Ser422 phosphorylation blocks human Tau cleavage by caspase-3: Biochemical implications to Alzheimer’s Disease

Proteolytic truncation of microtubule associated human (h) Tau protein by caspase-3 at the carboxy (C) terminus has been linked to the pathogenesis of Alzheimer’s Disease (AD). This cleavage likely occurs between Asp⁴²¹↓Ser⁴²² leading to the formation of 421-mer truncated Tau protein which has been found to be present as aggregate in high level after phosphorylation in mortal AD brain tissue compared to normal. At least 50 phosphorylation sites involving Ser, Thr and Tyr residues have been identified or proposed in hTau and a selected number of them have been implicated in hTau aggregation following latter’s proteolytic truncation. Interestingly, it is further noted that Ser⁴²² residue present in the P1′ position of hTau caspase-3 cleavage region is a potential phosphorylation site. So we became interested to examine in vitro the effect of phospho-Ser⁴²² residue on hTau cleavage by caspase-3 which is a crucial upstream event associated with hTau self-assembly leading to AD pathogenesis. The goal of this project is to study in vitro the caspase-3 cleavage site of hTau protein and to examine the kinetics of this cleavage following Ser⁴²² phosphorylation and treatment with caspase-3 inhibitors. This is achieved by designing peptides from the sequence of hTau protein containing the proposed caspase-3 cleavage region. Peptides were designed from 441-mer major human Tau protein sequence that encompasses the proposed caspase-3 cleavage site [Asp⁴²¹↓Ser⁴²²]. Corresponding phospho-, dextro-Ser⁴²² and dextro-Asp⁴²¹ analogs were also designed. Peptides were synthesized by solid phase chemistry, purified and fully characterized by mass spectrometry. These were then incubated with recombinant caspase-3 enzyme under identical condition for digestion and analyzed for cleavage by mass spectrometry and RP-HPLC chromatograms. Our results indicated that while the control peptide is efficiently cleaved by caspase-3 at Asp⁴²¹↓Ser⁴²² site producing the expected N- and C-terminal fragment peptides, the corresponding phospho-Ser⁴²² peptide remained completely resistant to the cleavage. Substitution of Asp⁴²¹ by its dextro isoform also blocks peptide cleavage by caspase-3. However substitution of Ser⁴²² by its dextro isoform in the peptide did not affect the cleavage significantly. The above results were further confirmed by caspase-3 digestion experiment in the presence of varying amounts of caspase-3 inhibitor (Ac-DQVD-aldehyde) which was found to block this cleavage in a highly effective manner. Our results highlighted the crucial significance of Ser⁴²² phosphorylation and suggest that the kinase associated with this Ser-phosphorylation may protect Tau from aggregation. Thus specific promoters/activators of this kinase may find useful therapeutic benefits in arresting Tau truncation by caspase-3 and the progression of AD. In addition our data demonstrated that Tau-peptides where Ser⁴²² or Asp⁴²¹ are substituted by their respective dextro isomers, exhibit different cleavage kinetics by caspase-3 and this may have important implications in therapeutic intervention of Tau aggregation and associated AD.     

Chloroplast tRNAAsp: nucleotide sequence and variation of in vivo levels during plastid maturation

Two chloroplast tRNA^Asp species from barley were purified by chromatography on benzoylated DEAE-cellulose and sequenced. They differ in the modification at position 34, where queuosine (Q) is present in one of the species. The same chromatographic procedure yielded only one tRNA^Glu species, corroborating the assumption that the same tRNA^Glu species participates in both protein and chlorophyll biosynthesis. The level of tRNA^Glu remains unchanged after light treatment of etiolated seedlings, whereas the amount of tRNA^Asp decreases to about 50% relative to the level of dark-grown plants.     

Fusion with Anticodon Binding Domain of GluRS is Not Sufficient to Alter the Substrate Specificity of a Chimeric Glu-Q-RS

