The yeast N(alpha)-acetyltransferase NatA is quantitatively anchored to the ribosome and interacts with nascent polypeptides

The majority of cytosolic proteins in eukaryotes contain a covalently linked acetyl moiety at their very N terminus. The mechanism by which the acetyl moiety is efficiently transferred to a large variety of nascent polypeptides is currently only poorly understood. Yeast N(alpha)-acetyltransferase NatA, consisting of the known subunits Nat1p and the catalytically active Ard1p, recognizes a wide range of sequences and is thought to act cotranslationally. We found that NatA was quantitatively bound to ribosomes via Nat1p and contained a previously unrecognized third subunit, the N(alpha)-acetyltransferase homologue Nat5p. Nat1p not only anchored Ard1p and Nat5p to the ribosome but also was in close proximity to nascent polypeptides, independent of whether they were substrates for N(alpha)-acetylation or not. Besides Nat1p, NAC (nascent polypeptide-associated complex) and the Hsp70 homologue Ssb1/2p interact with a variety of nascent polypeptides on the yeast ribosome. A direct comparison revealed that Nat1p required longer nascent polypeptides for interaction than NAC and Ssb1/2p. Delta nat1 or Delta ard1 deletion strains were temperature sensitive and showed derepression of silent mating type loci while Delta nat5 did not display any obvious phenotype. Temperature sensitivity and derepression of silent mating type loci caused by Delta nat1 or Delta ard1 were partially suppressed by overexpression of SSB1. The combination of data suggests that Nat1p presents the N termini of nascent polypeptides for acetylation and might serve additional roles during protein synthesis.     

Identification of the human N(alpha)-acetyltransferase complex B (hNatB): a complex important for cell-cycle progression

Protein N(alpha)-terminal acetylation is a conserved and widespread protein modification in eukaryotes. Several studies have linked it to normal cell function and cancer development, but nevertheless, little is known about its biological function. In yeast, protein N(alpha)-terminal acetylation is performed by the N-acetyltransferase complexes NatA, NatB and NatC. In humans, only the NatA complex has been identified and characterized. In the present study we present the components of hNatB (human NatB complex). It consists of the Nat3p homologue hNAT3 (human N-acetyltransferase 3) and the Mdm20p homologue hMDM20 (human mitochondrial distribution and morphology 20). They form a stable complex and in vitro display sequence-specific N(alpha)-acetyltransferase activity on a peptide with the N-terminus Met-Asp-. hNAT3 and hMDM20 co-sediment with ribosomal pellets, thus supporting a model where hNatB acts co-translationally on nascent polypeptides. Specific knockdown of hNAT3 and hMDM20 disrupts normal cell-cycle progression, and induces growth inhibition in HeLa cells and the thyroid cancer cell line CAL-62. hNAT3 knockdown results in an increase in G(0)/G(1)-phase cells, whereas hMDM20 knockdown decreased the fraction of cells in G(0)/G(1)-phase and increased the fraction of cells in the sub-G(0)/G(1)-phase. In summary, we show for the first time a vertebrate NatB protein N(alpha)-acetyltransferase complex essential for normal cell proliferation.     

Cysteine synthase (O-acetylserine (thiol) lyase) substrate specificities classify the mitochondrial isoform as a cyanoalanine synthase

A cyanoalanine synthase and two isoforms (A, cytosolic and B, chloroplastic) of cysteine synthase (O:-acetylserine (thiol) lyase) were isolated from spinach. N-terminal amino acid sequence analysis of the cyanoalanine synthase gave 100% homology for the determined 12 residues with a published sequence for the mitochondrial cysteine synthase isoform. All three enzymes catalysed both the cysteine synthesis and cyanoalanine synthesis reactions, although with different efficiencies. Michaelis-Menten kinetics were observed for all three enzymes when substrate saturation experiments were performed varying O:-acetylserine, chloroalanine and cysteine. Negative co-operative kinetics were observed for cysteine synthases A and B when substrate saturation experiments were performed varying sulphide and cyanide, compared with the Michaelis-Menten kinetics observed for cyanoalanine synthase. The exception was negative co-operativity observed towards sulphide for cyanoalanine synthase with O:-acetylserine as co-substrate. The optimum sulphide concentration was dependent on the alanyl co-substrate used. The amino acid sequence similarity places these three enzymes in the same gene family, and whilst the close kinetic similarities support this, they also indicate distinct roles for the isoforms.     

