Aromatic amino acid transamination and methionine recycling in trypanosomatids.

Although trypanosomatids are known to rapidly transaminate exogenous aromatic amino acids in vitro and in vivo, the physiological significance of this reaction is not understood. In postmitochondrial supernatants prepared from Trypanosoma brucei brucei and Crithidia fasciculata, we have found that aromatic amino acids were the preferred amino donors for the transamination of alpha-ketomethiobutyrate to methionine. Intact C. fasciculata grown in the presence of [15N]tyrosine were found to contain detectable [15N]methionine, demonstrating that this reaction occurs in situ in viable cells. This process is the final step in the recycling of methionine from methylthioadenosine, a product of decarboxylated S-adenosylmethionine from the polyamine synthetic pathway. Mammalian liver, in contrast, preferentially used glutamine for this reaction and utilized a narrower range of amino donors than seen with the trypanosomatids. Studies with methylthioadenosine showed that this compound was readily converted to methionine, demonstrating a fully functional methionine-recycling pathway in trypanosomatids.

Inducible nitric oxide synthase: identification of amino acid residues essential for dimerization and binding of tetrahydrobiopterin.

Nitric oxide synthases (NOSs) require tetrahydrobiopterin (BH4) for dimerization and NO production. Mutation analysis of mouse inducible NOS (iNOS; NOS2) identified Gly-450 and Ala-453 as critical for NO production, dimer formation, and BH4 binding. Substitutions at five neighboring positions were tolerated, and normal binding of heme, calmodulin, and NADPH militated against major distortions affecting the NH2-terminal portion, midzone, or COOH terminus of the inactive mutants. Direct involvement of residues 450 and 453 in the binding of BH4 is supported by the striking homology of residues 448-480 to a region extensively shared by the three BH4-utilizing aromatic amino acid hydroxylases and is consistent with the conservation of these residues among all 10 reported NOS sequences, including mammalian NOSs 1, 2, and 3, as well as avian and insect NOSs. Altered binding of BH4 and/or L-arginine may explain how the addition of a single methyl group to the side chain of residue 450 or the addition of three methylenes to residue 453 can each abolish an enzymatic activity that reflects the concerted function of 1143 other residues.

Aromatic-L-amino-acid decarboxylase, a pyridoxal phosphate-dependent enzyme, is a beta-cell autoantigen.

Different autoantigens are thought to be involved in the pathogenesis of insulin-dependent diabetes mellitus, and they may account for the variation in the clinical presentation of the disease. Sera from patients with autoimmune polyendocrine syndrome type I contain autoantibodies against the beta-cell proteins glutamate decarboxylase and an unrelated 51-kDa antigen. By screening of an expression library derived from rat insulinoma cells, we have identified the 51-kDa protein as aromatic-L-amino-acid decarboxylase (EC 4.1.1.28). In addition to the previously published full-length cDNA, forms coding for a truncated and an alternatively spliced version were identified. Aromatic L-amino acid decarboxylase catalyzes the decarboxylation of L-5-hydroxytryptophan to serotonin and that of L-3,4-dihydroxyphenylalanine to dopamine. Interestingly, pyridoxal phosphate is the cofactor of both aromatic L-amino acid decarboxylase and glutamate decarboxylase. The biological significance of the neurotransmitters produced by the two enzymes in the beta cells remains largely unknown.

