Dataset Information

Broadening the Utility of Farnesyltransferase-Catalyzed Protein Labeling Using Norbornene–Tetrazine Click Chemistry

ABSTRACT: Bio-orthogonal chemistry has gained widespread use in the study of many biological systems of interest, including protein prenylation. Prenylation is a post-translational modification in which a 15 or 20 carbon isoprenoid chain is transferred onto a cysteine near the C-terminus of a target protein. The three enzymes, protein farnesyltransferase (FTase), geranylgeranyl transferase I (GGTase I), and geranylgeranyl transferase II (GGTase II), that catalyze this process have been shown to exhibit some variability in substrate selection. This trait has been utilized in the past to transfer an array of farnesyl diphosphate analogues with a range of functionality, like an alkyne- containing analogue for copper-catalyzed bioconjugation reactions for example. Reported here is the synthesis of an analogue of the isoprenoid substrate embedded with norbornene functionality (C10NorOPP) for the study of prenylation and development of multifaceted protein-polymer-label conjugates. This analogue undergoes the inverse electron demand Diels Alder reaction with a tetrazine containing tags, allowing for copper-free labeling of proteins in the prenylome. This probe was synthesized in 7 steps with an overall yield of 7%. The use of C10NorOPP for the study of prenylation was explored in metabolic labeling of prenylated proteins in HeLa, COS-7, and astrocyte cells. Furthermore, in HeLa cells, these modified prenylated proteins were identified and quantified using LFQ proteomics, finding 24 enriched prenylated proteins. Additionally, here we utilize the unique chemistry of C10NorOPP in the construction of a multi- protein-polymer conjugate for the targeted labeling of cancer cells. This combines the specificity of the norbornene-tetrazine conjugation with traditional azide-alkyne cycloaddition for the construction of a polymer linked fluorescent trimer increasing cell binding.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell

SUBMITTER: Shelby Auger

LAB HEAD: Mark D.

PROVIDER: PXD045277 | Pride | 2024-05-21

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	HeLa_norC10OPP_combined.sf3	Other
	HeLa_norC10OPP_profiling_spectral_count.xlsx	Xlsx
	KS_HeLa_FPP_13.raw	Raw
	KS_HeLa_FPP_7.raw	Raw
	KS_HeLa_FPP_80.raw	Raw

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Publications

Broadening the Utility of Farnesyltransferase-Catalyzed Protein Labeling Using Norbornene-Tetrazine Click Chemistry.

Auger Shelby A SA Venkatachalapathy Sneha S Suazo Kiall Francis G KFG Wang Yiao Y Sarkis Alexander W AW Bernhagen Kaitlyn K Justyna Katarzyna K Schaefer Jonas V JV Wollack James W JW Plückthun Andreas A Li Ling L Distefano Mark D MD

Bioconjugate chemistry 20240424 7

Bioorthogonal chemistry has gained widespread use in the study of many biological systems of interest, including protein prenylation. Prenylation is a post-translational modification, in which one or two 15- or 20-carbon isoprenoid chains are transferred onto cysteine residues near the C-terminus of a target protein. The three main enzymes─protein farnesyltransferase (FTase), geranylgeranyl transferase I (GGTase I), and geranylgeranyl transferase II (GGTase II)─that catalyze this process have be ...[more]

PMID: 38654427

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