Metabolic labeling with an alkyne probe reveals similarities and differences in the prenylomes of several brain-derived cell lines and primary cells

Abstract

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.

In vivoprenylomic profiling in the brain of a transgenic mouse model of Alzheimer’s disease reveals increased prenylation of a key set of proteins

“…After 24 hours, the incorporation of the analogue was visualized using in-gel fluorescence. The protein labeling pattern in SH-SY5Y cells was similar to that previously reported in HeLa and COS-7 cells 28,35 with intense fluorescence bands in the 20-25 kDa region (Extended Data Fig. 1D).…”

“…This labeling was greater in the mouse that received the mixture of both SV and the analogue because statins increase the incorporation efficiency of exogenous analogues into target proteins by reducing endogenous FPP and GGPP levels. 28,35,39 However, surprisingly, there was no TAMRA fluorescence band in the 20-25 kDa region of the gel where many prenylated small-GTPases migrate, suggesting that C15AlkOPP did not efficiently label small GTPases in this experiment (Extended Data Fig. 1 C).…”

“…In this study, the profiling of the brain prenylome in live mice was tested using a synthetic alkyne-modified isoprenoid analogue, C15AlkOPP. Recently, the same analogue was employed to characterize the prenylome in neuronal and glial cell lines as well as primary astrocytes, 35 demonstrating its utility as a reporter for prenylation in multiple brain cell types. Due to the limited brain penetration of the analogue when administered via IP injection, a stereotaxic brain injection strategy was used to bypass the BBB.…”

“…47 Notably, the known prenylated protein Uchl1, 48,49 which was found to be enriched in all experiments here, had not been reported in previous prenylomic experiments. 31,35 Intriguingly, membrane-associated farnesylated Uchl1 promotes α-synuclein neurotoxicity and is implicated in the pathogenesis of Parkinson's disease (PD). 48 The down regulation and extensive oxidation of Uchl1 is observed in AD as well as PD patients; in particular in sporadic AD the levels of soluble cytosolic Uchl1 are inversely proportional to neurofibrillary tangle amounts.…”

“…[31][32][33][34] In earlier work, this method was used to profile prenylated proteins in brain-derived cell lines using both primary and immortalized cells. 35 Those experiments revealed similarities and differences in the identities and levels of prenylated proteins found between different cell types. However, such experiments using only one cell type cannot capture the complex pathology of a disease such as AD.…”