Analysis of the eukaryotic prenylome by isoprenoid affinity tagging

Abstract

Protein prenylation is a widespread phenomenon in eukaryotic cells that affects many important signaling molecules. We describe the structure-guided design of engineered protein prenyltransferases and their universal synthetic substrate, biotin-geranylpyrophosphate. These new tools allowed us to detect femtomolar amounts of prenylatable proteins in cells and organs and to identify their cognate protein prenyltransferases. Using this approach, we analyzed the in vivo effects of protein prenyltransferase inhibitors. Whereas some of the inhibitors displayed the expected activities, others lacked in vivo activity or targeted a broader spectrum of prenyltransferases than previously believed. To quantitate the in vivo effect of the prenylation inhibitors, we profiled biotin-geranyl-tagged RabGTPases across the proteome by mass spectrometry. We also demonstrate that sites of active vesicular transport carry most of the RabGTPases. This approach enables a quantitative proteome-wide analysis of the regulation of protein prenylation and its modulation by therapeutic agents.

Prenylome profiling reveals S-farnesylation is crucial for membrane targeting and antiviral activity of ZAP long-isoform

“…1D and Tables S1 and S2). In comparison with previous proteomic studies that targeted subsets of S-prenylated proteins (19)(20)(21)23), our proteomic analysis of alk-FOH-labeled proteins recovered both farnesylated and geranylgeranylated proteins, as well as many other candidate isoprenoid-modified proteins (Tables S1 and S2). To validate our alk-FOH-labeled proteins in our dataset, we biochemically characterized a canonical CaaX-containing farnesylated protein (DnaJA2) and an unpredicted substrate (Pcbp1).…”

“…More recently, alkynyl-isoprenoids have also been developed to enable more efficient labeling and detection of prenylated proteins in mammalian cells compared with their azide-counterparts using Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) (22,23). The application of these lipid chemical reporters has enabled the enrichment and identification of farnesylated (20) or geranylgeranylated protein subsets (19,21), but the general proteomic coverage of prenylated proteins has been limited. Using an alkyne-farnesol chemical reporter and improved bioorthogonal proteomic methods, we identified over 100 candidate isoprenoidlabeled proteins in macrophages, including small GTPases as well as previously unannotated S-prenylated substrates such as the zinc-finger antiviral protein (ZAP).…”

“…To this end, chemical reporters of prenylated proteins that provide better sensitivity than traditional radioactive labeling have now been developed (17,18). For example, a biotin geranyl diphosphate analog can be used with engineered prenyltransferases to visualize and profile geranylgeranylated proteins in vitro (19). Alternatively, azide-derivatives of farnesol or geranylgeraniol or their diphosphate analogs can be used to detect prenylated proteins after bioorthogonal labeling with fluorescent or affinity tags (20,21).…”

How the Golgi works: A cisternal progenitor model

“…For example, Rabs organize membrane domains that contain specific, so-called tethering factors and the SNARE proteins that mediate membrane docking and fusion events, respectively (24,25); Rabs also link to motor proteins to permit organelle motility. A given cell may contain more than 40 different Rab proteins (26), and each Rab will have distinct sets of binding partners that drive cargo collection into transport vesicles, link to molecular motors, and/or facilitate docking and fusion.…”

The Protein Lipidation and Its Analysis

“…The resulting prenylated proteins could be further modified by the Staudinger ligation or Diels Alder reaction [114]. Later, the authors used a biotin-geranylgeranyl (GerGer) pyrophosphate analog and an engineered protein prenyltransferases to analyse the in vivo effects of protein prenyltransferase inhibitors by quantifying by MS the amount of biotin-GerGer-tagged RabGTPases after treatment with inhibitors [115]. Synthesis of additional farnesyl diphosphate analogs bearing alkyne or azide groups [116,117] as well as in vitro studies to characterize them as substrates for the corresponding prenyl transferases have also been described by other groups [118,119].…”