Glutamyl-queuosine-tRNA^Asp synthetase (Glu-Q-RS) is a paralog of glutamyl-tRNA synthetase (GluRS) and is found in more than forty species of proteobacteria, cyanobacteria, and actinobacteria. Glu-Q-RS shows striking structural similarity with N-terminal catalytic domain of GluRS (NGluRS) but it lacks the C-terminal anticodon binding domain (CGluRS). In spite of structural similarities, Glu-Q-RS and NGluRS differ in their functional properties. Glu-Q-RS glutamylates the Q34 nucleotide of the anticodon of tRNA^Asp whereas NGluRS constitutes the catalytic domain of GluRS catalyzing the transfer of Glu on the acceptor end of tRNA^Glu. Since NGluRS is able to catalyze aminoacylation of only tRNA^Glu the glutamylation capacity of tRNA^Asp by Glu-Q-RS is surprising. To understand the substrate specificity of Glu-Q-RS we undertook a systemic approach by investigating the biophysical and biochemical properties of the NGluRS (1–301), CGluRS (314–471) and Glu-Q-RS-CGluRS, (1–298 of Glu-Q-RS fused to 314–471 from GluRS). Circular dichroism, fluorescence spectroscopy and differential scanning calorimetry analyses revealed absence of N-terminal domain (1–298 of Glu-Q-RS) and C-terminal domain (314–471 from GluRS) communication in chimera, in contrast to the native full length GluRS. The chimeric Glu-Q-RS is still able to aminoacylate tRNA^Asp but has also the capacity to bind tRNA^Glu. However the chimeric protein is unable to aminoacylate tRNA^Glu probably as a consequence of the lack of domain–domain communication.     

Identification of Functional Amino Acid Residues Involved in Polyamine and Agmatine Transport by Human Organic Cation Transporter 2

Polyamine (putrescine, spermidine and spermine) and agmatine uptake by the human organic cation transporter 2 (hOCT2) was studied using HEK293 cells transfected with pCMV6-XL4/hOCT2. The Km values for putrescine and spermidine were 7.50 and 6.76 mM, and the Vmax values were 4.71 and 2.34 nmol/min/mg protein, respectively. Spermine uptake by hOCT2 was not observed at pH 7.4, although it inhibited both putrescine and spermidine uptake. Agmatine was also taken up by hOCT2, with Km value: 3.27 mM and a Vmax value of 3.14 nmol/min/mg protein. Amino acid residues involved in putrescine, agmatine and spermidine uptake by hOCT2 were Asp⁴²⁷, Glu⁴⁴⁸, Glu⁴⁵⁶, Asp⁴⁷⁵, and Glu⁵¹⁶. In addition, Glu⁵²⁴ and Glu⁵³⁰ were involved in putrescine and spermidine uptake activity, and Glu⁵²⁸ and Glu⁵⁴⁰ were weakly involved in putrescine uptake activity. Furthermore, Asp⁵⁵¹ was also involved in the recognition of spermidine. These results indicate that the recognition sites for putrescine, agmatine and spermidine on hOCT2 strongly overlap, consistent with the observation that the three amines are transported with similar affinity and velocity. A model of spermidine binding to hOCT2 was constructed based on the functional amino acid residues.     

Roles of Two Ca2+-binding Domains in Regulation of the Cardiac Na+-Ca2+ Exchanger

We expressed full-length Na⁺-Ca²⁺ exchangers (NCXs) with mutations in two Ca²⁺-binding domains (CBD1 and CBD2) to determine the roles of the CBDs in Ca²⁺-dependent regulation of NCX. CBD1 has four Ca²⁺-binding sites, and mutation of residues Asp⁴²¹ and Glu⁴⁵¹, which primarily coordinate Ca²⁺ at sites 1 and 2, had little effect on regulation of NCX by Ca²⁺. In contrast, mutations at residues Glu³⁸⁵, Asp⁴⁴⁶, Asp⁴⁴⁷, and Asp⁵⁰⁰, which coordinate Ca²⁺ at sites 3 and 4 of CBD1, resulted in a drastic decrease in the apparent affinity of peak exchange current for regulatory Ca²⁺. Another mutant, M7, with 7 key residues of CBD1 replaced, showed a further decrease in apparent Ca²⁺ affinity but retained regulation, confirming a contribution of CBD2 to Ca²⁺ regulation. Addition of the mutation K585E (located in CBD2) into the M7 background induced a marked increase in Ca²⁺ affinity for both steady-state and peak currents. Also, we have shown previously that the CBD2 mutations E516L and E683V have no Ca²⁺-dependent regulation. We now demonstrate that introduction of a positive charge at these locations rescues Ca²⁺-dependent regulation. Finally, our data demonstrate that deletion of the unstructured loops between β-strands F and G of both CBDs does not alter the regulation of the exchanger by Ca²⁺, indicating that these segments are not important in regulation. Thus, CBD1 and CBD2 have distinct roles in Ca²⁺-dependent regulation of NCX. CBD1 determines the affinity of NCX for regulatory Ca²⁺, although CBD2 is also necessary for Ca²⁺-dependent regulation.     

