Protein prenylation involves the attachment of one or two isoprenoid group(s) onto cysteine residues positioned near the C-terminus. This modification is essential for many signal transduction processes. In this work, the use of the probe C15AlkOPP for metabolic labeling and identification of prenylated proteins in a variety of cell lines and primary cells is explored. Using a single isoprenoid analogue, 78 prenylated protein groups from the three classes of prenylation substrates were identified including three novel prenylation substrates in a single experiment. Applying this method to three brain-related cell lines including neurons, microglia, and astrocytes showed substantial overlap (25%) in the prenylated proteins identified. In addition, some unique prenylated proteins were identified in each type. Eight proteins were observed exclusively in neurons, five were observed exclusively in astrocytes and three were observed exclusively in microglia, suggesting their unique roles in these cells. Furthermore, inhibition of farnesylation in primary astrocytes revealed the differential responses of farnesylated proteins to an FTI. Importantly, these results provide a list of 19 prenylated proteins common to all the cell lines studied here that can be monitored using the C15AlkOPP probe as well as a number of proteins that were observed in only certain cell lines. Taken together, these results suggest that this chemical proteomic approach should be useful in monitoring the levels and exploring the underlying role(s) of prenylated proteins in various diseases.
Bibliographical noteFunding Information:
This work was supported in part by the National Institute of Health grants RF1AG056976 (LL and MDD), R01GM084152 (MDD), R01NS107442 (MDD), R21AG056025 (LL) and RF1AG058081 (LL), and the National Science Foundation grant CHE-1308655 (MDD). KFS was supported by a Doctoral Dissertation Fellowship from the University of Minnesota and CB by LANDO, a Summer Research Experiences for Undergraduates Program funded by the National Science Foundation (CHE-1851990). AJ was partly supported by the Kwanjeong Educational Foundation Overseas Scholarship from South Korea and by a Bighley Graduate Fellowship from the College of Pharmacy at the University of Minnesota. The authors would like to thank Dr. Hongwei Qin at the University of Alabama at Birmingham for providing BV2 cells, Dr. G. William Rebeck at Georgetown University for providing human APOE3 targeted-replacement immortalized mouse astrocytes, Elyse Froehling for generating the transcriptomic data using next-generation sequencing at the University of Minnesota Genomics Center, and Dr. Yingchun Zhao for the assistance in LC-MS analysis in the Mass Spectrometry Core Facility of the Masonic Cancer Center, designated by the National Cancer Institute and supported by P30 CA77598.
© 2021, The Author(s).
Copyright 2021 Elsevier B.V., All rights reserved.
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