Identification and characterisation of Toxoplasma gondii protein farnesyltransferase

Prenylated proteins are involved in the regulation of DNA replication and cell cycling and have important roles in the regulation of cell proliferation. Protein farnesyltransferase and protein geranylgeranyltransferase are the two enzymes responsible for catalysing isoprene lipid modifications. Recently these enzymes have been targets for the development of cancer chemotherapeutics. Using metabolic labelling we identified isoprenylated proteins which suggests the presence of protein farnesyltransferase in Toxoplasma gondii. T. gondii protein farnesyltransferase is heat-labile and requires Mg(2+) and Zn(2+) ions for full activity. Peptidomimetic analogues as well as short synthetic peptides were tested in vitro as possible competitors for farnesyltransferase substrates. We found that the synthetic peptide (KTSCVIA) specifically inhibited T. gondiiprotein farnesyltransferase but not mammalian (HeLa cells) farnesyltransferase. Therefore this study suggests the possible development of specific inhibitors of T. gondiiprotein farnesyltransferase as an approach to parasitic protozoa therapy.     

Synthesis of novel methyl jasmonate derivatives and evaluation of their biological activity in various cancer cell lines

Warburg hypothesized that the energy consumption of cancer cells is different than the normal cells. When compared to normal conditions, cancer cells do not undergo tricarboxylic acid (TCA) cycle therefore resulting in more lactate in the cells. Glycolysis pathway is a way of cancer cells to provide energy. The first step in glycolysis is the phosphorylation of glucose to glucose-6-phosphate. This reaction is catalyzed by the hexokinase-II enzyme (HK-II) which is known to be overexpressed in tumor cells. The feeding of cancer cells can be prevented by inhibiting the hexokinase-II enzyme in the first step of aerobic glycolysis. In literature, Methyl Jasmonate (MJ) is known as a Hexokinase-II inhibitor since it disposes VDAC and HK-II interaction on mitochondrial membrane. In our study, we aimed to increase the activity by synthesizing the novel MJ analogues with appropriate modifications. Here we report Hexokinase-2 enzyme and cell viability study results in different cancer cells. Based on the three different cancer cell lines we investigated, our novel MJ analogues proved to be more potent than the original molecule. Thus this research may provide more efficacious/novel HK-II inhibitors and may shed light to develop new anti-cancer agents.     

Conversion of Tyr361 beta to Leu in mammalian protein farnesyltransferase impairs product release but not substrate recognition

Protein farnesyltransferase catalyzes the lipid modification of protein substrates containing Met, Ser, Gln, or Ala at their C-terminus. A closely related enzyme, protein geranylgeranyltransferase type I, carries out a similar modification of protein substrates containing a C-terminal Leu residue. Analysis of a mutant of protein farnesyltransferase containing a Tyr-to-Leu substitution at position 361 in the beta subunit led to the conclusion that the side chain of this Tyr residue played a major role in recognition of the protein substrates. However, no interactions have been observed between this Tyr residue and peptide substrates in the crystal structures of protein farnesyltransferase. In an attempt to reconcile these apparently conflicting data, a thorough kinetic characterization of the Y361L variant of mammalian protein farnesyltransferase was performed. Direct binding measurements for the Y361L variant yielded peptide substrate binding that was actually some 40-fold tighter than that with the wild-type enzyme. In contrast, binding of the peptide substrate for protein geranylgeranyltransferase type I was very weak. The basis for the discrepancy was uncovered in a pre-steady-state kinetic analysis, which revealed that the Y361L variant catalyzed farnesylation of a normal peptide substrate at a rate similar to that of the wild-type enzyme in a single turnover, but that subsequent turnover was prevented. These and additional studies revealed that the Y361L variant does not "switch" protein substrate specificity as concluded from steady-state parameters; rather, this variant exhibits severely impaired product dissociation with its normal substrate, a situation resulting in a greatly compromised steady-state activity.     