Calcium modulates membrane association,positional specificity,and product distribution in dual positional specific maize lipoxygenase-1

This study investigates how calcium modulates the properties of dual positional specific maize lipoxygenase-1, including its interaction with substrate, association with subcellular membrane and alteration of product distribution. Bioinformatic analyses identified Asp³⁸, Glu¹²⁷ and Glu²⁰¹ as putative calcium binding residues and Leu³⁷ as a flanking hydrophobic residue also potentially involved in calcium-mediated binding of the enzyme to subcellular membranes. Asp³⁸ and Leu³⁷ were shown to be important but not essential for calcium-mediated association of maize lipoxygenase-1 to subcellular membranes in vitro. Kinetic studies demonstrate that catalytic efficiency (V_max/K_m) shows a bell-shaped dependence on log of the molar ratio of substrate to unbound calcium. Calcium also modulates product distribution of the maize lipoxygenase-1 reaction, favoring 13-positional specificity and increasing the relative amount of (E,Z)-isomeric products. The results suggest that calcium regulates the maize lipoxygenase-1 reaction by binding to substrate, and by promoting binding of substrate to enzyme and association of maize lipoxygenase-1 to subcellular membranes.     

Cyanine dye N744 inhibits tau fibrillization by blocking filament extension: implications for the treatment of tauopathic neurodegenerative diseases

Tau fibrillization is a potential therapeutic target for Alzheimer's and other neurodegenerative diseases. Small molecules capable of both inhibiting aggregation and promoting filament disaggregation have been discovered, but knowledge of their mechanism of action and potential for testing in biological models is fragmentary. To clarify these issues, the interaction between a small-molecule inhibitor of tau fibrillization, 3,3'-bis(beta-hydroxyethyl)-9-ethyl-5,5'-dimethoxythiacarbocyanine iodide (N744), and full-length four-repeat tau protein was characterized in vitro using transmission electron microscopy and fluorescence spectroscopy. Analysis of reaction time courses performed in the presence of anionic fibrillization inducers revealed that increasing concentrations of N744 decreased the total filament length without modulating lag time, indicating that filament extension but not nucleation was affected by inhibitor under the conditions that were investigated. Critical concentration measurements confirmed that N744 shifted equilibria at filament ends away from the fibrillized state, resulting in endwise filament disaggregation when it was added to synthetic filaments. Both increasing bulk tau concentrations and filament stabilizing modifications such as pseudophosphorylation and glycation antagonized N744 activity. The results illustrate the importance of mechanism for the design and interpretation of pharmacological studies in biological models of tau aggregation.     

Caspase cleavage of GFAP produces an assembly-compromised proteolytic fragment that promotes filament aggregation

IF (intermediate filament) proteins can be cleaved by caspases to generate proapoptotic fragments as shown for desmin. These fragments can also cause filament aggregation. The hypothesis is that disease-causing mutations in IF proteins and their subsequent characteristic histopathological aggregates could involve caspases. GFAP (glial fibrillary acidic protein), a closely related IF protein expressed mainly in astrocytes, is also a putative caspase substrate. Mutations in GFAP cause AxD (Alexander disease). The overexpression of wild-type or mutant GFAP promotes cytoplasmic aggregate formation, with caspase activation and GFAP proteolysis. In this study, we report that GFAP is cleaved specifically by caspase 6 at VELD²²⁵ in its L12 linker domain in vitro. Caspase cleavage of GFAP at Asp²²⁵ produces two major cleavage products. While the C-GFAP (C-terminal GFAP) is unable to assemble into filaments, the N-GFAP (N-terminal GFAP) forms filamentous structures that are variable in width and prone to aggregation. The effect of N-GFAP is dominant, thus affecting normal filament assembly in a way that promotes filament aggregation. Transient transfection of N-GFAP into a human astrocytoma cell line induces the formation of cytoplasmic aggregates, which also disrupt the endogenous GFAP networks. In addition, we generated a neo-epitope antibody that recognizes caspase-cleaved but not the intact GFAP. Using this antibody, we demonstrate the presence of the caspase-generated GFAP fragment in transfected cells expressing a disease-causing mutant GFAP and in two mouse models of AxD. These findings suggest that caspase-mediated GFAP proteolysis may be a common event in the context of both the GFAP mutation and excess.     

