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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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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Project description:Background: Prion diseases such as bovine spongiform encephalopathies (BSE) are transmissible neurodegenerative diseases which are presumably caused by an infectious conformational isoform of the cellular prion protein. Previous work has provided evidence that in murine prion disease the endogenous retrovirus (ERV) expression is altered in the brain. To determine if prion-induced changes in ERV expression are a general phenomenon we used a non-human primate model for prion disease. Results: Cynomolgus macaques (Macaca fasicularis) were infected intracerebrally with BSE-positive brain stem material from cattle and allowed to develop prion disease. Brain tissue from the basis pontis and vermis cerebelli of the six animals and the same regions from four healthy controls were subjected to ERV expression profiling using a retrovirus-specific microarray and quantitative real-time PCR. We could show that Class I gammaretroviruses HERV-E4-1, ERV-9, and MacERV-4 increase expression in BSE-infected macaques. In a second approach, we analysed ERV-K-(HML-2) RNA and protein expression in extracts from the same cynomolgus macaques. Here we found a significant downregulation of both, the macaque ERV-K-(HML-2) Gag protein and RNA in the frontal/parietal cortex of BSE-infected macaques. Conclusions: We provide evidence that dysregulation of ERVs in response to BSE-infection can be detected on both, the RNA and the protein level. To our knowledge, this is the first report on the differential expression of ERV-derived structural proteins in prion disorders. Our findings suggest that endogenous retroviruses may induce or exacerbate the pathological consequences of prion-associated neurodegeneration. Cynomolgus macaques (Macaca fasicularis) were infected intracerebrally with BSE-positive brain stem material from cattle and allowed to develop prion disease. Brain tissue from the basis pontis and vermis cerebelli of the six animals and the same regions from four healthy controls were subjected to ERV expression profiling using a retrovirus-specific microarray and quantitative real-time PCR. In a second approach, ERV-K-(HML-2) RNA and protein expression was analysed in extracts from the same cynomolgus macaques.

Dataset Information

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

Publications

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

Similar Datasets

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