High-performance liquid chromatographic method to measure protein L-isoaspartyl/D-aspartyl o-methyltransferase activity in cell lysates

Protein l-isoaspartyl/d-aspartyl o-methyltransferase (PIMT) is a widely expressed protein repair enzyme that restores isomerized aspartyl residues to their normal configuration. Current methods for measuring PIMT activity have limited sensitivity or require radioactivity. We have developed a highly sensitive new assay method to measure PIMT activity in cell lysates. As a substrate, we used a fluorescently labeled delta sleep-inducing peptide (DSIP) that contains an isoaspartyl residue: 7-nitro-2,1,3-benzoxadiazole (NBD)-DSIP(isoAsp). The PIMT-catalyzed transfer of a methyl group onto this substrate can be detected with a simple high-performance liquid chromatography (HPLC) procedure. After the enzyme reaction, the methylated form of the peptide is stable and can be reproducibly separated from the unmethylated form in an acidic solvent and fluorometrically detected by HPLC. The limit of detection was estimated to be approximately 1 pmol of NBD-DSIP(isoAsp) (signal/noise ratio [S/N] = 3), and the quantitation limit of the activity was approximately 18 μg of total cell lysate from HEK293 cells (10.7 pmol/min/mg protein). This assay method is sensitive enough to detect PIMT activity in biological samples without the use of radioisotopes, offering significant advantages over previously reported methods.     

Assay of tyrosine protein kinase activity from HL-60 by high-performance liquid chromatography for specificity studies

Using a partially purified HL-60 tyrosine protein kinase, we designed a new HPLC method for the measurement of tyrosylphosphorylation of angiotensin II. The present method uses reversed-phase chromatography and elution involving an acetonitrile gradient containing the counterion tetrabutylammonium phosphate. The peptide substrate, [gamma-32P]ATP, the cosubstrate, and 32P-labeled phosphorylated peptides were quantified online by measuring the Cerenkov effect. Injections, separation, and analysis were performed automatically. Furthermore, the method permits a direct visualization of peptide substrate phosphorylation and has a potentially universal application; i.e., it is usable with any kind of peptide in a given range of hydrophobicity. This assay was designed for specificity studies, which are of major importance at the molecular level, in order to understand active site topology and the biophysical requirements of tyrosine protein kinases. As examples, data on chromatography separations of angiotensin II analogs (five to ten amino acids in length) are presented, as well as for other peptide substrates such as RR-src, the pp60src autophosphorylation site-derived peptide, and minigastrin. We adapted our experimental conditions to accommodate crude extracts from HL-60 cells. Preliminary experiments clearly indicated that other biological sources can be used. Despite the existence of numerous methods published in the literature for the measurement of kinase activities, the method presented herein is the only one to the authors' knowledge that can be used in and has been assessed for specificity studies. Peptides do not require particular features such as charged residues (i.e., arginine) to be analyzed.     

Prioritizing candidate peptides for cancer vaccines through predicting peptide presentation by HLA-I proteins

Cancer (treatment) vaccines that are made of neoantigens, or peptides unique to tumor cells due to somatic mutations, have emerged as a promising method to reinvigorate the immune response against cancer. A key step to prioritizing neoantigens for cancer vaccines is computationally predicting which neoantigens are presented on the cell surface by a human leukocyte antigen (HLA). We propose to address this challenge by training a neural network using mass spectrometry (MS) data composed of peptides presented by at least one of several HLAs of a subject. We embed the neural network within a mixture model and train the neural network by maximizing the likelihood of the mixture model. After evaluating our method using data sets where the peptide presentation status was known, we applied it to analyze somatic mutations of 60 melanoma patients and identified a group of neoantigens more immunogenic in tumor cells than in normal cells. Moreover, neoantigen burden estimated by our method was significantly associated with a measurement of the immune system activity, suggesting these neoantigens could induce an immune response.     

