A mode of assembly of P0, P1, and P2 proteins at the GTPase-associated center in animal ribosome: in vitro analyses with P0 truncation mutants

Ribosomal P0, P1, and P2 proteins, together with the conserved domain of 28 S rRNA, constitute a major part of the GTPase-associated center in eukaryotic ribosomes. We investigated the mode of assembly in vitro by using various truncation mutants of silkworm P0. When compared with wild type (WT)-P0, the C-terminal truncation mutants CDelta65 and CDelta81 showed markedly reduced binding ability to P1 and P2, which was offset by the addition of an rRNA fragment covering the P0.P1-P2 binding site. The mutant CDelta107 lost the P1/P2 binding activity, whereas it retained the rRNA binding. In contrast, the N-terminal truncation mutants NDelta21-NDelta92 completely lost the rRNA binding, although they retained P1/P2 binding capability, implying an essential role of the N terminus of P0 for rRNA binding. The P0 mutants NDelta6, NDelta14, and CDelta18-CDelta81, together with P1/P2 and eL12, bound to the Escherichia coli core 50 S subunits deficient in L10.L7/L12 complex and L11. Analysis of incorporation of (32)P-labeled P1/P2 into the 50 S subunits with WT-P0 and CDelta81 by sedimentation analysis indicated that WT-P0 bound two copies of P1 and P2, but CDelta81 bound only one copy each. The hybrid ribosome with CDelta81 that appears to contain one P1-P2 heterodimer retained lower but considerable activities dependent on eukaryotic elongation factors. These results suggested that two P1-P2 dimers bind to close but separate regions on the C-terminal half of P0. The results were further confirmed by binding experiments using chimeric P0 mutants in which the C-terminal 81 or 107 amino acids were replaced with the homologous sequences of the archaebacterial P0.     

Functional characterization of the S41Y (C2755A) polymorphism of tryptophan hydroxylase 2

Human TPH2 (hTPH2) catalyzes the rate‐limiting step in CNS serotonin biosynthesis. We characterized a single‐nucleotide polymorphism (C2755A) in the hTPH2 gene that substitutes tyrosine for serine at position 41 in the regulatory domain of the enzyme. This polymorphism is associated with bipolar disorder and peripartum depression in a Chinese population. Recombinant h TPH2 human proteins were expressed in bacteria and also stably expressed in PC12 cells. Following bacterial expression and purification, the tyrosine for serine substitution at position 41 (S41Y) polymorphic enzyme displayed increased V_max with unchanged K_m values. By contrast, enzyme stability was decreased in vitro from 32 min to 4 min (37°C) for the S41Y enzyme (as compared to the wild‐type enzyme). The S41Y polymorphism decreased cyclic AMP‐dependent protein kinase A‐mediated phosphorylation ~ 50% relative to wild‐type hTPH2, suggesting that the S41Y mutation may disrupt the post‐translational regulation of this enzyme. Transfected PC12 cells expressed hTPH2 mRNA, active protein, and synthesized and released serotonin. Paradoxically, while S41Y‐transfected PC12 cells expressed higher levels of hTPH2 than wild type, they synthesized less serotonin. These findings suggest a modified regulation of the S41Y gene variant leading to altered regulation and reduced neurotransmitter synthesis that may contribute to association of the polymorphism with bipolar disorder and depression.

     

Characterization of wild-type and mutant forms of human tryptophan hydroxylase 2

Tryptophan hydroxylase (TPH) catalyses the rate-limiting step in the biosynthesis of serotonin. In vertebrates, the homologous genes tph1 and tph2 encode two different enzymes with distinct patterns of expression, enzyme kinetics and regulation. Variants of TPH2 have recently reported to be associated with reduced serotonin production and behavioural alterations in man and mice. We have produced the human forms of these enzymes in Esherichia coli and in human embryonic kidney cell lines (HEK293) and examined the effects of mutations on their heterologous expression levels, solubility, thermal stability, secondary structure, and catalytic properties. Pure human TPH2 P449R (corresponds to mouse P447R) had comparable catalytic activity (V(max)) and solubility relative to the wild type, but had decreased thermal stability; whereas human TPH2 R441H had decreased activity, solubility and stability. Thus, we consider the variations in kinetic values between wild-type and TPH2 mutants to be of secondary importance to their effects on protein stability and solubility. These findings provide potential molecular explanations for disorders related to the central serotonergic system, such as depression or suicidal behaviour.     