Method for determining protein kinase substrate specificities by the phosphorylation of peptide libraries on beads, phosphate-specific staining, automated sorting, and sequencing

A method is described for the elucidation of the peptide substrate phosphorylation specificity of a protein kinase. Peptide libraries with two to six degenerate positions and a length of seven or nine amino acids were generated directly on Sepharose beads by solid-phase peptide synthesis according to the split-and-mix procedure. The immobilized peptides were incubated with the catalytic subunit of the cyclic AMP-dependent protein kinase (PKA) as a model enzyme resulting in the phosphorylation of the beads that contain the recognition motif of the kinase. The beads were then stained with a new phosphate-monoester-specific fluorescent dye consisting of a complex of iron(III) with fluorescein-coupled iminodiacetic acid. A flow cytometer was used to analyze the phosphorylation efficiency and the beads with the highest phosphorylation degree were isolated by the use of a fluorescence-activated cell sorter. Pool sequencing of those beads revealed the preferred kinase motif. The results are in good agreement with data from the literature. The method lends itself to the rapid elucidation of the specificity of uncharacterized protein kinases.     

Altered spermidine/spermine N1-acetyltransferase activity as a mechanism of cellular resistance to bis(ethyl)polyamine analogues

To develop a model system to investigate mechanisms of antiproliferative action of bis(ethyl)polyamine analogues, intermittent analogue treatments followed by recovery periods in drug-free medium were used to select an N(1), N(12)-bis(ethyl)spermine-resistant derivative of the Chinese hamster ovary cell line C55.7. The resulting C55.7Res line was at least 10-fold resistant to N(1),N(12)-bis(ethyl)spermine and N(1), N(11)-bis(ethyl)norspermine. The stability of the resistance in the absence of selection pressure was >/=9 months, indicating that a heritable genotypic change was responsible for the resistance phenotype. Polyamine transport alterations and multi-drug resistance were eliminated as causes of the resistance. Spermidine/spermine N(1)-acetyltransferase (SSAT) activity and regulation were altered in C55.7Res cells as basal activity was decreased, and no activity induction resulted from exposure to analogue concentrations, which caused 300-fold enzyme induction in parental cells. SSAT mRNA levels in the absence and presence of analogue were unchanged, but no SSAT protein was detected in C55.7Res cells. A point mutation, which results in the change leucine156 (a fully conserved residue) to phenylalanine, was identified in the C55.7Res SSAT cDNA. Expression of wtSSAT activity in C55.7Res cells restored sensitivity to bis(ethyl)polyamines. These results provided definitive evidence that SSAT activity is a critical target of the cytotoxic action of these analogues.     

Characterization of the human nicotinic acetylcholine receptor subunit alpha (alpha) 9 (CHRNA9) and alpha (alpha) 10 (CHRNA10) in lymphocytes

Though the nicotinic acetylcholine receptor (nAChR) subunits alpha9 and alpha 10 have been thoroughly characterized within hair cells of the organ of Corti in the inner ear, prior studies have shown that they are also expressed in lymphocytes. In this report, we sought to more definitively characterize the nAChR subunits alpha9 and alpha10 within various populations of human lymphocytes. Using a combination of techniques, including RT-PCR, single-cell RT-PCR, Northern and western blot analysis, and immunofluorescence, expression of both alpha9 and alpha 10 was demonstrated in purified populations of T-cells (CD3+, CD4+, CD8+ and the Jurkat, MT2 and CEM T-cell lines) and B-cells (CD19+, CD80+ and EBV-immortalized B-cells). Single-lymphocyte recording techniques failed to identify an ionic current in response to applied acetylcholine in either T-cells or B-cells. These results clearly demonstrate the presence of these nicotinic receptor subunits within several populations of human lymphocytes, implicating their role in the immune response. However, a lack of demonstrated response to applied acetylcholine using standard single-cell recording techniques suggests a physiology different than that seen in hair cells of the inner ear.     