Probing the hydrophobic pocket of farnesyltransferase: aromatic substitution of CAAX peptidomimetics leads to highly potent inhibitors

Cysteine farnesylation at the carboxylate terminal tetrapeptide CAAX of Ras protein is catalyzed by farnesyltransferase. This lipid modification is necessary for regulatory function of both normal and oncogenic Ras. The high frequency of Ras mutation in human cancers has prompted an intensive study on finding ways of controlling oncogenic Ras function. Inhibition of farnesyltransferase is among the most sought after targets for cancer chemotherapy. We report here the design, synthesis and biological characterization of a series of peptidomimetics as farnesyltransferase inhibitors. These compounds are extremely potent towards farnesyltransferase with IC₅₀ values ranging from subnanomolar to low nanomolar concentrations. They have a high selectivity for farnesyltransferase over the closely related geranylgeranyltransferase-I. Structure–activity relationship studies demonstrated that a properly positioned hydrophobic group significantly enhanced inhibition potency, reflecting an improved complementarity to the large hydrophobic pocket in the CAAX binding site.     

cDNA cloning and expression of the peptide-binding beta subunit of rat p21ras farnesyltransferase, the counterpart of yeast DPR1/RAM1

Protein farnesyltransferase is a heterodimeric enzyme that attaches a farnesyl group to cysteine in ras proteins and other membrane-associated proteins. The beta subunit contains the recognition site for the peptide substrates, but is inactive in the absence of the alpha subunit. A cloned cDNA for the rat beta subunit predicts a protein of 437 amino acids whose mRNA is present in many tissues. Transfection of the beta subunit cDNA produced farnesyltransferase activity in human kidney cells, but only when it was transfected together with a cDNA encoding part of the alpha subunit. Each of the subunits appeared to be unstable in the transfected cells unless the other subunit was present. The rat beta subunit shows 37% sequence identity with the protein encoded by the yeast DPR1/RAM1 gene, indicating that DPR1/RAM1 is the yeast counterpart of the peptide-binding subunit of the mammalian farnesyltransferase.     

Enzymatic assays for NAD-dependent deacetylase activities

The NAD-dependent deacetylases are a new class of enzymes responsible for the removal of acetyl groups from lysines on proteins. Instead of water, the NAD-dependent deacetylases use a highly reactive ADP-ribose intermediate as a recipient for the acetyl group. The products of the reaction are nicotinamide, acetyl-ADP-ribose, and a deacetylated substrate. Many assays have been developed for the measurement of NAD-dependent deacetylase activity. In this review we present assays based on each of the two reactions catalyzed by these enzymes, deacetylation and NAD hydrolysis. First we describe methods for the production of acetylated protein and peptide substrates for use in deacetylation reactions. Then we describe four methods for assaying deacetylation, three of which directly measure the loss of acetyl groups from a protein or peptide substrate, and one that measures acetate production. We also describe two indirect methods for following enzyme activity, NAD hydrolysis and a novel NAD-nicotinamide exchange reaction. Finally, a quantitative method using a monoacetylated peptide as a substrate and HPLC to measure products is described.     

Characterization of SARS main protease and inhibitor assay using a fluorogenic substrate

SARS main protease is essential for life cycle of SARS coronavirus and may be a key target for developing anti-SARS drugs. Recently, the enzyme expressed in Escherichia coli was characterized using a HPLC assay to monitor the formation of products from 11 peptide substrates covering the cleavage sites found in the SARS viral genome. This protease easily dissociated into inactive monomer and the deduced Kd of the dimer was 100 microM. In order to detect enzyme activity, the assay needed to be performed at micromolar enzyme concentration. This makes finding the tight inhibitor (nanomolar range IC50) impossible. In this study, we prepared a peptide with fluorescence quenching pair (Dabcyl and Edans) at both ends of a peptide substrate and used this fluorogenic peptide substrate to characterize SARS main protease and screen inhibitors. The fluorogenic peptide gave extremely sensitive signal upon cleavage catalyzed by the protease. Using this substrate, the protease exhibits a significantly higher activity (kcat = 1.9 s(-1) and Km = 17 microM) compared to the previously reported parameters. Under our assay condition, the enzyme stays as an active dimer without dissociating into monomer and reveals a small Kd value (15 nM). This enzyme in conjunction with fluorogenic peptide substrate provides us a suitable tool for identifying potent inhibitors of SARS protease.     