Single Point Mutations in the Small Cytoplasmic Loop of ACA8, a Plasma Membrane Ca2+-ATPase of Arabidopsis thaliana, Generate Partially Deregulated Pumps

ACA8 is a type 2B Ca²⁺-ATPase having a regulatory N terminus whose auto-inhibitory action can be suppressed by binding of calmodulin (CaM) or of acidic phospholipids. ACA8 N terminus is able to interact with a region of the small cytoplasmic loop connecting transmembrane domains 2 and 3. To determine the role of this interaction in auto-inhibition we analyzed single point mutants produced by mutagenesis of ACA8 Glu²⁵² to Asn³⁴⁵ sequence. Mutation to Ala of any of six tested acidic residues (Glu²⁵², Asp²⁷³, Asp²⁹¹, Asp³⁰³, Glu³⁰², or Asp³³²) renders an enzyme that is less dependent on CaM for activity. These results highlight the relevance in ACA8 auto-inhibition of a negative charge of the surface area of the small cytoplasmic loop. The most deregulated of these mutants is D291A ACA8, which is less activated by controlled proteolysis or by acidic phospholipids; the D291A mutant has an apparent affinity for CaM higher than wild-type ACA8. Moreover, its phenotype is stronger than that of D291N ACA8, suggesting a more direct involvement of this residue in the mechanism of auto-inhibition. Among the other produced mutants (I284A, N286A, P289A, P322A, V344A, and N345A), only P322A ACA8 is less dependent on CaM for activity than the wild type. The results reported in this study provide the first evidence that the small cytoplasmic loop of a type 2B Ca²⁺-ATPase plays a role in the attainment of the auto-inhibited state.     

Asp and Thr are critical for cation exchange mediated by NhaD, Na/H antiporter of Vibrio cholerae

The Vc-NhaD is an Na⁺/H⁺ antiporter from Vibrio cholerae belonging to a new family of bacterial Na⁺/H⁺ antiporters, the NhaD family. In the present work we mutagenized five conserved Asp and Glu residues and one conserved Thr residue to Ala in order to identify amino acids that are critical for the antiport activity. All mutations fall into two distinct groups: (i) four variants, Glu¹⁰⁰Ala, Glu²⁵¹Ala, Glu³⁴²Ala, and Asp³⁹³Ala, did not abolish antiport activity but shifted the pH optimum to more alkaline pH, and (ii) variants Asp³⁴⁴Ala, Asp³⁴⁴Asn, and Thr³⁴⁵Ala caused a complete loss of both Na⁺/H⁺ and Li⁺/H⁺ antiport activity whereas the Asp³⁴⁴Glu variant exhibited reduced Na⁺/H⁺ and Li⁺/H⁺ antiport activity. This is the first mutational analysis of the antiporter of NhaD type and the first demonstration of Thr residue being indispensable for Na⁺/H⁺ antiport. We discuss the possible role of Asp³⁴⁴ and Thr³⁴⁵ in the functioning of Vc-NhaD.     

A minimalist glutamyl-tRNA synthetase dedicated to aminoacylation of the tRNAAsp QUC anticodon

Escherichia coli encodes YadB, a protein displaying 34% identity with the catalytic core of glutamyl-tRNA synthetase but lacking the anticodon-binding domain. We show that YadB is a tRNA modifying enzyme that evidently glutamylates the queuosine residue, a modified nucleoside at the wobble position of the tRNA^Asp QUC anticodon. This conclusion is supported by a variety of biochemical data and by the inability of the enzyme to glutamylate tRNA^Asp isolated from an E.coli tRNA-guanosine transglycosylase minus strain deprived of the capacity to exchange guanosine 34 with queuosine. Structural mimicry between the tRNA^Asp anticodon stem and the tRNA^Glu amino acid acceptor stem in prokaryotes encoding YadB proteins indicates that the function of these tRNA modifying enzymes, which we rename glutamyl-Q tRNA^Asp synthetases, is conserved among prokaryotes.     

Identification of a spermidine excretion protein complex (MdtJI) in Escherichia coli

A spermidine excretion protein in Escherichia coli was looked for among 33 putative drug exporters thus far identified. Cell toxicity and inhibition of growth due to overaccumulation of spermidine were examined in an E. coli strain deficient in spermidine acetyltransferase, an enzyme that metabolizes spermidine. Toxicity and inhibition of cell growth by spermidine were recovered in cells transformed with pUCmdtJI or pMWmdtJI, encoding MdtJ and MdtI, which belong to the small multidrug resistance family of drug exporters. Both mdtJ and mdtI are necessary for recovery from the toxicity of overaccumulated spermidine. It was also found that the level of mdtJI mRNA was increased by spermidine. The spermidine content in cells cultured in the presence of 2 mM spermidine was decreased, and excretion of spermidine from cells was enhanced by MdtJI, indicating that the MdtJI complex can catalyze excretion of spermidine from cells. It was found that Tyr⁴, Trp⁵, Glu¹⁵, Tyr⁴⁵, Tyr⁶¹, and Glu⁸² in MdtJ and Glu⁵, Glu¹⁹, Asp⁶⁰, Trp⁶⁸, and Trp⁸¹ in MdtI are involved in the excretion activity of MdtJI.     

