Investigation of S-Farnesyl Transferase Substrate Specificity with Combinatorial Tetrapeptide Libraries

Boutin, Jean A.; Marande, William; Petit, Laurence; Loynel, Armelle; Desmet, Christine; Canet, Emmanuel; Fauchère, Jean‐Luc

doi:10.1016/s0898-6568(98)00032-1

Cited by 22 publications

(14 citation statements)

References 49 publications

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“…To both limit the number of peptides in the library and bias the library toward potential FTase substrates, we started by predicting which of the remaining proteins are most likely to be farnesylated using a model based on the sequence of known farnesylated proteins (Table S1) and peptides10; 18; 20; 27; 31; 32. We selected all of the proteins that have amino acids at each of the a 1 , a 2 and X positions that were represented in known substrates.…”

Section: Resultsmentioning

confidence: 99%

Identification of Novel Peptide Substrates for Protein Farnesyltransferase Reveals Two Substrate Classes with Distinct Sequence Selectivities

Hougland

Hicks

Hartman

et al. 2010

Journal of Molecular Biology

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Prenylation is a post-translational modification essential for the proper localization and function of many proteins. Farnesylation, the attachment of a 15-carbon farnesyl group near the C-terminus of protein substrates, is catalyzed by protein farnesyltransferase (FTase). Farnesylation has received significant interest as a target for pharmaceutical development and farnesyltransferase inhibitors (FTIs) are in clinical trials as cancer therapeutics. However, as the total complement of prenylated proteins is unknown, the FTase substrates responsible for FTI efficacy are not yet understood. Identifying novel prenylated proteins within the human proteome constitutes an important step towards understanding prenylation-dependent cellular processes. Based on sequence preferences for FTase derived from analysis of known farnesylated proteins, we selected and screened a library of small peptides representing the C-termini of 213 human proteins for activity with FTase. We identified 77 novel FTase substrates that exhibit multiple-turnover reactivity within this library; our library also contained 85 peptides that can be farnesylated by FTase only under singleturnover conditions. Based on these results, a second library was designed that yielded an additional 29 novel multiple-turnover FTase substrates and 45 single-turnover substrates. The two classes of substrates exhibit different specificity requirements. Efficient multiple-turnover reactivity correlates with the presence of a nonpolar amino acid at the a 2 position and a Phe, Met, or Gln at the terminal X residue, consistent with the proposed Ca 1 a 2 X sequence model. In contrast, the sequences of the single-turnover substrates vary significantly more at both the a 2 and X residues and are not well-described by current farnesylation algorithms. These results improve the definition of prenyltransferase substrate specificity, test the efficacy of substrate algorithms, and provide valuable information about therapeutic targets. Finally, these data illuminate the potential for in vivo regulation of prenylation through modulation of single-versus multipleturnover peptide reactivity with FTase.

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Section: Resultsmentioning

confidence: 99%

Identification of Novel Peptide Substrates for Protein Farnesyltransferase Reveals Two Substrate Classes with Distinct Sequence Selectivities

Hougland

Hicks

Hartman

et al. 2010

Journal of Molecular Biology

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“…However, because of earlier interest in developing peptide-based inhibitors of FT and GGT1 as anticancer treatments, the kinetics of the enzymes with various tetrapeptide substrates already modified with lipid anchors by the enzymes have been measured [15]. Hence, a protein homologous to a verified prenylated protein can be included in the learning set if its CaaX box has already been shown to interact productively with one of the prenyltransferases at least as a tetrapeptide.…”

Section: Known Substrates and Their Motif-compliant Homologs As Learnmentioning

confidence: 99%

Untitled

2005

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Refinement and prediction of protein prenylation motifs

Three prenylation motif predictions are presented that allow discrimination between proteins that are unique substrates of farnesyltransferase (FT) and those that can be alternatively processed by geranylgeranyltransferase I (GGT1).