Proper sorting of the cation-dependent mannose 6-phosphate receptor in endosomes depends on a pair of aromatic amino acids in its cytoplasmic tail

The 67-amino acid cytoplasmic tail of the cation-dependent mannose 6-phosphate receptor (CD-MPR) contains a signal(s) that prevents the receptor from entering lysosomes where it would be degraded. To identify the key residues required for proper endosomal sorting, we analyzed the intracellular distribution of mutant forms of the receptor by Percoll density gradients. A receptor with a Trp19 → Ala substitution in the cytoplasmic tail was highly missorted to lysosomes whereas receptors with either Phe18 → Ala or Phe13 → Ala mutations were partially defective in avoiding transport to lysosomes. Analysis of double and triple mutants confirmed the key role of Trp19 for sorting of the CD-MPR in endosomes, with Phe18, Phe13, and several neighboring residues contributing to this function. The addition of the Phe18-Trp19 motif of the CD-MPR to the cytoplasmic tail of the lysosomal membrane protein Lamp1 was sufficient to partially impair its delivery to lysosomes. Replacing Phe18 and Trp19 with other aromatic amino acids did not impair endosomal sorting of the CD-MPR, indicating that two aromatic residues located at these positions are sufficient to prevent the receptor from trafficking to lysosomes. However, alterations in the spacing of the diaromatic amino acid sequence relative to the transmembrane domain resulted in receptor accumulation in lysosomes. These findings indicate that the endosomal sorting of the CD-MPR depends on the correct presentation of a diaromatic amino acid-containing motif in its cytoplasmic tail. Because a diaromatic amino acid sequence is also present in the cytoplasmic tail of other receptors known to be internalized from the plasma membrane, this feature may prove to be a general determinant for endosomal sorting.

Mass spectrometric amino acid sequencing of a mixture of seed storage proteins (napin) from Brassica napus, products of a multigene family.

The amino acid sequences of a number of closely related proteins ("napin") isolated from Brassica napus were determined by mass spectrometry without prior separation into individual components. Some of these proteins correspond to those previously deduced (napA, BngNAP1, and gNa), chiefly from DNA sequences. Others were found to differ to a varying extent (BngNAP1', BngNAP1A, BngNAP1B, BngNAP1C, gNa', and gNaA). The short chains of gNa and gNa' and of BngNAP1 and BngNAP1' differ by the replacement of N-terminal proline by pyroglutamic acid; the long chains of gNaA and BngNAP1B contain a six amino acid stretch, MQGQQM, which is present in gNa (according to its DNA sequence) but absent from BngNAP1 and BngNAP1C. These alternations of sequences between napin isoforms are most likely due to homologous recombination of the genetic material, but some of the changes may also be due to RNA editing. The amino acids that follow the untruncated C termini of those napin chains for which the DNA sequences are known (napA, BngNAP1, and gNa) are aromatic amino acids. This suggests that the processing of the proprotein leading to the C termini of the two chains is due to the action of a protease that specifically cleaves a G/S-F/Y/W bond.

A chimeric light-regulated amino acid transport system allows the isolation of blue light regulator (blr) mutants of Neurospora crassa.

We have developed a system for the isolation of Neurospora crassa mutants that shows altered responses to blue light. To this end we have used the light-regulated promoter of the albino-3 gene fused to the neutral amino acid permease gene mtr. The product of the mtr gene is required for the uptake of neutral aliphatic and aromatic amino acids, as well as toxic analogs such as p-flurophenylalanine or 4-methyltryptophan. mtr trp-2-carrying cells were transformed with the al-3 promoter-mtr wild-type gene (al-3p-mtr+) to obtain a strain with a light-regulated tryptophan uptake. This strain is sensitive to p-fluorophenylalanine when grown under illumination and resistant when grown in the dark. UV mutagenesis of the al-3p-mtr(+)-carrying strain allowed us to isolate two mutant strains, BLR-1 and BLR-2 (blue light regulator), that are light-resistant to p-fluorophenylalanine and have lost the ability to grow on tryptophan. These two strains have a pale-orange phenotype and show down-regulation of all the photoregulated genes tested (al-3, al-1, con-8, and con-10). Mutations in the BLR strains are not allelic with white collar 1 or white collar 2, regulatory genes that are also involved in the response to blue light.

On the distribution of amino acid residues in transmembrane alpha-helix bundles.

