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Proximity-labeling chemoproteomics defines the subcellular cysteinome and inflammation-responsive mitochondrial redoxome

Tianyang Yan, Ashley R. Julio, Miranda Villanueva, Anthony E. Jones, Andréa B. Ball, Lisa M. Boatner, Alexandra C. Turmon, Stephanie L. Yen, Heta S. Desai, Ajit S. Divakaruni, View ORCID ProfileKeriann M. Backus
doi: https://doi.org/10.1101/2023.01.22.525042
Tianyang Yan
1Biological Chemistry Department, David Geffen School of Medicine, UCLA, Los Angeles, CA, 90095, USA
2Department of Chemistry and Biochemistry, UCLA, Los Angeles, CA, 90095, USA
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Ashley R. Julio
1Biological Chemistry Department, David Geffen School of Medicine, UCLA, Los Angeles, CA, 90095, USA
2Department of Chemistry and Biochemistry, UCLA, Los Angeles, CA, 90095, USA
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Miranda Villanueva
1Biological Chemistry Department, David Geffen School of Medicine, UCLA, Los Angeles, CA, 90095, USA
4Molecular Biology Institute, UCLA, Los Angeles, CA, 90095, USA
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Anthony E. Jones
3Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, UCLA, Los Angeles, CA, 90095, USA
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Andréa B. Ball
3Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, UCLA, Los Angeles, CA, 90095, USA
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Lisa M. Boatner
1Biological Chemistry Department, David Geffen School of Medicine, UCLA, Los Angeles, CA, 90095, USA
2Department of Chemistry and Biochemistry, UCLA, Los Angeles, CA, 90095, USA
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Alexandra C. Turmon
1Biological Chemistry Department, David Geffen School of Medicine, UCLA, Los Angeles, CA, 90095, USA
2Department of Chemistry and Biochemistry, UCLA, Los Angeles, CA, 90095, USA
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Stephanie L. Yen
1Biological Chemistry Department, David Geffen School of Medicine, UCLA, Los Angeles, CA, 90095, USA
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Heta S. Desai
1Biological Chemistry Department, David Geffen School of Medicine, UCLA, Los Angeles, CA, 90095, USA
4Molecular Biology Institute, UCLA, Los Angeles, CA, 90095, USA
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Ajit S. Divakaruni
3Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, UCLA, Los Angeles, CA, 90095, USA
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Keriann M. Backus
1Biological Chemistry Department, David Geffen School of Medicine, UCLA, Los Angeles, CA, 90095, USA
2Department of Chemistry and Biochemistry, UCLA, Los Angeles, CA, 90095, USA
4Molecular Biology Institute, UCLA, Los Angeles, CA, 90095, USA
5DOE Institute for Genomics and Proteomics, UCLA, Los Angeles, CA, 90095, USA
6Jonsson Comprehensive Cancer Center, UCLA, Los Angeles, CA, 90095, USA
7Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, UCLA, Los Angeles, CA, 90095, USA
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  • ORCID record for Keriann M. Backus
  • For correspondence: kbackus@mednet.ucla.edu
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Abstract

Proteinaceous cysteines function as essential sensors of cellular redox state. Consequently, defining the cysteine redoxome is a key challenge for functional proteomic studies. While proteome-wide inventories of cysteine oxidation state are readily achieved using established, widely adopted proteomic methods such as OxiCat, Biotin Switch, and SP3-Rox, they typically assay bulk proteomes and therefore fail to capture protein localization-dependent oxidative modifications. To obviate requirements for laborious biochemical fractionation, here, we develop and apply an unprecedented two step cysteine capture method to establish the Local Cysteine Capture (Cys-LoC), and Local Cysteine Oxidation (Cys-LOx) methods, which together yield compartment-specific cysteine capture and quantitation of cysteine oxidation state. Benchmarking of the Cys-LoC method across a panel of subcellular compartments revealed more than 3,500 cysteines not previously captured by whole cell proteomic analysis. Application of the Cys-LOx method to LPS stimulated murine immortalized bone marrow-derived macrophages (iBMDM), revealed previously unidentified mitochondria-specific inflammation-induced cysteine oxidative modifications including those associated with oxidative phosphorylation. These findings shed light on post-translational mechanisms regulating mitochondrial function during the cellular innate immune response.

Competing Interest Statement

The authors have declared no competing interest.

Footnotes

  • Abstract and methods modified. PRIDE dataset identifier added.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted January 31, 2023.
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Proximity-labeling chemoproteomics defines the subcellular cysteinome and inflammation-responsive mitochondrial redoxome
Tianyang Yan, Ashley R. Julio, Miranda Villanueva, Anthony E. Jones, Andréa B. Ball, Lisa M. Boatner, Alexandra C. Turmon, Stephanie L. Yen, Heta S. Desai, Ajit S. Divakaruni, Keriann M. Backus
bioRxiv 2023.01.22.525042; doi: https://doi.org/10.1101/2023.01.22.525042
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Proximity-labeling chemoproteomics defines the subcellular cysteinome and inflammation-responsive mitochondrial redoxome
Tianyang Yan, Ashley R. Julio, Miranda Villanueva, Anthony E. Jones, Andréa B. Ball, Lisa M. Boatner, Alexandra C. Turmon, Stephanie L. Yen, Heta S. Desai, Ajit S. Divakaruni, Keriann M. Backus
bioRxiv 2023.01.22.525042; doi: https://doi.org/10.1101/2023.01.22.525042

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