A regulatory domain in the N terminus of tryptophan hydroxylase 2 controls enzyme expression

Serotonin is involved in a variety of physiological processes in the central nervous system and the periphery. As the rate-limiting enzyme in serotonin synthesis, tryptophan hydroxylase plays an important role in modulating these processes. Of the two variants of tryptophan hydroxylase, tryptophan hydroxylase 2 (TPH2) is expressed predominantly in the central nervous system, whereas tryptophan hydroxylase 1 (TPH1) is expressed mostly in peripheral tissues. Although the two enzymes share considerable sequence homology, the regulatory domain of TPH2 contains an additional 41 amino acids at the N terminus that TPH1 lacks. Here we show that the extended TPH2 N-terminal domain contains a unique sequence involved in the regulation of enzyme expression. When expressed in cultured mammalian cells, TPH2 is synthesized less efficiently and is also less stable than TPH1. Removal of the unique portion of the N terminus of TPH2 results in expression of the enzyme at a level similar to that of TPH1, whereas protein chimeras containing this fragment are expressed at lower levels than their wild-type counterparts. We identify a region centered on amino acids 10-20 that mediates the bulk of this effect. We also demonstrate that phosphorylation of serine 19, a protein kinase A consensus site located in this N-terminal domain, results in increased TPH2 stability and consequent increases in enzyme output in cell culture systems. Because this domain is unique to TPH2, these data provide evidence for selective regulation of brain serotonin synthesis.     

Conversion of a Golgi apparatus sialyltransferase to a secretory protein by replacement of the NH2-terminal signal anchor with a signal peptide

The beta-galactoside alpha 2,6 sialyltransferase, an integral membrane protein localized to the trans-region of the Golgi apparatus, has been converted into a catalytically active secreted protein by the replacement of the NH2-terminal signal-anchor domain with the cleavable signal peptide of human gamma-interferon. Pulse-chase analysis of the wild type and recombinant proteins expressed in stably transfected Chinese hamster ovary cells showed that the wild type sialyltransferase (47 kDa) remained cell-associated. In contrast, the signal peptide-sialyltransferase fusion protein yielded an enzymatically active 41-kDa polypeptide which was secreted with a half-time of 2-3 h, consistent with cleavage of the signal peptide. The data indicate that the catalytic domain does not contain sufficient information for retention in the Golgi apparatus and that retention signals are likely to be found in the NH2-terminal 57 amino acids of the wild type enzyme.     

Mouse tryptophan hydroxylase isoform 2 and the role of proline 447 in enzyme function

Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in the synthesis of the neurotransmitter serotonin (5-HT). Once thought to be a single gene product, TPH is now known to exist in two isoforms. Isoform 1 (TPH1) is found in the pineal gland and gut, and isoform 2 (TPH2) is selectively expressed in brain. A single-nucleotide polymorphism in TPH2 results in a proline-to-arginine mutation at residue 447 and substantially lowers catalytic activity. In view of the importance of TPH in determining brain 5-HT function, we cloned TPH2 and produced the P447R mutant to assess the importance of this proline in enzyme function. Catalytically active TPH2 and the P447R mutant were expressed at the predicted subunit molecular mass of 56 kDa. The P447R mutant expressed less than 50% of the activity of TPH2. Mutation of this conserved proline in TPH1 (P403R) also resulted in an enzyme with significantly lower activity than the wild-type enzyme. The P447R mutant had a V(max) 50% lower than that of TPH2. The P447R mutation did not alter the oligomeric assembly of the protein, nor change its responsiveness to cysteine modification. The P447R mutation did not alter enzyme substrate specificity or stability, but conferred slightly enhanced sensitivity to inhibition by dopamine and diminished sensitivity to iron in catalysis. The conserved proline in TPH (residue 447 in TPH2 and 403 in TPH1) plays an important role in enzyme function by regulating V(max) of the catalytic reaction.     