Role of metals in the reaction catalyzed by protein farnesyltransferase

Protein farnesyltransferase catalyzes the posttranslational farnesylation of several proteins involved in signal transduction, including Ras, and is a target enzyme for antitumor therapies. Efficient product formation catalyzed by protein farnesyltransferase requires an enzyme-bound zinc cation and high concentrations of magnesium ions. In this work, we have measured the pH dependence of the chemical step of product formation, determined under single-turnover conditions, and have demonstrated that the prenylation rate constant is enhanced by two deprotonations. Substitution of the active site zinc by cadmium demonstrated that one of the ionizations reflects deprotonation of the metal-coordinated thiol of the peptide "CaaX" motif, pK(a1) = 6.0. These data provide additional evidence for the direct involvement of a metal-coordinated sulfur nucleophile in catalysis. The second ionization was assigned to a hydroxyl on the pyrophosphate moiety of farnesyl pyrophosphate, pK(a2) = 7.4. Deprotonation of this group is important for binding of magnesium. This second ionization is not observed for catalysis in the absence of magnesium or when the substrate is farnesyl monophosphate. These data indicate that the maximal rate constant for prenylation requires formation of a zinc-coordinated thiolate nucleophile and enhancement of the electrophilic character at C1 of the farnesyl chain by magnesium ion coordination of the pyrophosphate leaving group.     

Substrate-Based Design of Human Farnesyltransferase Peptide-like Pain Antagonists

Human protein farnesyltransferase is a key enzyme for the lipid modification of a large and important number of proteins, which has been recognized as the promising therapeutic target of pain disorder and other diseases such as inflammation and cancer. In this study, we systematically investigated the binding behavior of existing peptide substrates and antagonists to farnesyltransferase at structural level and revealed that peptide’s C-terminus is primarily responsible for the binding, while exposing N-terminus to solvent. The amino acid property preference profile at each of the six core N-terminal residue positions of a cocrystallized chimera peptide substrate was defined, based on which a combinatorial library that contains more than twenty thousands of peptide-like compounds (PLCs) was generated using sixteen structurally diverse non-natural amino acids as building blocks. Subsequently, a systematic protocol was exhaustively carried out to perform virtual screening against the library, in order to discover those PLCs that match well the property preference profile and simultaneously exhibit high binding potency to farnesyltransferase. Consequently, ninety hits were identified from the library, in which five structurally diverse PLCs with high consensus scores were determined to have potent or moderate affinity to the active site of farnesyltransferase through nonbonded/coordination interactions. These identified PLCs can be considered as promising lead molecular entities to further develop peptidomimetic farnesyltransferase antagonists combating pain, inflammation and cancer.

     

Assessment of the bioactive conformation of the farnesyltransferase protein binding recognition motif by computational methods.

Ras farnesyltransferase catalyzes the carboxyl-terminal farnesylation of Ras as well as other proteins involved in signal transduction processes. Previous studies demonstrated that its inhibition suppresses the activity of Ras transformed phenotypes in cultured cells, causing tumor regression in animal models. This observation led to the consideration of farnesyltransferase as a target for cancer therapy. In the present work we report the results of a computational study aimed at assessing the bioactive conformation of the peptide Cys-Val-Phe-Met, known to be the minimum peptide sequence that inhibits farnesyltransferase. For this purpose the conformational preferences of four analogs of the peptide were assessed by means of thorough searches of their respective conformational spaces, using a simulated annealing protocol as sampling technique. Specifically, two active analogs: Cys-Val-Tic-Met and Cys-Val-psi(CH2NH)Tic-Met and two inactive analogs: Cys-Val-Tic-psi(CH2NH)Met and Cys-Val-Aic-Met were selected for the present study. Low energy conformations of the four analogs were classified according to their structural motifs. The putative bioactive conformation of the minimum farnesyltransferase recognition motif was assessed by cross-comparison of the different classes of conformations obtained for the two active and the two inactive analogs. The putative bioactive conformation is characterized by two structural motifs: i) a C14 pseudo-ring stabilized by a hydrogen bond between the amino group of Cys1 and the carboxylate group of Met4 and a C11 pseudo-ring involving the residues Cys1 and Tic3. In addition, the thiol group of Cys1 side chain of the bioactive conformation points to the carboxylate moiety of Met4.     