Quantitative analysis of formyl peptide receptor coupling to g(i)alpha(1), g(i)alpha(2), and g(i)alpha(3)

The human formyl peptide receptor (FPR) is a prototypical G(i) protein-coupled receptor, but little is known about quantitative aspects of FPR-G(i) protein coupling. To address this issue, we fused the FPR to G(i)alpha(1), G(i)alpha(2), and G(i)alpha(3) and expressed the fusion proteins in Sf9 insect cells. Fusion of a receptor to Galpha ensures a defined 1:1 stoichiometry of the signaling partners. By analyzing high affinity agonist binding, the kinetics of agonist- and inverse agonist-regulated guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) binding and GTP hydrolysis and photolabeling of Galpha, we demonstrate highly efficient coupling of the FPR to fused G(i)alpha(1), G(i)alpha(2), and G(i)alpha(3) without cross-talk of the receptor to insect cell G proteins. The FPR displayed high constitutive activity when coupled to all three G(i)alpha isoforms. The K(d) values of high affinity agonist binding were approximately 100-fold lower than the EC(50) (concentration that gives half-maximal stimulation) values of agonist for GTPase activation. Based on the B(max) values of agonist saturation binding and ligand-regulated GTPgammaS binding, it was previously proposed that the FPR activates G proteins catalytically, i.e. one FPR activates several G(i) proteins. Analysis of agonist saturation binding, ligand-regulated GTPgammaS saturation binding and quantitative immunoblotting with membranes expressing FPR-G(i)alpha fusion proteins and nonfused FPR now reveals that FPR agonist binding greatly underestimates the actual FPR expression level. Our data show the following: (i) the FPR couples to G(i)alpha(1), G(i)alpha(2), and G(i)alpha(3) with similar efficiency; (ii) the FPR can exist in a state of low agonist affinity that couples efficiently to G proteins; and (iii) in contrast to the previously held view, the FPR appears to activate G(i) proteins linearly and not catalytically.     

L-plastin peptide activation of alpha(v)beta(3)-mediated adhesion requires integrin conformational change and actin filament disassembly

L-plastin (LPL) is a leukocyte actin binding protein previously implicated in the activation of the integrin alpha(M)beta(2) on polymorphonuclear neutrophils. To determine the role for LPL in integrin activation, K562 cell adhesion to vitronectin via alpha(v)beta(3), a well-studied model for activable integrins, was examined. Cell permeant versions of peptides based on the N-terminal sequence of LPL and the LPL headpiece domain both activated alpha(v)beta(3)-mediated adhesion. In contrast to adhesion induced by treatment with phorbol 12-myristate 13-acetate (PMA), LPL peptide-activated adhesion was independent of integrin beta(3) cytoplasmic domain tyrosines and was not inhibited by cytochalasin D. Also in contrast to PMA, LPL peptides synergized with RGD ligand or Mn(2+) for generation of a conformational change in alpha(v)beta(3) associated with the high affinity state of the integrin, as determined by binding of a ligand-induced binding site antibody. Although LPL and ligand showed synergy for ligand-induced binding site expression when actin depolymerization was inhibited by jasplakinolide, LPL peptide-induced adhesion was inhibited. Thus, both actin depolymerization and ligand-induced integrin conformational change are required for LPL peptide-induced adhesion. We hypothesize that the critical steps of increased integrin diffusion and affinity enhancement may be linked via modulation of the function of the actin binding protein L-plastin.     

Purification and characterization of EpiA, the peptide substrate for post-translational modifications involved in epidermin biosynthesis

Abstract For the investigation of enzymes involved in epidermin biosynthesis it is necessary to produce sufficient amounts of preepidermin (EpiA) as a substrate and to design EpiA detection systems. Therefore, EpiA was expressed in Escherichia coli using a malE-epiA fusion. The identity of purified EpiA was confirmed by ion spray mass spectrometry and amino acid sequencing. For EpiA detection, anti-EpiA antisera were raised. Upon prolonged incubation, factor Xa not only cleaved EpiA from the fusion protein, but also less efficiently cleaved EpiA internally between R⁻¹ and I⁺¹. The internal factor Xa cleavage site of EpiA was masked by altering the sequence -A⁻⁴-E-P-R⁻¹- to -A⁻⁴-E-P-Q⁻¹- by site-directed mutagenesis.     

