Towards the systematic mapping and engineering of the protein prenylation machinery in Saccharomyces cerevisiae.

Protein prenylation is a widespread and highly conserved eukaryotic post-translational modification that endows proteins with the ability to reversibly attach to intracellular membranes. The dynamic interaction of prenylated proteins with intracellular membranes is essential for their signalling functions and is frequently deregulated in disease processes such as cancer. As a result, protein prenylation has been pharmacologically targeted by numerous drug discovery programs, albeit with limited success. To a large extent, this can be attributed to an insufficient understanding of the interplay of different protein prenyltransferases and the combinatorial diversity of the prenylatable sequence space. Here, we report a high-throughput, growth-based genetic selection assay in Saccharomyces cerevisiae based on the Ras Recruitment System which, for the first time, has allowed us to create a comprehensive map of prenylatable protein sequences in S. cerevisiae. We demonstrate that potential prenylatable space is sparsely (6.2%) occupied leaving room for creation of synthetic orthogonal prenylatable sequences. To experimentally demonstrate that, we used the developed platform to engineer mutant farnesyltransferases that efficiently prenylate substrate motives that are not recognised by endogenous protein prenyltransferases. These uncoupled mutants can now be used as starting points for the systematic engineering of the eukaryotic protein prenylation machinery

Voltammetry with microelectrodes in wine: determination of total acidity.

Protein prenylation is a widespread and highly conserved eukaryotic post-translational modification that endows proteins with the ability to reversibly attach to intracellular membranes. The dynamic interaction of prenylated proteins with intracellular membranes is essential for their signalling functions and is frequently deregulated in disease processes such as cancer. As a result, protein prenylation has been pharmacologically targeted by numerous drug discovery programs, albeit with limited success. To a large extent, this can be attributed to an insufficient understanding of the interplay of different protein prenyltransferases and the combinatorial diversity of the prenylatable sequence space. Here, we report a high-throughput, growth-based genetic selection assay in Saccharomyces cerevisiae based on the Ras Recruitment System which, for the first time, has allowed us to create a comprehensive map of prenylatable protein sequences in S. cerevisiae. We demonstrate that potential prenylatable space is sparsely (6.2%) occupied leaving room for creation of synthetic orthogonal prenylatable sequences. To experimentally demonstrate that, we used the developed platform to engineer mutant farnesyltransferases that efficiently prenylate substrate motives that are not recognised by endogenous protein prenyltransferases. These uncoupled mutants can now be used as starting points for the systematic engineering of the eukaryotic protein prenylation machinery

Placental fibroblast growth factor 21 is not altered in late-onset preeclampsia

Background: Preeclampsia (PE) is associated with alterations of placental function. The incidence of PE is higher in insulin resistant states. Women with PE have high circulating levels of the metabolic regulator fibroblast growth factor 21 (FGF21). FGF21 is synthesized in the placenta. The aim of this study was to compare the expression of FGF21, its receptors, downstream targets and transcriptional regulators in placental tissue from pregnancies with and without late-onset PE. Circulating FGF21 in maternal and cord blood was also studied

Summary of constructs employed in this study.

<p>Summary of constructs employed in this study.</p

Engineering FTases with altered substrate specificities.

<p>(A) CaaX-box motives with positively charged residues in the anchoring position X cannot rescue growth in the RRS and thus provide poor substrates for endogenous FTases in <i>Saccharomyces cerevisiae</i>. (B) Structural model of the αβ-FTase heterodimer derived from <i>Rattus norvegicus</i> (PDB: 1KZO). The C-terminus of the α-subunit (highlighted in blue) is separated by 40 Å from the N-terminus of the β-subunit (highlighted in red). (C) Western blot analysis of GFP-αβ-FTase fusion proteins derived from <i>R</i>. <i>norvegicus</i> expressed in <i>Leishmania tarantolae</i> cell-free expression system. The linker connecting α- and β-subunits contained a TEV protease cleavage site that is cleaved with exogenously added TEV protease. L1: Protein Ladder; L2: Uncleaved GFP-αβ-FTase; L3: GFP-αβ-FTase cleaved with TEV Protease. (D) Fluorescent scan of SDS–PAGE loaded with mCherry-K-Ras <i>in vitro</i> prenylation reaction containing single-chain GFP-αβ-FTase fusion proteins and fluorescent phosphoisoprenoid NBD-GPP [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120716#pone.0120716.ref035" target="_blank">35</a>]. Addition of FPP to the reaction prevents formation of the fluorescent reaction product due to competition with the fluorescent lipid donor. L1: Protein Ladder; L2: GFP-αβ-FTase bound to GFP-Cap beads, 5 μM mCherry-K-Ras, 5 μM NBP-GPP; L3: GFP-αβ-FTase bound to GFP-Cap beads, 5 μM mCherry-K-Ras, 5 μM NBD-GPP, 25 μM FPP. (E) To facilitate expression and prevent cross-heterodimerisation between yeast and exogenous FTase subunits, a single-chain αβ-FTase was created based on mutant β-W102T while introducing negative charges at the bottom of the active site at β-G142D and β-G142E enabling FTase to farnesylate a CaaX-box motif with a positive charge in X and thus rescue growth in the RRS. Controls: pYES2 denotes vector control and β-W102T the unmodified, single-chain αβ-FTase^β-W102T mutant neither of which can prenylate the orthogonal CaaX-box motif.</p

Mapping CaaX-box dependent membrane recruitment space in yeast.

<p>(A) Flow chart of the mapping experiment. The CaaX-box library was transformed into the RRS screening strain, grown for 4 days under permissive conditions at 25°C, replica plated and then grown for another 4–6 days under restrictive and permissive conditions at 37°C and 25°C. CaaX-box coding plasmid DNA was then isolated and analysed for the two different library sets by NGS with the Ion Torrent system. The enrichment was determined for each of the 8000 different CaaX-box motives by measuring the frequency of each peptide under restrictive conditions and normalising it over its frequency under permissive conditions. (B) Graphic representation of the enrichment factors of 8000 different CaaX-box motives is summarised in a 4D plot: Each axis represents the 20 different amino acids while the size of each dot is proportional to the enrichment of a specific CaaX-box motif. Only CaaX-box motives that have been enriched >3 are shown. (C) Cross-sectional views along the a₂-a₁, X-a₁ and X-a₂ axis illustrate that a₂ exerts the greatest specificity on substrate specificity with small hydrophobic residues highly preferred followed by the anchoring position X and a₁.</p

Summary of NGS counts and library coverage.

<p>¹Based on the observation that a sequence motif is detected at least twice in a particular NGS run.</p><p>²Based on the observation that a sequence motif is detected at least twice in the Naïve, 25°C or 37°C data set.</p><p>³Based on the observation that a sequence motif is detected at least twice in either the 25°C or 37°C data set.</p><p>Summary of NGS counts and library coverage.</p