Page 51 - Annual report 2021-22

Annual Report 2021-22 |

Kausik Chakraborty

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Kausik Chakraborty’s laboratory studies various factors that govern protein folding, stability and in
vivo evolution of proteins using yeast and bacterial systems. In one project, they have generated ~50
thermotolerant S. cerevisiae strains by repeated dilutions. These strains have been evolved to grow at
40°C, 10 degrees above the physiological growth temperature of S. cerevisiae. They have done
competitive fitness assays for the evolved strains to check if they have evolved thermotolerance. Most
of the strains are fitter than the unevolved strains at high temperature. They have also checked if
these strains have better proteostasis by assays where the cells were treated with chemicals that
induce misfolding (azetidine-2-carboxylic acid(AZC) or tunicamycin). The evolved strains were fitter in
the presence of these misfolding inducing chemicals. They also checked the activity of a misfolding
mutant of NAT-R that had been reported previously. The mutant protein was more active in some of
the evolved strains, while less active in the other evolved strains. They showed that the
thermotolerant evolved strains depended on the central carbon metabolism for enhanced
proteostasis and thermotolerance.

The evolved strains also exhibited better capacity to degrade misfolded proteins at physiological
temperatures. Using the degradation rate of the misfolded proteins they checked if the degradation
rate is glucose dependent. The evolved strains and the WT unevolved lines behaved similarly in the
presence of glucose. Competitive fitness experiments in the presence of glucose in the growth
medium rely on the property that glucose activates glycolysis and removes the repression of glycolysis
in the evolved lines. The evolved strains lost their capability to outcompete the unevolved WT strains
at 47°C. Forced upregulation of glycolysis led to decreased degradation in the evolved strains. Thus,
glycolysis repression may be a central pathway that regulates cellular proteostasis in E. coli. Since
metabolic alterations were seen in the evolved strains, they explored if E. coli responds to misfolding
by changing metabolism. Trehalose and Arginine metabolite levels were altered indicating that the
evolved strains use the same metabolic pathway that is used by unevolved strains in responding to
misfolding stress. Thus, this study provided evidence that misfolding changes metabolite levels in E.
coli and evolved E. coli strains have used metabolic alteration as the preferred pathway for bolstering
proteostasis.

To check the role of protein chaperones in increasing the proteostasis capacity of the evolved lines,
transcriptomics of 14 of the strains and proteomics of two of the strains was performed at
physiological temperatures. Except one strain none of the other strains showed increase in the
transcripts or proteins encoding chaperones. Few of the chaperones were checked by immunoblotting
that confirmed the results obtained from proteomic and transcriptomic studies. Thus, these strains
use pathways other than canonical molecular chaperones to reinforce proteostasis.

To check how mitochodrial Hsp60s help in cellular proteostasis Kausik Chakraborty has collaborated
with Koyeli Mapa, Shiv Nadar University and Arjun Ray, IIITD to generate a substrate of E. coli
GroEL/ES, characterize the folding landscape of the substrate in the presence and absence of
GroEL/ES. Differences between GroEL/ES and the mammalian homolog hHsp60/10 in terms of their
capability to refold this GroEL/ES substrate in vitro and in vivo were studied and a comprehensive