Influence of human tryptophan hydroxylase 2 N- and C-terminus on enzymatic activity and oligomerization

Tryptophan hydroxylase (TPH) catalyses the first and rate limiting step in the biosynthesis of the neurotransmitter serotonin. There are two TPH isoenzymes in humans, encoded by two different genes: TPH1 and the recently described TPH2. We have expressed both human enzymes and various deletion mutants of TPH2 (DeltaN44, DeltaC17, DeltaC19, DeltaC51) in COS7 cells. TPH1 and 2 displayed different kinetic properties with a lower K(m) value of TPH1. Removal of 44 amino acids from the N-terminus of TPH2 resulted in a 3-4-fold increased V(max), which indicates a strong inhibitory function of this part on the enzymes activity. TPH1 and 2 were able to form homooligomers and also heterooligomers with each other. The different deletion mutants (DeltaC17, DeltaC19 and DeltaC51), which lack the putative C-terminal leucine zipper tetramerization domain, existed as monomeric enzymes. While short deletions (DeltaC17 and DeltaC19) hardly changed V(max) values, the DeltaC51 mutant lost 99% of TPH activity. These data identify a region between the C-terminal oligomerization domain and the catalytic domain, which is indispensable for TPH2 activity.     

Evidence that the iron-sulfur cluster of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase determines stability of the enzyme to degradation in vivo

Bacillus subtilis glutamine P-Rib-PP amidotransferase contains a [4Fe-4S] cluster which is essential for activity. The enzyme also undergoes removal of 11 NH2-terminal residues from the primary translation product in vivo to form the active enzyme. It has been proposed that oxidative inactivation of the FeS cluster in vivo is the first step in degradation of the enzyme in starving cells. Four mutants of amidotransferases that alter cysteinyl ligands to the FeS cluster or residues adjacent to them have been prepared by site-directed mutagenesis, expressed in Escherichia coli, and characterized (Makaroff, C. A., Paluh, J. L., and Zalkin, H. (1986) J. Biol. Chem. 261, 11416-11423). These mutations were integrated into the B. subtilis chromosome in place of the normal purF gene. Inactivation and degradation in vivo of wild type and mutant amidotransferases were characterized in these integrants. Mutants FeS1 (C448S) and FeS2 (C451S) failed to form active enzyme, assemble FeS clusters, or undergo NH2-terminal processing. The immunochemically cross-reactive protein produced by both mutants was degraded rapidly (t1/2 = 16 min) in exponentially growing cells. In contrast the wild type enzyme was stable in growing cells, and activity and cross-reactive protein were lost from glucose-starved cells with a t1/2 of 57 min. Mutant FeS3 (F394V) contained an FeS cluster and was processed normally, but had only about 40% of normal specific activity. The FeS3 enzyme was also inactivated by reaction with O2 in vitro about twice as fast as the wild type. The amidotransferase produced by the FeS3 integrant was stable in growing cells but was inactivated and degraded in glucose-starved cells more rapidly (t1/2 = 35 min) than the wild type enzyme. Mutant FeS4 (C451S, D442C) also contained an FeS cluster and was processed; the enzyme had about 50% of wild type-specific activity and reacted with O2 in vitro at the same rate as the wild type. Inactivation and degradation of the FeS4 mutant in vivo in glucose-starved cells proceeded at a rate (t1/2 = 45 min) that was somewhat faster than normal. The correlation between absence of an FeS cluster or enhanced lability of the cluster to O2 and increased degradation rates in vivo supports the conclusions that stability of the enzyme in vivo requires an intact FeS cluster and that O2-dependent inactivation is the rate-determining step in degradation of the enzyme. The fact that mutant FeS3 was processed normally but degraded rapidly argues against a role for NH2-terminal processing in controlling degradation rates.     