Folate biosynthesis in higher plants: purification and molecular cloning of a bifunctional 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase/7,8-dihydropteroate synthase localized in mitochondria.

In pea leaves, the synthesis of 7,8-dihydropteroate, a primary step in folate synthesis, was only detected in mitochondria. This reaction is catalyzed by a bifunctional 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase/7,8-dihydropteroate synthase enzyme, which represented 0.04-0.06% of the matrix proteins. The enzyme had a native mol. wt of 280-300 kDa and was made up of identical subunits of 53 kDa. The reaction catalyzed by the 7,8-dihydropteroate synthase domain of the protein was Mg2+-dependent and behaved like a random bireactant system. The related cDNA contained an open reading frame of 1545 bp and the deduced amino acid sequence corresponded to a polypeptide of 515 residues with a calculated M(r) of 56,454 Da. Comparison of the deduced amino acid sequence with the N-terminal sequence of the purified protein indicated that the plant enzyme is synthesized with a putative mitochondrial transit peptide of 28 amino acids. The calculated M(r) of the mature protein was 53,450 Da. Southern blot experiments suggested that a single-copy gene codes for the enzyme. This result, together with the facts that the protein is synthesized with a mitochondrial transit peptide and that the activity was only detected in mitochondria, strongly supports the view that mitochondria is the major (unique?) site of 7,8-dihydropteroate synthesis in higher plant cells.     

Identification and partial purification of a thiol endothelin-converting enzyme from porcine aortic endothelial cells.

Endothelin is a potent peptide vasoconstrictor. The final step in the processing of endothelin has been postulated to be the cleavage of the Trp21-Val22 peptide bond in proendothelin by a putative endothelin-converting enzyme. A soluble extract of primary porcine aortic endothelial cells was found to contain an enzyme activity that converted proendothelin-1 (proET-1) to an endothelin-1 (ET-1)-like peptide as determined by the rabbit aortic ring contraction assay. This enzyme was partially purified by DE52 ion-exchange chromatography. Incubation of proET-1 with the partially purified enzyme generated a product which had a retention time on HPLC identical to that of authentic ET-1. Further analysis of the product showed that it caused contraction of rabbit aortic rings, had a molecular weight identical to ET-1 as measured by fast atom bombardment mass spectrometry, and competed for [125I]ET-1 binding in an RIA using specific antibodies which recognize the carboxy terminal tryptophan of ET-1. The enzyme activity could be inhibited by thiol protease inhibitors such as Z-phe-pheCHN2 and p-hydroxymercuribenzoate, but not by serine- or metalloprotease inhibitors. The optimal pH for the enzymatic activity was between 7.0 and 7.5, and no activity was detected at pH 4.0. These results demonstrate that this thiol protease is a potential endothelin-converting enzyme.     

