Improved synthesis and application of an alkyne-functionalized isoprenoid analogue to study the prenylomes of motor neurons, astrocytes and their stem cell progenitors

Kiall F Suazo; Vartika Mishra; Sanjay Maity; Shelby A Auger; Katarzyna Justyna; Alexandru M Petre; Linda Ottoboni; Jessica Ongaro; Stefania P Corti; Francesco Lotti; Serge Przedborski; Mark D Distefano

doi:10.1016/j.bioorg.2024.107365

Improved synthesis and application of an alkyne-functionalized isoprenoid analogue to study the prenylomes of motor neurons, astrocytes and their stem cell progenitors

Bioorg Chem. 2024 Jun:147:107365. doi: 10.1016/j.bioorg.2024.107365. Epub 2024 Apr 16.

Authors

Affiliations

¹ Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA. Electronic address: [email protected].
² Center for Motor Neuron Biology and Diseases, Department of Neurology. Columbia University Irving Medical Center. New York, NY 10032, USA; Department of Pathology & Cell Biology. Columbia University Irving Medical Center. New York, NY 10032, USA. Electronic address: [email protected].
³ Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA.
⁴ Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA. Electronic address: [email protected].
⁵ Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA. Electronic address: [email protected].
⁶ Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA. Electronic address: [email protected].
⁷ Department of Pathophysiology and Transplantation, Dino Ferrari Center, Università degli Studi di Milano, Milan, Italy. Electronic address: [email protected].
⁸ Neurology Unit, Foundation IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy.
⁹ Department of Pathophysiology and Transplantation, Dino Ferrari Center, Università degli Studi di Milano, Milan, Italy; Neurology Unit, Foundation IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy; Neuromuscular and Rare Diseases Unit, Department of Neuroscience, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy. Electronic address: [email protected].
¹⁰ Center for Motor Neuron Biology and Diseases, Department of Neurology. Columbia University Irving Medical Center. New York, NY 10032, USA; Department of Pathology & Cell Biology. Columbia University Irving Medical Center. New York, NY 10032, USA. Electronic address: [email protected].
¹¹ Center for Motor Neuron Biology and Diseases, Department of Neurology. Columbia University Irving Medical Center. New York, NY 10032, USA; Department of Pathology & Cell Biology. Columbia University Irving Medical Center. New York, NY 10032, USA; Department of Neuroscience, Pathology, and Cell Biology, Columbia University Irving Medical Center, New York, NY 10032, USA. Electronic address: [email protected].
¹² Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA. Electronic address: [email protected].

PMID: 38636436
DOI: 10.1016/j.bioorg.2024.107365

Abstract

Protein prenylation is one example of a broad class of post-translational modifications where proteins are covalently linked to various hydrophobic moieties. To globally identify and monitor levels of all prenylated proteins in a cell simultaneously, our laboratory and others have developed chemical proteomic approaches that rely on the metabolic incorporation of isoprenoid analogues bearing bio-orthogonal functionality followed by enrichment and subsequent quantitative proteomic analysis. Here, several improvements in the synthesis of the alkyne-containing isoprenoid analogue C15AlkOPP are reported to improve synthetic efficiency. Next, metabolic labeling with C15AlkOPP was optimized to obtain useful levels of metabolic incorporation of the probe in several types of primary cells. Those conditions were then used to study the prenylomes of motor neurons (ES-MNs), astrocytes (ES-As), and their embryonic stem cell progenitors (ESCs), which allowed for the identification of 54 prenylated proteins from ESCs, 50 from ES-MNs, and 84 from ES-As, representing all types of prenylation. Bioinformatic analysis revealed specific enriched pathways, including nervous system development, chemokine signaling, Rho GTPase signaling, and adhesion. Hierarchical clustering showed that most enriched pathways in all three cell types are related to GTPase activity and vesicular transport. In contrast, STRING analysis showed significant interactions in two populations that appear to be cell type dependent. The data provided herein demonstrates that robust incorporation of C15AlkOPP can be obtained in ES-MNs and related primary cells purified via magnetic-activated cell sorting allowing the identification and quantification of numerous prenylated proteins. These results suggest that metabolic labeling with C15AlkOPP should be an effective approach for investigating the role of prenylated proteins in primary cells in both normal cells and disease pathologies, including ALS.

Keywords: Click chemistry; Farnesylation; Geranylgeranylation; Metabolic labeling; Prenylation; Proteomics; Synthetic probe.

MeSH terms

Alkynes* / chemical synthesis
Alkynes* / chemistry
Animals
Astrocytes* / cytology
Astrocytes* / metabolism
Cells, Cultured
Mice
Molecular Structure
Motor Neurons* / cytology
Motor Neurons* / metabolism
Protein Prenylation*
Terpenes / chemical synthesis
Terpenes / chemistry
Terpenes / metabolism

Grants and funding

R21 NS101575/NS/NINDS NIH HHS/United States