Mutational analysis of protein substrate presentation in the post-translational attachment of biotin to biotin domains

Biotinylation in vivo is an extremely selective post-translational event where the enzyme biotin protein ligase (BPL) catalyzes the covalent attachment of biotin to one specific and conserved lysine residue of biotin-dependent enzymes. The biotin-accepting lysine, present in a conserved Met-Lys-Met motif, resides in a structured domain that functions as the BPL substrate. We have employed phage display coupled with a genetic selection to identify determinants of the biotin domain (yPC-104) of yeast pyruvate carboxylase 1 (residues 1075-1178) required for interaction with BPL. Mutants isolated using this strategy were analyzed by in vivo biotinylation assays performed at both 30 degrees C and 37 degrees C. The temperature-sensitive substrates were reasoned to have structural mutations, leading to compromised conformations at the higher temperature. This interpretation was supplemented by molecular modeling of yPC-104, since these mutants mapped to residues involved in defining the structure of the biotin domain. In contrast, substitution of the Met residue N-terminal to the target lysine with either Val or Thr produced mutations that were temperature-insensitive in the in vivo assay. Furthermore, these two mutant proteins and wild-type yPC-104 showed identical susceptibility to trypsin, consistent with these substitutions having no structural effect. Kinetic analysis of enzymatic biotinylation using purified Met --> Thr/Val mutant proteins with both yeast and Escherichia coli BPLs revealed that these substitutions had a strong effect upon K(m) values but not k(cat). The Met --> Thr mutant was a poor substrate for both BPLs, whereas the Met --> Val substitution was a poor substrate for bacterial BPL but had only a 2-fold lower affinity for yeast BPL than the wild-type peptide. Our data suggest that substitution of Thr or Val for the Met N-terminal of the biotinyl-Lys results in mutants specifically compromised in their interaction with BPL.     

A post-translational modification of nuclear proteins, N(G),N(G)-dimethyl-Arg, found in a natural HLA class I peptide ligand

Presentation of peptides derived from endogenous proteins by class I major histocompatibility complex molecules is essential both for immunological self-tolerance and induction of cytotoxic T-cell responses against intracellular parasites. Despite frequent and diverse post-translational modification of eukaryotic cell proteins, very few class I-bound peptides with post-translationally modified residues are known. Here we describe a natural dodecamer ligand of HLA-B39 (B*3910) derived from an RNA-binding nucleoprotein that carried N(G),N(G)-dimethyl-Arg. Although common among RNA-binding proteins, this modification was not previously known among natural class I ligands. The sequence of this peptide was determined by Edman degradation and electrospray ion trap mass spectrometry. The fragmentation pattern of the dimethyl-Arg side chain observed with this latter technique allowed us to unambiguously assign the isomeric form of the modified residue. The post-translationally modified ligand was a prominent component (1-2%) of the B*3910-bound peptide repertoire. The dimethyl-Arg residue was located in a central position of the peptide, amenable to interacting with T-cell receptors, and most other residues in the middle region of the peptide were Gly. These structural features strongly suggest that the post-translationally modified residue may have a major influence on the antigenic properties of this natural ligand.     

Synthesis of positional-scanning libraries of fluorogenic peptide substrates to define the extended substrate specificity of plasmin and thrombin