The periodic distribution of residues in the sequence of 469 putative transmembrane alpha-helices from eukaryotic plasma membrane polytopic proteins has been analyzed with correlation matrices. The method does not involve any a priori assumption about the secondary structure of the segments or about the physicochemical properties of individual amino acid residues. Maximal correlation is observed at 3.6 residues per period, characteristic of alpha-helices. A scale extracted from the data describes the propensity of the various residues to lie on the same or on opposite helix faces. The most polar face of transmembrane helices, presumably that buried in the protein core, shows a strong enrichment in aromatic residues, while residues likely to face the fatty acyl chains of lipids are largely aliphatic.

A synthetic all d-amino acid peptide corresponding to the N-terminal sequence of HIV-1 gp41 recognizes the wild-type fusion peptide in the membrane and inhibits HIV-1 envelope glycoprotein-mediated cell fusion

Recent studies demonstrated that a synthetic fusion peptide of HIV-1 self-associates in phospholipid membranes and inhibits HIV-1 envelope glycoprotein-mediated cell fusion, presumably by interacting with the N-terminal domain of gp41 and forming inactive heteroaggregates [Kliger, Y., Aharoni, A., Rapaport, D., Jones, P., Blumenthal, R. & Shai, Y. (1997) J. Biol. Chem. 272, 13496–13505]. Here, we show that a synthetic all d-amino acid peptide corresponding to the N-terminal sequence of HIV-1 gp41 (D-WT) of HIV-1 associates with its enantiomeric wild-type fusion (WT) peptide in the membrane and inhibits cell fusion mediated by the HIV-1 envelope glycoprotein. D-WT does not inhibit cell fusion mediated by the HIV-2 envelope glycoprotein. WT and D-WT are equally potent in inducing membrane fusion. D-WT peptide but not WT peptide is resistant to proteolytic digestion. Structural analysis showed that the CD spectra of D-WT in trifluoroethanol/water is a mirror image of that of WT, and attenuated total reflectance–fourier transform infrared spectroscopy revealed similar structures and orientation for the two enantiomers in the membrane. The results reveal that the chirality of the synthetic peptide corresponding to the HIV-1 gp41 N-terminal sequence does not play a role in liposome fusion and that the peptides’ chirality is not necessarily required for peptide–peptide interaction within the membrane environment. Furthermore, studies along these lines may provide criteria to design protease-resistant therapeutic agents against HIV and other viruses.

Synthetic amino acid copolymers that bind to HLA-DR proteins and inhibit type II collagen-reactive T cell clones

Copolymer 1 [poly(Y,E,A,K)] is a random synthetic amino acid copolymer of l-tyrosine, l-glutamic acid, l-alanine, and l-lysine that is effective both in suppression of experimental allergic encephalomyelitis and in the treatment of relapsing forms of multiple sclerosis. Copolymer 1 binds promiscuously and very efficiently to purified HLA-DR molecules within the peptide-binding groove. In the present study, YEAK and YEAK-related copolymers and type II collagen (CII) peptide 261–273, a candidate autoantigen in rheumatoid arthritis (RA), competed for binding to RA-associated HLA-DR molecules encoded by DRB1*0101 and DRB1*0401. Moreover, these copolymers (particularly YEAK, YAK, and YEK) inhibited the response of DR1- and DR4-restricted T cell clones to the CII epitope 261–273 by >50%. This direct evidence both for competitive interactions of these copolymers and CII peptide with RA-associated HLA-DR molecules and for inhibition of CII-specific T cell responses suggests that these compounds should be evaluated in animal models for rheumatoid arthritis.

