Abstract
Cytotoxic protein aggregation-induced impairment of cell function and homeostasis are hallmarks of age-related neurodegenerative pathologies. As proteasomal degradation represents the major clearance pathway for oxidatively damaged proteins, a detailed understanding of the molecular events underlying its stress response is critical for developing strategies to maintain cell viability and function. Although the 26S proteasome has been shown to disassemble during oxidative stress, its conformational dynamics remains unclear. To this end, we have developed a new quantitative cross-linking mass spectrometry (QXL-MS) workflow to explore the structural dynamics of proteasome complexes in response to oxidative stress. This strategy comprises SILAC-based metabolic labeling, HB tag-based affinity purification, a 2-step cross-linking reaction consisting of mild in vivo formaldehyde and on-bead DSSO cross-linking, and multi-stage tandem mass spectrometry (MSn) to identify and quantify cross-links. This integrated workflow has been successfully applied to explore the molecular events underlying oxidative stress-dependent proteasomal regulation by comparative analyses of proteasome complex topologies from treated and untreated cells. Our results show that H2O2 treatment weakens the 19S-20S interaction within the 26S proteasome, along with reorganizations within the 19S and 20S subcomplexes. Altogether, this work sheds light on the mechanistic response of the 26S to acute oxidative stress, suggesting an intermediate proteasomal state(s) before H2O2-mediated dissociation of the 26S. The QXL-MS strategy presented here can be applied to study conformational changes of other protein complexes under different physiological conditions.
Highlights
Oxidative stress has been implicated in a multitude of human pathologies, ranging from neurodegenerative disorders and cancers to the general aging process [1, 2]
We have developed a new quantitative cross-linking mass spectrometry (QXL-MS) workflow to explore the structural dynamics of proteasome complexes in response to oxidative stress
Given the effectiveness of XL-MS methods in their unique ability to provide structural insight on conformational ensembles of protein complexes and quantitatively determine their changes under different physiological conditions, we have developed and employed a QXL-MS strategy by integrating QTAX with DSSO-based XL-MS strategy to delineate in vivo interaction and structural dynamics of the human 26S proteasome in response to H2O2-induced oxidative stress
Summary
Oxidative stress has been implicated in a multitude of human pathologies, ranging from neurodegenerative disorders and cancers to the general aging process [1, 2]. Further understanding of stress-induced impairment of proteasome structure and function may prove useful in developing new strategies for ameliorating proteasomal down-regulation and preventing cytotoxic aggregation of oxidized proteins. Ecm deletion in yeast and knockdown in human cells prevented oxidative stress-mediated disassembly of the 26S, suggesting the critical role of Ecm in proteasome regulation [15, 16]. It seems that the dissociation of the 19S from the 20S is important as the blockage of this event makes cells much more susceptible to acute oxidative stress. Further studies are needed to define stress-triggered conformational changes of the 26S to fully dissect mechanisms underlying its regulation
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