An Alkyne-Containing Isoprenoid Analogue Based on a Farnesyl Diphosphate Scaffold Is a Biologically Functional Universal Probe for Proteomic Analysis. Biochemistry 2025 Jan 07;64(1):138-155
Date
12/09/2024Pubmed ID
39652878Pubmed Central ID
PMC11706708DOI
10.1021/acs.biochem.4c00558Scopus ID
2-s2.0-85211597943 (requires institutional sign-in at Scopus site)Abstract
Prenylation consists of the modification of proteins with either farnesyl diphosphate (FPP) or geranylgeranyl diphosphate (GGPP) at a cysteine near the C-terminus of target proteins to generate thioether-linked lipidated proteins. In recent work, metabolic labeling with alkyne-containing isoprenoid analogues including C15AlkOPP has been used to identify prenylated proteins and track their levels in different diseases. Here, a systematic study of the impact of isoprenoid length on proteins labeled with these probes was performed. Chemical synthesis was used to generate two new analogues, C15hAlkOPP and C20AlkOPP, bringing the total number of compounds to eight used in this study. Enzyme kinetics performed in vitro combined with metabolic labeling in cellulo, resulted in the identification of 8 proteins for C10AlkOPP, 70 proteins for C15AlkOPP, 41 proteins for C15hAlkOPP, and 7 proteins for C20AlkOPP. While C10AlkOPP was the most selective for farnesylated proteins and C20AlkOPP was most selective for geranylgeranylated proteins, the number of proteins identified using those probes was relatively small. In contrast, C15AlkOPP labeled the most proteins including representatives from all classes of prenylated proteins. Functional analysis of these analogues demonstrated that C15AlkOPP was particularly well suited for biological studies since it was efficiently incorporated in cellulo, was able to confer correct plasma membrane localization of H-Ras protein and complement the effects of GGPP depletion in macrophages to yield correct cell polarization and filopodia. Collectively, these results indicate that C15AlkOPP is a biologically functional, universal probe for metabolic labeling experiments that has minimal effects on cellular physiology.
Author List
Auger SA, Pedersen JS, Maity S, Sprague-Getsy AM, Lorimer EL, Koehn OJ, Krauklis SA, Berns B, Murphy KM, Hussain J, Thao P, Bernhagen K, Justyna K, Sundaresan AP, McKim DB, Williams CL, Hougland JL, Li L, Distefano MDAuthor
Carol L. Williams PhD Professor in the Pharmacology and Toxicology department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
AlkynesAnimals
Humans
Mice
Molecular Probes
Polyisoprenyl Phosphates
Protein Prenylation
Proteomics
RAW 264.7 Cells
Sesquiterpenes
Terpenes