Proteolytic modification of the amino-terminal and carboxyl-terminal regions of rat hepatic phenylalanine hydroxylase

Activation of rat liver phenylalanine hydroxylase by limited proteolysis catalyzed by chymotrypsin was investigated with the use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high pressure gel filtration. Both activation and proteolysis were decreased by the addition of the natural cofactor, (6R)-tetrahydrobiopterin. From chymotryptic digests of the hydroxylase carried out in the presence and absence of (6R)-tetrahydrobiopterin, several different enzyme species were isolated by high pressure gel filtration. One species (subunit Mr = 47,000) with unchanged hydroxylase activity was isolated from the chymotryptic digest in the presence of (6R)-tetrahydrobiopterin; it was derived from the native enzyme (Mr = 52,000) by cleavage of the COOH-terminal Mr = 5,000 portion of the native enzyme. In the absence of (6R)-tetrahydrobiopterin, another species (subunit Mr = 36,000) was isolated. In addition to modification at the COOH-terminal end of the molecule, this species also had lost a Mr = 11,000 fragment from the NH2-terminal end of the hydroxylase. The Mr = 11,000 fragment was shown to include the phosphorylation site of the enzyme. This Mr = 36,000 species was 30-fold more active than the native phenylalanine hydroxylase when assayed in the presence of tetrahydrobiopterin. These results suggest that the regulatory domain that inhibits hydroxylase activity in the basal state may be located at the NH2 terminus of the phenylalanine hydroxylase subunit.     

Amino-Terminal Analysis of Tryptophan Hydroxylase: Protein Kinase Phosphorylation Occurs at Serine-58

Abstract: Tryptophan hydroxylase (TPH) catalyzes the rate-limiting and committed step in serotonin biosynthesis. Within this enzyme, two distinct domains have been hypothesized to exist, an amino-terminal regulatory domain and a carboxyl-terminal catalytic domain. In the present experiments, the functional boundary between the putative domains was defined using deletion muta-genesis. A full-length cDNA clone for rabbit TPH was engineered for expression in bacteria. Five amino-terminal deletions were constructed using PCR, i.e., NΔ50, NΔ60, NΔ90, NΔ106, and NΔ116 (referring to the number of amino acids deleted from the amino terminus). Enzymatic activity was determined for each mutant after expression in bacteria. Whereas deletion of 116 amino acids (NΔ116) abolished enzyme activity, all of the other amino-terminal deletions exhibited increased specific activity relative to the recombinant wild-type TPH. The ability of the cyclic AMP-dependent protein kinase (PKA) to phosphorylate members of the deletion series was also examined. Deletion of the first 60 amino-terminal residues abolished the ability of the enzyme to serve as a substrate for PKA, yet the native and NΔ50 enzymes were phosphorylated. Moreover, a serine-58 point mutant (S58A) was not phosphorylated by PKA. In conclusion, the first 106 amino acids comprise a regulatory domain that is phosphorylated by PKA at serine-58. In addition, the boundary between regulatory and catalytic domains is analogous to the domain structure observed for the related enzyme tyrosine hydroxylase.     

Phosphorylation and activation of tryptophan hydroxylase 2: identification of serine-19 as the substrate site for calcium, calmodulin-dependent protein kinase II

Tryptophan hydroxylase (TPH) is the initial and rate-limiting enzyme in the biosynthesis of serotonin. TPH was once thought to be a single-gene product but it is now known to exist in two isoforms. TPH1 is found in the periphery and pineal gland whereas TPH2 is expressed specifically in the CNS. Both TPH isoforms are known to be regulated by protein kinase-dependent phosphorylation and the sites of modification of TPH1 by protein kinase A have been identified. While TPH2 is activated by calcium, calmodulin-dependent protein kinase II (CaMKII), the sites at which this isoform is modified are not known. Treatment of wild-type TPH2 with CaMKII followed by mass spectrometry analysis revealed that the enzyme was activated and phosphorylated at a single site, serine-19. Mutagenesis of serine-19 to alanine did not alter the catalytic function of TPH2 but this mutant enzyme was neither activated nor phosphorylated by CaMKII. A phosphopeptide bracketing phosphoserine-19 in TPH2 was used as an antigen to generate polyclonal antibodies against phosphoserine-19. The antibodies are highly specific for phosphoserine-19 in TPH2. The antibodies do not react with wild-type TPH2 or TPH1 and they do not recognize phophoserine-58 or phosphoserine-260 in TPH1. These results establish that activation of TPH2 by CaMKII is mediated by phosphorylation of serine-19 within the regulatory domain of the enzyme. Production of a specific antibody against the CaMKII phosphorylation site in TPH2 represents a valuable tool to advance the study of the mechanisms regulating the function of this important enzyme.     