Isolation and characterization of an amino acid-selective channel protein present in the chloroplastic outer envelope membrane

The reconstituted pea chloroplastic outer envelope protein of 16 kDa (OEP16) forms a slightly cation-selective, high-conductance channel with a conductance of Λ = 1,2 nS (in 1 M KCl). The open probability of OEP16 channel is highest at 0 mV (Popen = 0.8), decreasing exponentially with higher potentials. Transport studies using reconstituted recombinant OEP16 protein show that the OEP16 channel is selective for amino acids but excludes triosephosphates or uncharged sugars. Crosslinking indicates that OEP16 forms a homodimer in the membrane. According to its primary sequence and predicted secondary structure, OEP16 shows neither sequence nor structural homologies to classical porins. The results indicate that the intermembrane space between the two envelope membranes might not be as freely accessible as previously thought.

The interaction of the general anesthetic etomidate with the γ-aminobutyric acid type A receptor is influenced by a single amino acid

The γ-aminobutyric acid type A (GABAA) receptor is a transmitter-gated ion channel mediating the majority of fast inhibitory synaptic transmission within the brain. The receptor is a pentameric assembly of subunits drawn from multiple classes (α1–6, β1–3, γ1–3, δ1, and ɛ1). Positive allosteric modulation of GABAA receptor activity by general anesthetics represents one logical mechanism for central nervous system depression. The ability of the intravenous general anesthetic etomidate to modulate and activate GABAA receptors is uniquely dependent upon the β subunit subtype present within the receptor. Receptors containing β2- or β3-, but not β1 subunits, are highly sensitive to the agent. Here, chimeric β1/β2 subunits coexpressed in Xenopus laevis oocytes with human α6 and γ2 subunits identified a region distal to the extracellular N-terminal domain as a determinant of the selectivity of etomidate. The mutation of an amino acid (Asn-289) present within the channel domain of the β3 subunit to Ser (the homologous residue in β1), strongly suppressed the GABA-modulatory and GABA-mimetic effects of etomidate. The replacement of the β1 subunit Ser-290 by Asn produced the converse effect. When applied intracellularly to mouse L(tk−) cells stably expressing the α6β3γ2 subunit combination, etomidate was inert. Hence, the effects of a clinically utilized general anesthetic upon a physiologically relevant target protein are dramatically influenced by a single amino acid. Together with the lack of effect of intracellular etomidate, the data argue against a unitary, lipid-based theory of anesthesia.

Thermus thermophilus: A link in evolution of the tRNA-dependent amino acid amidation pathways

Thermus thermophilus possesses an aspartyl-tRNA synthetase (AspRS2) able to aspartylate efficiently tRNAAsp and tRNAAsn. Aspartate mischarged on tRNAAsn then is converted into asparagine by an ω amidase that differs structurally from all known asparagine synthetases. However, aspartate is not misincorporated into proteins because the binding capacity of aminoacylated tRNAAsn to elongation factor Tu is only conferred by conversion of aspartate into asparagine. T. thermophilus additionally contains a second aspartyl-tRNA synthetase (AspRS1) able to aspartylate tRNAAsp and an asparaginyl-tRNA synthetase able to charge tRNAAsn with free asparagine, although the organism does not contain a tRNA-independent asparagine synthetase. In contrast to the duplicated pathway of tRNA asparaginylation, tRNA glutaminylation occurs in the thermophile via the usual pathway by using glutaminyl-tRNA synthetase and free glutamine synthesized by glutamine synthetase that is unique. T. thermophilus is able to ensure tRNA aminoacylation by alternative routes involving either the direct pathway or by conversion of amino acid mischarged on tRNA. These findings shed light on the interrelation between the tRNA-dependent and tRNA-independent pathways of amino acid amidation and on the processes involved in fidelity of the aminoacylation systems.

