Reactive oxygen species (ROS) are a significant byproduct of many intracellular and extracellular events. Cellular systems exist to limit ROS accumulation within cells; a loss of fidelity for these pathways over time is proposed to contribute towards the excessive intracellular ROS levels associated with a variety of disease pathologies. Protein folding is particularly susceptible to disruption upon elevated ROS, and a loss of folding homeostasis has also been linked to the accumulation of misfolded aggregates in various neurodegenerative disorders. We have uncovered two novel redox events that make use of ROS to alter the activity of molecular chaperones of the Hsp70 class during oxidative stress. We propose that these pathways normally serve to maintain folding homeostasis upon conditions of elevated intracellular ROS. Both events make use of sulfur-based post-translational events, but each system is unique in the means by which it influences chaperone activity and cell physiology. Our data suggest that both systems help to maintain normal cell function during overly oxidizing conditions. This proposal focuses on elucidating the mechanistic features of these two cellular redox systems.
In Aim 1, we continue our characterization of the redox-signaling pathway we uncovered in the endoplasmic reticulum centered on a thiol-based redox switch in the Hsp70 BiP. We propose to address how adduct removal from BiP post-stress is achieved and regulated to maintain folding homeostasis.
In Aim 2, we aim to describe the mechanistic details for a redox switch in the cytoplasmic Hsp70 co-chaperone Fes1. We will characterize how redox-dependent inactivation of Fes1 via methionine oxidation influences cytoplasmic Hsp70 function, and how inactivation of Fes1 is advantageous during overly oxidizing conditions. Successful completion of the proposed studies will provide insight into the basic cell functions used to manage cellular ROS and avert cellular damage.

Public Health Relevance

Accumulation of reactive oxygen species (ROS) within cells is associated with the pathology of many degenerative disorders, including Alzheimer's disease, Parkinson's disease, aging, diabetes, and atherosclerosis. It is now appreciated that ROS can elicit beneficial signaling pathways that serve to limit the damage associated with elevated intracellular ROS. Identification and characterization of the regulatory pathways that help manage ROS within healthy cells is key to understand what triggers the loss of ROS homeostasis associated with disease progression.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Membrane Biology and Protein Processing Study Section (MBPP)
Program Officer
Barski, Oleg
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Cornell University
Other Basic Sciences
Schools of Veterinary Medicine
United States
Zip Code
Ellgaard, Lars; Sevier, Carolyn S; Bulleid, Neil J (2018) How Are Proteins Reduced in the Endoplasmic Reticulum? Trends Biochem Sci 43:32-43
O'Donnell, John P; Marsh, Heather M; Sondermann, Holger et al. (2018) Disrupted Hydrogen-Bond Network and Impaired ATPase Activity in an Hsc70 Cysteine Mutant. Biochemistry 57:1073-1086
Siegenthaler, Kevin D; Pareja, Kristeen A; Wang, Jie et al. (2017) An unexpected role for the yeast nucleotide exchange factor Sil1 as a reductant acting on the molecular chaperone BiP. Elife 6:
Brison, Yoann; Malbert, Yannick; Czaplicki, Georges et al. (2016) Structural Insights into the Carbohydrate Binding Ability of an ?-(1?2) Branching Sucrase from Glycoside Hydrolase Family 70. J Biol Chem 291:7527-40
Wang, Jie; Sevier, Carolyn S (2016) Formation and Reversibility of BiP Protein Cysteine Oxidation Facilitate Cell Survival during and post Oxidative Stress. J Biol Chem 291:7541-57
Xu, Mengni; Marsh, Heather M; Sevier, Carolyn S (2016) A Conserved Cysteine within the ATPase Domain of the Endoplasmic Reticulum Chaperone BiP is Necessary for a Complete Complement of BiP Activities. J Mol Biol 428:4168-4184
Wang, Jie; Pareja, Kristeen A; Kaiser, Chris A et al. (2014) Redox signaling via the molecular chaperone BiP protects cells against endoplasmic reticulum-derived oxidative stress. Elife 3:e03496