We have developed a strategy for the synthesis of positional-scanning synthetic combinatorial libraries (PS-SCL) that does not depend on the identity of the P1 substituent. To demonstrate the strategy, we synthesized a tetrapeptide positional library in which the P1 amino acid is held constant as a lysine and the P4-P3-P2 positions are positionally randomized. The 6,859 members of the library were synthesized on solid support with an alkane sulfonamide linker, and then displaced from the solid support by condensation with a fluorogenic 7-amino-4-methylcoumarin-derivatized lysine. This library was used to determine the extended substrate specificities of two trypsin-like enzymes, plasmin and thrombin, which are involved in the blood coagulation pathway. The optimal P4 to P2 substrate specificity for plasmin was P4-Lys/Nle (norleucine)/Val/Ile/Phe, P3-Xaa, and P2-Tyr/Phe/Trp. This cleavage sequence has recently been identified in some of plasmin's physiological substrates. The optimal P4 to P2 extended substrate sequence determined for thrombin was P4-Nle/Leu/Ile/Phe/Val, P3-Xaa, and P2-Pro, a sequence found in many of the physiological substrates of thrombin. Single-substrate kinetic analysis of plasmin and thrombin was used to validate the substrate preferences resulting from the PS-SCL. By three-dimensional structural modeling of the substrates into the active sites of plasmin and thrombin, we identified potential determinants of the defined substrate specificity. This method is amenable to the incorporation of diverse substituents at the P1 position for exploring molecular recognition elements in proteolytic enzymes.     

Calcium-dependent cell-cell adhesion molecules (cadherins): subclass specificities and possible involvement of actin bundles

Cadherins are a family of cell-cell adhesion molecules and are divided into subclasses with distinct adhesive specificities and tissue distribution. Here we examined the distribution of cadherins at contact sites between cells expressing the same or different cadherin subclasses. Each cadherin was concentrated at the boundary between cells expressing an identical cadherin subclass, irrespective of the cell types connected. However, such localization decreased or disappeared at the boundary between cells containing different cadherin subclasses. We also found that the localization of cadherins precisely coincided with that of actin bundles; both were detected at the apical region of cell sheets. This co-localization was retained even after cells were either treated with cytochalasin D or extracted with the detergent NP40. These results suggest that each cadherin subclass preferentially interacts with its own molecular type at intercellular boundaries, and that cadherin molecules may be associated with actin- based cytoskeletal elements.     

Photoaffinity labeling of Ras converting enzyme 1 (Rce1p) using a benzophenone-containing peptide substrate

Isoprenylation is a post-translational modification that increases protein hydrophobicity and helps target certain proteins to membranes. Ras converting enzyme 1 (Rce1p) is an endoprotease that catalyzes the removal of a three residue fragment from the C-terminus of isoprenylated proteins. To obtain structural information about this membrane protein, photoaffinity labeling agents are being prepared and employed. Here, we describe the synthesis of a benzophenone-containing peptide substrate analogue for Rce1p. Using a continuous spectrofluorometric assay, this peptide was shown to be a substrate for Rce1p. Mass spectrometry was performed to confirm the site of cleavage and structure of the processed probe. Photolysis of the biotinylated compound in the presence of membranes containing Rce1p followed by streptavidin pull-down and Western blot analysis indicated that Rce1p had been labeled by the probe. Photolysis in the presence of both the biotinylated, benzophenone-containing probe and a farnesylated peptide competitor reduced the extent of labeling, suggesting that labeling is occurring in the active site.     

High resolution analysis of snake venom metalloproteinase (SVMP) peptide bond cleavage specificity using proteome based peptide libraries and mass spectrometry

Both serine and metalloproteinases have been shown to play the role of toxins in the venoms of many snakes. Determination of the natural protein substrates of these toxins is an important feature in the toxinological characterization of these proteinases. Furthermore, characterization of their peptide bond specificity is of value for understanding active site preference of the proteinase associated with effective proteolysis as well as of use in the design of peptide substrates and inhibitor lead compounds. Typically the determination of peptide bond cleavage specificity of snake venom serine proteinases (SVSPs) and snake venom metalloproteinases (SVMPs) has been performed using limited sets of peptides or small oligopeptides as experimental substrates. Although this approach has yielded valuable data it is generally limited in scope due to the relatively small sets of substrates used to generate the consensus specificity sequences for these proteinases. In this study we use a large, plasma based, proteome-derived peptide library as substrates along with mass spectrometry to explore the peptide bond specificity of three PI SVMPs and one PIII SVMP to determine their individual peptide cleavage consensus sequences. All of the proteinases assayed displayed a clear preference for a leucine residue in the P1' site. Careful analysis of the specificity profiles of the SVMPs examined showed interesting differences in the preferences at the other P and P' sites suggesting functional differences between these proteinases. The PI SVMPs, leucurolysin-a, atrolysin C, and BaP1, showed preferences across the full P4 to P4' range whereas the PIII SVMP bothropasin showed a narrower range of preferences across the sites. In silico docking experiments with the experimentally derived consensus sequences as well as with comparison of the results to those in the literature regarding peptide bond specificity based on both peptide and protein substrates give rise to a fresh understanding of the specificity of these SVMPS and may serve as a foundation for future experiments to better elucidate their mechanism of action in the complex pathophysiology of snakebite envenomation.     

