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Molecular & Cellular Proteomics

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Technological Innovation and Resources

Probing H2O2-mediated Structural Dynamics of the Human 26S Proteasome Using Quantitative Cross-linking Mass Spectrometry (QXL-MS)

Clinton Yu, Xiaorong Wang, Alexander S Huszagh, Rosa Viner, Eric J Novitsky, Scott D. Rychnovsky and Lan Huang  Correspondence email
Molecular & Cellular Proteomics February 5, 2019, mcp.TIR119.001323; https://doi.org/10.1074/mcp.TIR119.001323
Clinton Yu
University of California, Irvine, United States of America
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Xiaorong Wang
University of California, Irvine
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Alexander S Huszagh
University of California, Irvine, United States of America
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Rosa Viner
Thermo Fisher Scientific, San Jose
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Eric J Novitsky
University of California, Irvine, United States of America
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Scott D. Rychnovsky
University of California, Irvine
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Lan Huang
Physiology & Biophysics, University of California-Irvine, United States of America
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  • For correspondence: lanhuang@uci.edu
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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 facilitates a weakening of 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) prior to 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.

  • 26S proteasome
  • structural dynamics
  • quantitative cross-linking mass spectrometry
  • Oxidative stress
  • Protein complex analysis
  • Protein Conformation*
  • Protein Cross-linking*
  • Protein-Protein Interactions*
  • Quantification

Footnotes

  • Author contributions: C.Y., X.W., and L.H. designed research; C.Y., X.W., and R.V. performed research; C.Y. and A.S.H. analyzed data; C.Y. and L.H. wrote the paper; E.J.N. and S.D.R. contributed new reagents/analytic tools.

  • Received January 4, 2019.
  • Accepted February 5, 2019.
  • Published under license by The American Society for Biochemistry and Molecular Biology, Inc.
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Probing H2O2-mediated Structural Dynamics of the Human 26S Proteasome Using Quantitative Cross-linking Mass Spectrometry (QXL-MS)
Clinton Yu, Xiaorong Wang, Alexander S Huszagh, Rosa Viner, Eric J Novitsky, Scott D. Rychnovsky, Lan Huang
Molecular & Cellular Proteomics February 5, 2019, mcp.TIR119.001323; DOI: 10.1074/mcp.TIR119.001323

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Probing H2O2-mediated Structural Dynamics of the Human 26S Proteasome Using Quantitative Cross-linking Mass Spectrometry (QXL-MS)
Clinton Yu, Xiaorong Wang, Alexander S Huszagh, Rosa Viner, Eric J Novitsky, Scott D. Rychnovsky, Lan Huang
Molecular & Cellular Proteomics February 5, 2019, mcp.TIR119.001323; DOI: 10.1074/mcp.TIR119.001323
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