AbstractWe refined the motifs for carboxy-terminal protein prenylation by analysis of known substrates for farnesyltransferase (FT), geranylgeranyltransferase I (GGT1) and geranylgeranyltransferase II (GGT2). In addition to the CaaX box for the first two enzymes, we identify a preceding linker region that appears constrained in physicochemical properties, requiring small or flexible, preferably hydrophilic, amino acids. Predictors were constructed on the basis of sequence and physical property profiles, including interpositional correlations, and are available as the Prenylation Prediction Suite (PrePS, http://mendel.imp.univie.ac.at/sat/PrePS) which also allows evaluation of evolutionary motif conservation. PrePS can predict partially overlapping substrate specificities, which is of medical importance in the case of understanding cellular action of FT inhibitors as anticancer and anti-parasite agents. RationalePrenylation refers to the posttranslational modification of proteins with isoprenyl anchors [1][2][3]. These lipid moieties are typically involved in mediating not only protein-membrane but also protein-protein interactions. Three eukaryotic enzymes are known to catalyze the lipid transfer. The first two, farnesyltransferase (FT) and geranylgeranyltransferase 1 (GGT1), recognize the so-called CaaX box in the carboxy termini of substrate proteins and attach farnesyl (15-carbon polyisoprene) or geranylgeranyl (20-carbon polyisoprene), respectively, to a required and spatially fixed cysteine in that motif. The third enzyme, geranylgeranyltransferase 2 (GGT2 or RabGGT) recognizes the complex [4] of Rab GTPase substrate proteins with a specific Rab escort protein (REP) to attach one or two geranylgeranyl anchors to cysteines in a more flexible but also carboxy-terminal motif.The CaaX box was initially understood to consist of a cysteine (C), followed by two aliphatic residues (aa) and a terminal residue (X) that would direct modification by either FT or GGT1, but newly found substrates and kinetic studies of mutated substrate peptides and enzyme inhibitors have shown that the motif recognized by the enzymes appears to be more flexible [2]. Furthermore, the determination of preference for FT or GGT1 is more complex and a function of the overall sequence context rather than specific amino acids at single positions. Whereas GGT2 appears to be specific to Rab GTPases as substrates, the recognition mechanism is not well understood. Overlapping substrate specificities between all three prenylating enzymes further complicate the understanding of the lipid modification process [5,6].An unsolved problem so far is accounting for the complexity of the prenylation substrate recognition motifs in theoretical models in order to ident...

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“…We became interested in peptide combinatorial libraries at the very beginning of this approach and studied several aspects of those libraries: numbers [100], quality control [101], and activity [102,103]. For solid-phase synthesis approaches, the actual number of peptides in a given library is limited by the availability of amino acids on the resin, and that number is limited by the number of individual peptide repeats in the initial mixture, which is obviously by far the most complex one.…”

Section: Peptide Librariesmentioning

confidence: 99%

On the Organization of a Drug Discovery Platform

Nosjean¹,

Ferry²

2018

Drug Discovery - Concepts to Market

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Some of the most exciting parts of work in the pharmaceutical industry are the steps leading up to drug discovery. This process can be oversimplified by describing it as a screening campaign involving the systematic testing of many compounds in a test relevant to a given pathology. This naïve description takes place without taking into consideration the numerous key steps that led up to the screening or the steps that might follow. The present chapter describes this whole process as it was conducted in our company during our early drug discovery activities. First, the purpose of the procedures is described and rationalized. Next follows a series of mostly published examples from our own work illustrating the various steps of the process from cloning to biophysics, including expression systems and membrane-bound protein purifications. We believe that what is described here presents an example of how pharmaceutical industry research can organize its platform(s) when the goal is to find and qualify a new preclinical drug candidate using cutting-edge technologies and a lot of hard work.

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Investigation of S-Farnesyl Transferase Substrate Specificity with Combinatorial Tetrapeptide Libraries

Cited by 22 publications

References 49 publications

Identification of Novel Peptide Substrates for Protein Farnesyltransferase Reveals Two Substrate Classes with Distinct Sequence Selectivities

Identification of Novel Peptide Substrates for Protein Farnesyltransferase Reveals Two Substrate Classes with Distinct Sequence Selectivities

Untitled

On the Organization of a Drug Discovery Platform

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