C-terminal region of the active domain enhances enzymatic activity in dinoflagellate luciferase.

The dinoflagellate luciferase of Lingulodinium polyedrum has three catalytic domains in its single polypeptide chain (M(r) = 137 kDa), and each 42 kDa domain is enzymatically active. Deletion mutants for N- or C-terminal regions of domain 3 of the luciferase, ranging from 29 to 38 kDa, were constructed and expressed in E. coli cells. The activities of N-terminal deleted mutants were above 20% of wild type, but showed different pH-activity profiles. By contrast, the activities of C-terminal deleted mutants decreased drastically to below 1% of wild type, although their pH-activity profiles and spectra were identical to those of wild type L. polyedrum luciferase. These results indicate that the C-terminal region of this enzyme could be important for the bioluminescence reaction, although based on crystal structure of the luciferase domain, this region does not contain active or regulatory sites.     

Structure and heterologous expression of a gene encoding fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase from Arabidopsis thaliana

A full-length cDNA clone encoding fructose-6-phosphate, 2-kinase/fructose-2,6-bisphosphatase from Arabidopsis thaliana (AtF2KP) was isolated. The encoded protein is composed of two different regions: (i) a 400 amino acid COOH-terminal region, covering the catalytic region of the protein which is homologous to enzymes from other eukaryotes. This region is highly conserved among plant species (88% identity to spinach F2KP). (ii) A 345 amino acid plant-specific NH(2)-terminal region, with 59% identity to spinach F2KP, which is composed of homologous motifs and intermittent variable sequences. Western blots show that F2KP from several plant species migrates in sodium dodecyl sulphate-polyacrylamide gel electrophoresis as a similar sized (93 kDa) protein. AtF2KP was expressed in Escherichia coli as a full length and a truncated (without the NH(2)-terminal region) fusion protein. Both forms had kinase as well as phosphatase activity, but presence of the NH(2)-terminal region influenced the ratio between the two activities. It is suggested that the NH(2)-terminal region represents a regulatory region, which defines specific properties of the plant enzymes. A genomic clone for the corresponding gene, AtF2KP, was isolated. The clone (9519 bp) included 23 exons, 22 introns and the promoter sequence. Southern blot analysis showed only one copy of the gene in the A. thaliana genome.     

Primary structure of beta-galactoside alpha 2,6-sialyltransferase. Conversion of membrane-bound enzyme to soluble forms by cleavage of the NH2-terminal signal anchor

This report describes the primary structure of a rat liver beta-galactoside alpha 2,6-sialyltransferase (EC 2.4.99.1), a Golgi apparatus enzyme involved in the terminal sialylation of N-linked carbohydrate groups of glycoproteins. The complete amino acid sequence was deduced from the nucleotide sequence of cDNA clones of the enzyme. The primary structure suggests that the topology of the enzyme in the Golgi apparatus consists of a short NH2-terminal cytoplasmic domain, a 17-residue hydrophobic sequence which serves as the membrane anchor and signal sequence, and a large lumenal, catalytic domain. NH2-terminal sequence analysis of a truncated form of the enzyme, obtained by purification from tissue homogenates, reveals that it is missing a 63-residue NH2-terminal peptide which includes the membrane binding domain. These and supporting results show that soluble forms of the sialyltransferase can be generated by proteolytic cleavage between the NH2-terminal signal-anchor and the catalytic domain.     

