Biology subjects

Chaphalkar, A.

Publications and source records attributed to Chaphalkar, A..

3 recordsLinked to original sources

bioRxiv2022

Live visualization of cytosolic and endoplasmic reticulum redox potential in zebrafish embryos reveals region-specific differences

Redox homeostasis is an integral part of many cellular processes, and its perturbation is associated with conditions such as diabetes, aging, and neurodegenerative disorders. Redox homeostasis or redox potential in organelles is maintained within a particular range to facilitate the organelle specific cellular redox reactions. Previous studies using yeast, cell systems, and nematodes have demonstrated that the Endoplasmic Reticulum (ER) has a more oxidizing environment while the cytosol exhibits a reducing redox potential. However, we know very little about how universal this phenomenon is. We created transgenic zebrafish (Danio rerio) lines with roGFP sensors targeted to the ER and cytosol for studying physiological redox potential at the systems level. In the process, we also characterized the ER-targeting signal sequence in D. rerio for the first time. Measurements of the redox state in live embryos found that the endoplasmic reticulum exhibits deviations from its expected oxidizing redox state in different regions of the developing embryos. The ER is far more reducing than expected in certain tissues of the embryo. Cytosol also exhibited unexpected redox states in some parts of the embryo. Notably, the brain showed regions with unexpected redox states in both the ER and the cytosol. Tissue-specific differences in ER-redox potential became even more evident in a transgenic line expressing the more sensitive roGFPiE variant. Thus, live monitoring of redox potential across the developing zebrafish embryos revealed unanticipated redox states of the ER that will require new biological definitions.

biochemistry↗

bioRxiv2020

GroEL/ES Buffers Entropic Traps In Folding Pathway During Evolution Of A Model Substrate

The folding landscape of proteins can change during evolution with the accumulation of mutations that may introduce entropic or enthalpic barriers in the protein folding pathway, making it a possible substrate of molecular chaperones in vivo. Can the nature of such physical barriers of folding dictate the feasibility of chaperone-assistance? To address this, we have simulated the evolutionary step to chaperone-dependence keeping GroEL/ES as the target chaperone and GFP as a model protein in an unbiased screen. We find that the mutation conferring GroEL/ES dependence in vivo and in vitro encode an entropic trap in the folding pathway rescued by the chaperonin. Additionally, GroEL/ES can edit the formation of non-native contacts similar to DnaK/J/E machinery. However, this capability is not utilized by the substrates in vivo. As a consequence, GroEL/ES caters to buffer mutations that predominantly cause entropic traps, despite possessing the capacity to edit both enthalpic and entropic traps in the folding pathway of the substrate protein.

molecular biology↗

bioRxiv2019

Distinct metabolic states of a cell guide alternate fates of mutational buffering through altered proteostasis

Changes in metabolism can alter the cellular milieu; can this also change intracellular proteostasis? Since proteostasis can modulate mutational buffering, if change in metabolism has the ability to change proteostasis, arguably, it should also alter mutational buffering. Building on this, we find that altered cellular metabolic states in E. coli buffer distinct mutations. Buffered-mutants had folding problems in vivo and were differently chaperoned in different metabolic states. Notably, this assistance was dependent upon the metabolites and not on the increase in canonical chaperone machineries. Additionally, we were able to reconstitute the folding assistance afforded by metabolites in vitro and propose that changes in metabolite concentrations have the potential to alter proteostasis. Collectively, we unravel that the metabolite pools are bona fide members of proteostasis and aid in mutational buffering. Given the plasticity in cellular metabolism, we posit that metabolic alterations may play an important role in the positive or negative regulation of proteostasis.

biochemistry↗