MALDI-MS Analysis of Peptide Libraries Expands the Scope of Substrates for Farnesyltransferase

Abstract

Protein farnesylation is a post-translational modification where a 15-carbon farnesyl isoprenoid is appended to the C-terminal end of a protein by farnesyltransferase (FTase). This modification typically causes proteins to associate with the membrane and allows them to participate in signaling pathways. In the canonical understanding of FTase, the isoprenoids are attached to the cysteine residue of a four-amino-acid CaaX box sequence. However, recent work has shown that five-amino-acid sequences can be recognized, including the pentapeptide CMIIM. This paper describes a new systematic approach to discover novel peptide substrates for FTase by combining the combinatorial power of solid-phase peptide synthesis (SPPS) with the ease of matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS). The workflow consists of synthesizing focused libraries containing 10–20 sequences obtained by randomizing a synthetic peptide at a single position. Incubation of the library with FTase and farnesyl pyrophosphate (FPP) followed by mass spectrometric analysis allows the enzymatic products to be clearly resolved from starting peptides due to the increase in mass that occurs upon farnesylation. Using this method, 30 hits were obtained from a series of libraries containing a total of 80 members. Eight of the above peptides were selected for further evaluation, reflecting a mixture that represented a sampling of diverse substrate space. Six of these sequences were found to be bona fide substrates for FTase, with several meeting or surpassing the in vitro efficiency of the benchmark sequence CMIIM. Experiments in yeast demonstrated that proteins bearing these sequences can be efficiently farnesylated within live cells. Additionally, a bioinformatics search showed that a variety of pentapeptide CaaaX sequences can be found in the mammalian genome, and several of these sequences display excellent farnesylation in vitro and in yeast cells, suggesting that the number of farnesylated proteins within mammalian cells may be larger than previously thought.

A comprehensivein vivoscreen of yeast farnesyltransferase activity reveals broad reactivity across a majority of CXXX sequences

“…Gel-shift assay: The assay was performed as previously described (Hildebrandt, Cheng et al 2016, Berger, Kim et al 2018, Schey, Buttery et al 2021. Whole cell lysates of mid log cells were prepared and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 6% stacking with 9.5% resolving gel) then transferred onto nitrocellulose.…”

“…Most of the in vivo reporters used to probe CaaX space have relied on CaaX protein reporters requiring 3-step modification and the superposition of 3 enzyme specificities for their activities (Boyartchuk, Ashby et al 1997, Stein, Kubala et al 2015). Moreover, recent reports demonstrating FTase activity on shortened and extended sequences highlight the flexibility of CaaX substrate lengths, additionally complicating the recognized model of FTase CaaX specificity (Ashok, Hildebrandt et al 2020, Schey, Buttery et al 2021).…”

“…Moreover, recent reports demonstrating FTase activity on shortened and extended sequences highlight the flexibility of CaaX substrate lengths, additionally complicating the recognized model of FTase CaaX specificity (Ashok, Hildebrandt et al 2020, Schey, Buttery et al 2021).…”

Farnesyltransferase inhibitor LNK-754 attenuates axonal dystrophy and reduces amyloid pathology in mice

“…FTIs were initially designed to block oncogenic Ras activity by inhibiting its prenylation, but were discontinued in clinical trials for cancer treatment following the discovery of alternate Ras prenylation pathways [21,22]. However, many FTase substrates exist [23][24][25], leading to the evaluation of FTIs for a wide range of disorders, including cardiovascular disease, hepatitis D and progeria [20,26]. Additionally, FTIs have shown promise towards neurodegenerative diseases characterized by the accumulation of protein aggregates.…”

Thinking outside the CaaX-box: an unusual reversible prenylation on ALDH9A1

“…However, recent studies have shown that proteins terminating in CXXXX or CXX may also potentially be substrates of prenylation. [3][4][5] The third type of prenylation involves geranylgeranyltransferase type II (GGTase-II or RabGGTase) that usually appends two geranylgeranyl units on dual cysteine motifs on proteins, particularly those that belong to the Rab family. These substrates are typically recognized first by the rab escort protein 1 or 2 (REP-1/2) and modified by GGTase-II at their C-terminal cysteines in their -CCXX, -CXC, or -XXCC canonical sequences.…”