A single amino acid substitution converts a carboxylesterase to an organophosphorus hydrolase and confers insecticide resistance on a blowfly

Resistance to organophosphorus (OP) insecticides is associated with decreased carboxylesterase activity in several insect species. It has been proposed that the resistance may be the result of a mutation in a carboxylesterase that simultaneously reduces its carboxylesterase activity and confers an OP hydrolase activity (the “mutant ali-esterase hypothesis”). In the sheep blowfly, Lucilia cuprina, the association is due to a change in a specific esterase isozyme, E3, which, in resistant flies, has a null phenotype on gels stained using standard carboxylesterase substrates. Here we show that an OP-resistant allele of the gene that encodes E3 differs at five amino acid replacement sites from a previously described OP-susceptible allele. Knowledge of the structure of a related enzyme (acetylcholinesterase) suggests that one of these substitutions (Gly137 → Asp) lies within the active site of the enzyme. The occurrence of this substitution is completely correlated with resistance across 15 isogenic strains. In vitro expression of two natural and two synthetic chimeric alleles shows that the Asp137 substitution alone is responsible for both the loss of E3’s carboxylesterase activity and the acquisition of a novel OP hydrolase activity. Modeling of Asp137 in the homologous position in acetylcholinesterase suggests that Asp137 may act as a base to orientate a water molecule in the appropriate position for hydrolysis of the phosphorylated enzyme intermediate.

Evidence that the 42- and 40-amino acid forms of amyloid β protein are generated from the β-amyloid precursor protein by different protease activities

Cerebral deposition of the amyloid β protein (Aβ) is an early and invariant feature of Alzheimer disease (AD). Whereas the 40-amino acid form of Aβ (Aβ40) accounts for ≈90% of all Aβ normally released from cells, it appears to contribute only to later phases of the pathology. In contrast, the longer more amyloidogenic 42-residue form (Aβ42), accounting for only ≈10% of secreted Aβ, is deposited in the earliest phase of AD and remains the major constituent of most amyloid plaques throughout the disease. Moreover, its levels have been shown to be increased in all known forms of early-onset familial AD. Thus, inhibition of Aβ42 production is a prime therapeutic goal. The same protease, γ-secretase, is assumed to generate the C termini of both Aβ40 and Aβ42. Herein, we analyze the effect of the compound MDL 28170, previously suggested to inhibit γ-secretase, on β-amyloid precursor protein processing. By immunoprecipitating conditioned medium of different cell lines with various Aβ40- and Aβ42-specific antibodies, we demonstrate a much stronger inhibition of the γ-secretase cleavage at residue 40 than of that at residue 42. These data suggest that different proteases generate the Aβ40 and Aβ42 C termini. Further, they raise the possibility of identifying compounds that do not interfere with general β-amyloid precursor protein metabolism, including Aβ40 production, but specifically block the generation of the pathogenic Aβ42 peptide.

Inorganic polyphosphate kinase is required to stimulate protein degradation and for adaptation to amino acid starvation in Escherichia coli

Inorganic polyphosphate (polyP) kinase was studied for its roles in physiological responses to nutritional deprivation in Escherichia coli. A mutant lacking polyP kinase exhibited an extended lag phase of growth, when shifted from a rich to a minimal medium (nutritional downshift). Supplementation of amino acids to the minimal medium abolished the extended growth lag of the mutant. Levels of the stringent response factor, guanosine 5′-diphosphate 3′-diphosphate, increased in response to the nutritional downshift, but, unlike in the wild type, the levels were sustained in the mutant. These results suggested that the mutant was impaired in the induction of amino acid biosynthetic enzymes. The expression of an amino acid biosynthetic gene, hisG, was examined by using a transcriptional lacZ fusion. Although the mutant did not express the fusion in response to the nutritional downshift, Northern blot analysis revealed a significant increase of hisG-lacZ mRNA. Amino acids generated by intracellular protein degradation are very important for the synthesis of enzymes at the onset of starvation. In the wild type, the rate of protein degradation increased in response to the nutritional downshift whereas it did not in the mutant. Supplementation of amino acids at low concentrations to the minimal medium enabled the mutant to express the hisG-lacZ fusion. Thus, the impaired regulation of protein degradation results in the adaptation defect, suggesting that polyP kinase is required to stimulate protein degradation.