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Evaluating Calmodulin–Protein Interactions by Rapid Photoactivated Cross-Linking in Live Cells Metabolically Labeled with Photo-Methionine

  • DJ Black
    DJ Black
    Division of Molecular Biology and Biochemistry, University of Missouri-Kansas City, Kansas City, Missouri 64110-2499, United States
    More by DJ Black
  • Quang-Kim Tran
    Quang-Kim Tran
    Division of Molecular Biology and Biochemistry, University of Missouri-Kansas City, Kansas City, Missouri 64110-2499, United States
    Division of Molecular Biology and Biochemistry, University of Missouri-Kansas City, 5007 Rockhill Road, Kansas City, Missouri 64110-2499, United States
  • Andrew Keightley
    Andrew Keightley
    Division of Molecular Biology and Biochemistry, University of Missouri-Kansas City, Kansas City, Missouri 64110-2499, United States
  • Ameya Chinawalkar
    Ameya Chinawalkar
    Division of Molecular Biology and Biochemistry, University of Missouri-Kansas City, Kansas City, Missouri 64110-2499, United States
  • Cole McMullin
    Cole McMullin
    Division of Molecular Biology and Biochemistry, University of Missouri-Kansas City, Kansas City, Missouri 64110-2499, United States
  • , and 
  • Anthony Persechini*
    Anthony Persechini
    Division of Molecular Biology and Biochemistry, University of Missouri-Kansas City, 5007 Rockhill Road, Kansas City, Missouri 64110-2499, United States
    *E-mail: [email protected]. Phone: 816-235-6076. Fax: 816-235-5595.
Cite this: J. Proteome Res. 2019, 18, 10, 3780–3791
Publication Date (Web):September 4, 2019
https://doi.org/10.1021/acs.jproteome.9b00510
Copyright © 2019 American Chemical Society

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    Supporting Info (4)»

    Abstract

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    This work addresses the question of how the Ca2+ sensor protein calmodulin shapes cellular responses to Ca2+ signals. Proteins interacting with affinity tagged calmodulin were captured by rapid (t1/2 ≈ 7 s) photoactivated cross-linking under basal conditions, after brief removal of extracellular Ca2+ and during a cytosolic [Ca2+] transient in cells metabolically labeled with a photoreactive methionine analog. Tagged adducts were stringently enriched, and captured proteins were identified and quantified by LC–MS/MS. A set of 489 proteins including 27 known calmodulin interactors was derived. A threshold for fractional capture was applied to define a high specificity group of 170 proteins, including 22 known interactors, and a low specificity group of 319 proteins. Capture of ∼60% of the high specificity group was affected by manipulations of Ca2+, compared with ∼20% of the low specificity group. This suggests that the former is likely to contain novel interactors of physiological significance. The capture of 29 proteins, nearly all high specificity, was decreased by the removal of extracellular Ca2+, although this does not affect cytosolic [Ca2+]. Capture of half of these was unaffected by the cytosolic [Ca2+] transient, consistent with high local [Ca2+]. These proteins are hypothesized to reside in or near microdomains of high [Ca2+] supported by the Ca2+ influx.

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    The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jproteome.9b00510.

    • Histogram plots of iBAQ values for filtered capture data, comparison of known CaM interactors captured in adducts with tagged CaM and identified among a set of 4228 proteins derived from whole HEK293 cell lysates, volcano plots of −log p values versus log2 BAPTA/Basal or Iono/Basal ratios, and list of no photolysis (No Photo) contaminants (PDF)

    • Worksheet for analysis of fractional levels of photo-Met or its photolysis products in methionine containing peptides (XLSX)

    • AllPeptides output from the MaxQuant analysis of LC–MS/MS data files 1601–1615 (ZIP)

    • Full listing of proteins in the filtered capture data set (XLSX)

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    Cited By

    This article is cited by 10 publications.

    1. Tomas Jecmen, Roman Tuzhilkin, Miroslav Sulc. Photo-Methionine, Azidohomoalanine and Homopropargylglycine Are Incorporated into Newly Synthesized Proteins at Different Rates and Differentially Affect the Growth and Protein Expression Levels of Auxotrophic and Prototrophic E. coli in Minimal Medium. International Journal of Molecular Sciences 2023, 24 (14) , 11779. https://doi.org/10.3390/ijms241411779
    2. Ryan B. Williams, Md Nure Alam Afsar, Svetlana Tikunova, Yongjun Kou, Xuan Fang, Radha P. Somarathne, Rita F. Gyawu, Garrett M. Knotts, Taylor A. Agee, Sara A. Garcia, Luke D. Losordo, Nicholas C. Fitzkee, Peter M. Kekenes-Huskey, Jonathan P. Davis, Christopher N. Johnson. Human disease-associated calmodulin mutations alter calcineurin function through multiple mechanisms. Cell Calcium 2023, 113 , 102752. https://doi.org/10.1016/j.ceca.2023.102752
    3. Jennifer Giles, Vanessa Lopez, Elizabeth McConnaha, Matthew Hayden, Caleb Kragenbring, David Carli, Eric Wauson, Quang‐Kim Tran. Regulation of basal autophagy by calmodulin availability. The FEBS Journal 2022, 289 (17) , 5322-5340. https://doi.org/10.1111/febs.16432
    4. Sunidhi Jaiswal, Yufan He, H. Peter Lu. Probing functional conformation-state fluctuation dynamics in recognition binding between calmodulin and target peptide. The Journal of Chemical Physics 2022, 156 (5) https://doi.org/10.1063/5.0074277
    5. Kyle Kaster, John Patton, Sarah Clayton, Eric Wauson, Jennifer Giles, Quang-Kim Tran. A novel assay to assess the effects of estrogen on the cardiac calmodulin binding equilibrium. Life Sciences 2022, 290 , 120247. https://doi.org/10.1016/j.lfs.2021.120247
    6. Anthony Persechini, Hailey Armbruster, Andrew Keightley. Investigating the landscape of intracellular [Ca2+] in live cells by rapid photoactivated cross-linking of calmodulin-protein interactions. Cell Calcium 2021, 98 , 102450. https://doi.org/10.1016/j.ceca.2021.102450
    7. Thushara N. Samarasinghe, Yong Zeng, Carey K. Johnson. Microchip electrophoresis assay for calmodulin binding proteins. Journal of Separation Science 2021, 44 (4) , 895-902. https://doi.org/10.1002/jssc.202000884
    8. William D. Kim, Shyong Q. Yap, Robert J. Huber. A Proteomics Analysis of Calmodulin-Binding Proteins in Dictyostelium discoideum during the Transition from Unicellular Growth to Multicellular Development. International Journal of Molecular Sciences 2021, 22 (4) , 1722. https://doi.org/10.3390/ijms22041722
    9. Eduardo Izquierdo-Torres, Andrés Hernández-Oliveras, Gabriela Fuentes-García, Ángel Zarain-Herzberg. Calcium signaling and epigenetics: A key point to understand carcinogenesis. Cell Calcium 2020, 91 , 102285. https://doi.org/10.1016/j.ceca.2020.102285
    10. Anna Goehring, Irina Michin, Tina Gerdes, Nina Schulze, Mike Blueggel, Edisa Rehic, Farnusch Kaschani, Markus Kaiser, Peter Bayer. Targeting of parvulin interactors by diazirine mediated cross-linking discloses a cellular role of human Par14/17 in actin polymerization. Biological Chemistry 2020, 401 (8) , 955-968. https://doi.org/10.1515/hsz-2019-0423