Soluble peptidase isozymes of the Japanese medaka (Oryzias latipes): Tissue distributions and substrate specificities

Peptidases catalyze the hydrolysis of di- and tripeptidases to their constituent amino acids. Five isozymes (PEP A, B, C, D, and S) were shown to be the products of independent genetic loci by several criteria including distinct adult tissue and substrate specificities, non-cross-reacting immunochemical properties, and independent genetic variation at three of the loci. Four of the peptidases had at least one substrate against which they contributed over 95% of the activity. These substrates were used for isozyme-specific assays. In adult tissues, three of the peptidases had higher activities in liver and intestine than in other tissues (PEP A, B, and S). PEP C had a 10-fold higher specific activity in brain than in other tissues.     

Extracellular matrix-derived peptide binds to alpha(v)beta(3) integrin and inhibits angiogenesis

Angiogenesis is associated with several pathological disorders as well as with normal physiological maintenance. Components of vascular basement membrane are speculated to regulate angiogenesis in both positive and negative manner. Recently, we reported that tumstatin (the NC1 domain of alpha 3 chain of type IV collagen) and its deletion mutant tum-5 possess anti-angiogenic activity. In the present study, we confirm that the anti-angiogenic activity of tumstatin and tum-5 is independent of disulfide bond requirement. This property of tum-5 allowed us to use overlapping synthetic peptide strategy to identify peptide sequence(s) which possess anti-angiogenic activity. Among these peptides, only the T3 peptide (69-88 amino acids) and T7 peptide (74-98 amino acids) inhibited proliferation and induced apoptosis specifically in endothelial cells. The peptides, similar to tumstatin and the tum-5 domain, bind and function via alpha(v)beta(3) in an RGD-independent manner. Restoration of a disulfide bond between two cysteines within the peptide did not alter the anti-angiogenic activity. Additionally, these studies show that tumstatin peptides can inhibit proliferation of endothelial cells in the presence of vitronectin, fibronectin, and collagen I. Anti-angiogenic effect of the peptides was further confirmed in vivo using a Matrigel plug assay in C57BL/6 mice. Collectively, these experiments suggest that the anti-angiogenic activity of tumstatin is localized to a 25-amino acid region of tumstatin and it is independent of disulfide bond linkage. Structural features and potency of the tumstatin peptide make it highly feasible as a potential anti-cancer drug.     

Phe(208) and Ile(199) in human monoamine oxidase A and B do not determine substrate and inhibitor specificities as in rat

It has been reported previously that reciprocally switching Phe(208) and Ile(199) in rat monoamine oxidase (MAO) A and B, respectively, was sufficient to switch their substrate and inhibitor preferences. In this study, the same mutants were made in the human forms of MAO. When compared with MAO A, MAO A-F208I showed a sixfold decrease in the specificity constant k(cat)/K(m) for both the MAO A- and the MAO B-preferring substrates 5-hydroxytryptamine and beta-phenylethylamine, respectively. The reciprocal point mutant MAO B-I199F had no effect on substrate affinity. To investigate if the region neighboring these two residues is responsible for conferring preferences, we have also made chimeric constructs by reciprocally switching the corresponding amino acid segments 159-214 in MAO A and 150-205 in MAO B. Chimerics MAO AB(159-214)A and MAO BA(150-205)B had small changes in K(m) and IC(50) values when compared with MAO A and B, respectively, but did not exhibit a preference switch. The results suggest that Phe(208) in MAO A and amino acid segments 159-214 and 150-205 in MAO A and B, respectively, influence the enzyme active site. However, substrate and inhibitor preferences of human MAO A and B are not determined by the respective residues Phe(108) and Ile(199) as in rat MAO nor by their neighboring regions.