Chromatographic Assay and Peptide Substrate Characterization of Partially Purified Farnesyl- and Geranylgeranyltransferases from Rat Brain Cytosol

Boutin, Jean A.; Marande, William; Goussard, Marion; Loynel, Armelle; Canet, Emmanuel; Fauchère, Jean‐Luc

doi:10.1006/abbi.1998.0678

Cited by 12 publications

(12 citation statements)

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“…in the millimolar range for serotonin and tryptamine), with the sole exception of PEA, while millimolar K m values toward acetyl-CoA had also been recorded in other species (33). However, K m values in the same range are not rare for other transferases toward their respective substrates (57)(58)(59). For the AANAT of other origin, various levels of K m for serotonin have been reported in sheep, rat, and even in fishes as follows: 170 (17), 20 (29) and 85 M (15) for sheep, 2000 M (15) for rat, 20 M for pike (62) but 1400 M for zebrafish (63).…”

Section: Discussionmentioning

confidence: 98%

Substrate Specificity and Inhibition Studies of Human SerotoninN-Acetyltransferase

Ferry¹,

Loynel²,

Kucharczyk³

et al. 2000

Journal of Biological Chemistry

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Arylalkylamine N-acetyltransferase (AANAT) catalyzes the reaction of serotonin with acetyl-CoA to form N-acetylserotonin and plays a major role in the regulation of the melatonin circadian rhythm in vertebrates. In the present study, the human cloned enzyme has been expressed in bacteria, purified, cleaved, and characterized. The specificity of the human enzyme toward substrates (natural as well as synthetic arylethylamines) and cosubstrates (essentially acyl homologs of acetylCoA) has been investigated. Peptide combinatorial libraries of tri-, tetra-, and pentapeptides with various amino acid compositions were also screened as potential sources of inhibitors. We report the findings of several peptides with low micromolar inhibitory potency. For activity measurement as well as for specificity studies, an original and rapid method of analysis was developed. The assay was based on the separation and detection of N-[ 3 H]acetylarylethylamine formed from various arylethylamines and tritiated acetyl-CoA, by means of high performance liquid chromatography with radiochemical detection. The assay proved to be robust and flexible, could accommodate the use of numerous synthetic substrates, and was successfully used throughout this study. We also screened a large number of pharmacological bioamines among which only one, tranylcypromine, behaved as a substrate. The synthesis and survey of simple arylethylamines also showed that AANAT has a large recognition pattern, including compounds as different as phenyl-, naphthyl-, benzothienyl-, or benzofuranyl-ethylamine derivatives. An extensive enzymatic study allowed us to pinpoint the amino acid residue of the pentapeptide inhibitor, S 34461, which interacts with the cosubstrate-binding site area, in agreement with an in silico study based on the available coordinates of the hAANAT crystal.Melatonin (5-methoxy-N-acetyltryptamine) is a pineal hormone that modulates a variety of endocrinological, neurophysiological, and behavioral functions in vertebrates (1). It is involved in the regulation of circadian rhythms and in the reproduction of photoperiodic species (2). The chronobiotic effects of melatonin in humans have been mainly studied in circadian rhythm sleep disorders (3). Moreover, alterations of the melatonin profiles have been reported in other biological rhythm disorders (3). Melatonin exerts its effects through at least three targets: 2 receptor subtypes, mt 1 and MT 2 , and a binding site, MT 3 (4). The two first ones have been cloned (5-6) and their pharmacological effects largely studied, and several specific and potent ligands (7-9) discovered. The MT 3 subtype is still a putative binding site under intensive research from purification attempts to pharmacological characterizations (10 -11). Since melatonin is implicated in several types of mild to severe pathologies, including mood disorders (3, 12), it is considered a valuable therapeutic target. Beside the classical search for agonists and antagonists of the melatonin receptors, a series of programs was launched t...

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Section: Discussionmentioning

confidence: 98%

Substrate Specificity and Inhibition Studies of Human SerotoninN-Acetyltransferase

Ferry¹,

Loynel²,

Kucharczyk³

et al. 2000

Journal of Biological Chemistry

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“…However, these rules are not absolute: for example, a CaaX protein with a C-terminal phenylalanine can be farnesylated or geranylgeranylated 181 . And although GGT1 clearly prefers X to be leucine 182 , some CaaL motifs can also be farnesylated by FT 183 . Furthermore, at least one protein, RHOB (CaaX box sequence: CKVL), is naturally both farnesylated and geranylgeranylated 35 .…”

Section: Figurementioning

confidence: 99%

Targeting protein prenylation for cancer therapy

2011

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Protein farnesylation and geranylgeranylation, together referred to as prenylation, are lipid post-translational modifications that are required for the transforming activity of many oncogenic proteins, including some RAS family members. This observation prompted the development of inhibitors of farnesyltransferase (FT) and geranylgeranyltransferase 1 (GGT1) as potential anticancer drugs. In this Review, we discuss the mechanisms by which FT and GGT1 inhibitors (FTIs and GGTIs, respectively) affect signal transduction pathways, cell cycle progression, proliferation and cell survival. In contrast to their preclinical efficacy, only a small subset of patients responds to FTIs. Identifying tumours that depend on farnesylation for survival remains a challenge, and strategies to overcome this are discussed. One GGTI has recently entered the clinic, and the safety and efficacy of GGTIs await results from clinical trials.

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“…This view has not changed but it has become clear that several substrates with leucine at position +3 can also be modified (if only to a lesser extent) by FT and not only GGT1. For example, in vitro studies have shown that motifs like CVIL, CVLL, CAIL and CCIL (single-letter amino-acid code) are valid for FT as well [28]. Mutation of the CVIA motif of yeast A-factor to CVIL results in geranylgeranylated as well as farnesylated proteins in vivo [29].…”

Section: Prediction Function and Validationmentioning

confidence: 99%

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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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Chromatographic Assay and Peptide Substrate Characterization of Partially Purified Farnesyl- and Geranylgeranyltransferases from Rat Brain Cytosol

Cited by 12 publications

References 71 publications

Substrate Specificity and Inhibition Studies of Human SerotoninN-Acetyltransferase

Substrate Specificity and Inhibition Studies of Human SerotoninN-Acetyltransferase

Targeting protein prenylation for cancer therapy

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