Pyruvate:NADP+ oxidoreductase from Euglena gracilis: limited proteolysis of the enzyme with trypsin

Pyruvate:NADP+ oxidoreductase from Euglena gracilis, a homodimeric protein with a molecular weight of 309 kDa, is an iron-sulfur flavoenzyme that contains thiamin pyrophosphate (TPP). The functional structure of the enzyme was studied by a limited proteolysis experiment using trypsin. The evidence obtained shows that the enzyme consists of two functional domains, one of which contains an iron-sulfur cluster, which can be isolated as a homodimeric fragment of approximately 220 kDa by proteolysis. The other domain that contains FAD is released as a monomeric fragment of approximately 55 kDa. The pyruvate dehydrogenase reaction is still catalyzed by the large fragment when NADP+ is substituted by methyl viologen, while the small fragment retains a diaphorase-like electron-transfer activity from NADPH to MV. It is thus shown that pyruvate is oxidized in a CoA-dependent reaction to form CO2 and acetyl-CoA in the iron-sulfur domain, and that the two electrons formed are transferred to the FAD domain in which NADP+ is reduced. TPP is considered to be associated in the iron-sulfur domain. The NH2-terminal sequences of the enzyme and its proteolytic fragments reveal that the iron-sulfur domain occurs in the NH2-terminal side of the enzyme. For elucidation of the O2 instability of the enzyme, limited proteolysis was attempted in air. The tryptic fragment derived from the iron-sulfur domain, similar to the native enzyme, appears to be inactivated by direct contact with O2. In contrast, the FAD domain, when separated from the other domain, is quite stable in air, although the diaphorase activity decays when the native enzyme is exposed to O2.     

A soluble NH(2)-terminally truncated catalytically active form of rat cytochrome P450 2E1 targeted to liver mitochondria(1).

The role of the NH(2)-terminus of cytochrome P450 2E1 (CYP2E1) in intracellular targeting was investigated. Two NH(2)-terminal CYP2E1 mutants, Delta(2-29)2E1, lacking the transmembrane domain, and N(++)2E1, having Ala2Lys and Val3Arg substitutions, were generated and expressed in the H2.35 mouse hepatoma cell line. In cells transfected with both constructs, a 40 kDa fragment of CYP2E1 (Delta2E1) was found to be localized to mitochondria as evidenced from immunofluorescence microscopy and subcellular fractionation studies. Delta2E1 was shown to be a soluble protein localized inside the mitochondria, displayed catalytic activity when reconstituted with adrenodoxin and adrenodoxin reductase, and was also present in mitochondria isolated from rat liver. It is concluded that in the absence of the hydrophobic NH(2)-terminal sequence, a putative mitochondrial import signal is exposed which targets CYP2E1 to this organelle where it is further processed.     

Papain-inhibitory activity of oryzacystatin, a rice seed cysteine proteinase inhibitor, depends on the central Gln-Val-Val-Ala-Gly region conserved among cystatin superfamily members

Oryzacystatin, a cysteine proteinase inhibitor occurring in rice seeds, contains a particular glycine residue (Gly5) near the NH2-terminal position, and the sequence Gln53-Val54-Val55-Ala56-Gly57 in a central part of the molecule. Both are conserved among most members of the cystatin superfamily. We have found from Escherichia coli expression studies that the NH2-terminal 21 residues of oryzacystatin are not essential for its papain-inhibitory activity, and that the conserved pentapeptide region may be indispensable [Abe, K., Emori, Y., Kondo, H., Arai, S., & Suzuki, K. (1988) J. Biol. Chem. 263, 7655-7659]. Here we present more detailed data based on quantitative analyses of the inhibitory activities of NH2- and COOH-terminally truncated oryzacystatin and site-directed mutants at the Gln-Val-Val-Ala-Gly region. The data indicate the following results. (1) The truncated mutants lacking the NH2-terminal 21 residues or the COOH-terminal 11 residues exhibit potent papain-inhibitory activity equivalent to the activity of wild oryzacystatin. (2) However, neither the mutant lacking the NH2-terminal 38 residues nor that lacking the COOH-terminal 35 residues is completely able to inhibit papain. (3) Site-directed mutants at the Gln residue of the Gln-Val-Val-Ala-Gly region have drastically reduced papain-inhibitory activities: the Gln----Pro mutant is completely inactive and the Gln----Leu mutant has an approximately 150 times higher Ki value than wild-type oryzacystatin.(ABSTRACT TRUNCATED AT 250 WORDS)