Recombinant production and purification of novel antisense antimicrobial peptide in Escherichia coli

A fusion protein was genetically engineered that contains an antimicrobial peptide, designated P2, at its carboxy terminus and bovine prochymosin at its amino terminus. Bovine prochymosin was chosen as the fusion partner because of its complete insolubility in Escherichia coli, a property utilized to protect the cells from the toxic effects of the antimicrobial peptide. This fusion protein was purified by centrifugation as an insoluble inclusion body. A methionine linker between prochymosin and the P2 peptide enabled P2 to be released by digestion with cyanogen bromide. Cation exchange HPLC followed by reversed-phase HPLC were used to purify the P2 peptide. The recombinant P2 peptide's molecular mass was confirmed by mass spectrometry to within 0.1% of the theoretical value (2480.9 Da), and the antimicrobial activity of the purified recombinant P2 against E. coli D31 was determined to be identical to that of the chemically synthesized peptide (minimal inhibitory concentration of 5 mg/mL). Although the yield of the fusion protein after expression by the cells was high (16% of the total cell protein), the percentage recovery of the P2 peptide in the inclusion bodies was relatively low, which appears to be due to losses in the cyanogen bromide digestion step.     

Effect of inositol starvation on the in vitro syntheses of mannan and N-acetylglucosaminylpyrophosphoryldolichol in Saccharomyces cerevisiae.

An early consequence of starvation for inositol in yeast is inhibition of synthesis of the major cell wall components mannan and glucan. In looking for the mechanism of this inhibition, we found that the activity of the enzyme catalyzing the synthesis of N-acetylglucosaminylpyrophosphoryldolichol was diminished in particular membrane preparations from cells starved for inositol. This loss of reactivity was observed under a variety of in vitro assay conditions and could be restored by the addition of phosphatidylinositol but not by other phosphoinositol-containing sphingolipids known to occur in yeast. When assayed in the presence of high concentrations of Triton X-100, enzyme preparations from both control and inositol-starved cells required phosphatidylinositol for maximal activity. Since this enzyme catalyzed an early step in the synthesis of mannan that is N-linked to protein, a reasonable hypothesis is that inhibition of mannan synthesis in inositol-starved cells results from the depletion of the necessary cofactor phosphatidylinositol.     

Unraveling Entropic Rate Acceleration Induced by Solvent Dynamics in Membrane Enzymes

Enzyme catalysis evolved in an aqueous environment. The influence of solvent dynamics on catalysis is, however, currently poorly understood and usually neglected. The study of water dynamics in enzymes and the associated thermodynamical consequences is highly complex and has involved computer simulations, nuclear magnetic resonance (NMR) experiments, and calorimetry. Water tunnels that connect the active site with the surrounding solvent are key to solvent displacement and dynamics. The protocol herein allows for the engineering of these motifs for water transport, which affects specificity, activity and thermodynamics. By providing a biophysical framework founded on theory and experiments, the method presented herein can be used by researchers without previous expertise in computer modeling or biophysical chemistry. The method will advance our understanding of enzyme catalysis on the molecular level by measuring the enthalpic and entropic changes associated with catalysis by enzyme variants with obstructed water tunnels. The protocol can be used for the study of membrane-bound enzymes and other complex systems. This will enhance our understanding of the importance of solvent reorganization in catalysis as well as provide new catalytic strategies in protein design and engineering.     

Biosynthesis of epsilon-poly-L-lysine in a cell-free system of Streptomyces albulus

epsilon-Poly-L-lysine (epsilon-PL) is a homo-poly-amino acid characterized by a peptide bond between carboxyl and epsilon-amino groups of L-lysine. Here we report the cell-free synthesis of epsilon-PL by a sensitive radioisotopic epsilon-PL assay system. In vitro epsilon-PL synthesis depended on ATP and was not affected by ribonuclease, kanamycin, or chloramphenicol. epsilon-PL synthesizing activity was detected in the membrane fraction. The reaction product, epsilon-PL, from L-lysine was identified by MALDI-TOF MS and the number of lysine residues of the epsilon-PL products was apparently 11-34. These results suggest that the biosynthesis of epsilon-PL is nonribosomal peptide synthesis and is catalyzed by membrane bound enzyme(s). The enzyme preparation showing the epsilon-PL synthesizing activity also catalyzed lysine-dependent AMP production and an ATP-PPi exchange reaction, suggesting that L-lysine is adenylated in the first step of epsilon-PL biosynthesis.