaquone, supports that topoisomerase VI is the mitochondrial topoisomerase of the malaria parasite. Our study underlines topoisomerase VI as a novel anti-malaria target.
Priyanka Singh et al., Microbiology Spectrum, 2023
We have developed an economic, yeast-based assay system to characterise PfTopoVI biochemically. We generated a Δtop2 yeast strain harbouring PfTopoVIB and PfSpo11. Using the yeast cell free extract, we show that PfTopoVIB-PfSpo11 together can decatenate DNA in an ATP and Mg2+ dependent manner. Using this biochemical assay, we screened inhibitors of PfTopoVI and found Radicicol as a specific inhibitor of its decatenation activity.
Sureshkumar Chalapareddy et al., mSphere, 2016
We have characterised a Type IA Topoisomerase, namely Topoisomerase III from Plasmodium falciparum. We have identified a unique charged 85-amino acids long stretch, within domain II. This unique region is absent in all eukaryotic topoisomerase III including human. We used several genetic studies to prove that this region stabilises the DNA binding property of PfTopoIII. Our study shows that deletion of this region completely abolishes the function of PfTopoIII.
Shephali Bansod et al., Biochemical Journal, 2021
We are presently working to understand the mechanism behind the intracellular stability and activity of another important replication protein ribonucleotide reductase (RNR). Initial characterisation of this enzyme showed the presence of one large (PfR1) and two small subunits (PfR2 and PfR4). PfR2 subunit harbours the di-ferric tyrosyl radical, which is vital for catalytic activity of the enzyme complex. It was reported that benzo-hydroxamate, the inhibitor of PfRNR, can inhibit growth of parasite (IC50 = 17 μM) at 20-fold lower doses than that required for the purified human RNR (IC50 = 400 μM). This result suggests that there is a difference between the structures of Plasmodium and human RNRs, and hence, PfRNR qualifies as an important anti-malaria target. We are currently working to decipher the molecular determinants that are essential to maintain the cellular homeostasis of RNR.