Pseudophosphorylation of tau protein directly modulates its aggregation kinetics

Hyperphosphorylation of tau protein is associated with neurofibrillary lesion formation in Alzheimer's disease and other tauopathic neurodegenerative diseases. It fosters lesion formation by increasing the concentration of free tau available for aggregation and by directly modulating the tau aggregation reaction. To clarify how negative charge incorporation into tau directly affects aggregation behavior, the fibrillization of pseudophosphorylation mutant T212E prepared in a full-length four-repeat tau background was examined in vitro as a function of time and submicromolar tau concentrations using electron microscopy assay methods. Kinetic constants for nucleation and extension phases of aggregation were then estimated by direct measurement and mathematical simulation. Kinetic analysis revealed that pseudophosphorylation increased tau aggregation rate by increasing the rate of filament nucleation. In addition, it increased aggregation propensity by stabilizing mature filaments against disaggregation. The data suggest that incorporation of negative charge into the T212 site can directly promote tau filament formation at multiple steps in the aggregation pathway.     

The antigenic structure and topography of bacteriorhodopsin in purple membranes as determined by interaction with monoclonal antibodies

The spatial organization and the antigenic structure of the bacteriorhodopsin molecule in the purple membrane were studied by immunochemical techniques. Five monoclonal antibodies directed against exposed parts of the protein molecule in the membrane were prepared and characterized. Antigenic determinants were localized in the bacteriorhodopsin polypeptide chain by analysis of the interaction between monoclonal antibodies and protein fragments. The structure of antigenic determinants was revealed by the interaction of monoclonal antibodies with (i) isolated bacteriorhodopsin fragments further modified by sequential Edman degradation and (ii) derivatives of bacteriorhodopsin obtained biosynthetically or by selective chemical modification. Five antigenic determinants were localized in the following parts of bacteriorhodopsin: < Glu'-Met²⁰ involving one of the 3 amino acid residues of the N-terminal part; Gly³³-Met⁵⁶ involving Asp³⁶ and/or Asp³⁸ and Phe⁴²; Phe¹⁵⁶-Met¹⁶³ involving Phe¹⁵⁶; Glu¹⁹⁴-Leu²⁰⁷ involving Glu¹⁹⁴; Pro²⁰⁰-Leu²⁰⁷.     

Critical Role of Zinc Ion on E. coli Glutamyl-Queuosine-tRNAAsp Synthetase (Glu-Q-RS) Structure and Function

Glutamyl-queuosine-tRNA^Asp synthetase (Glu-Q-RS) and glutamyl-tRNA synthetase (GluRS), differ widely by their function although they share close structural resemblance within their catalytic core of GluRS. In particular both Escherichia coli GluRS and Glu-Q-RS contain a single zinc-binding site in their putative tRNA acceptor stem-binding domain. It has been shown that the zinc is crucial for correct positioning of the tRNA^Glu acceptor-end in the active site of E. coli GluRS. To address the role of zinc ion in Glu-Q-RS, the C101S/C103S Glu-Q-RS variant is constructed. Energy dispersive X-ray fluorescence show that the zinc ion still remained coordinated but the variant became structurally labile and acquired aggregation capacity. The extent of aggregation of the protein is significantly decreased in presence of the small substrates and more particularly by adenosine triphosphate. Addition of zinc increased significantly the solubility of the variant. The aminoacylation assay reveals a decrease in activity of the variant even after addition of zinc as compared to the wild-type, although the secondary structure of the protein is not altered as shown by the Fourier transform infrared spectroscopy study.     

Novel dextranase catalyzing cycloisomaltooligosaccharide formation and identification of catalytic amino acids and their functions using chemical rescue approach

A novel endodextranase from Paenibacillus sp. (Paenibacillus sp. dextranase; PsDex) was found to mainly produce isomaltotetraose and small amounts of cycloisomaltooligosaccharides (CIs) with a degree of polymerization of 7–14 from dextran. The 1,696-amino acid sequence belonging to the glycosyl hydrolase family 66 (GH-66) has a long insertion (632 residues; Thr⁴⁵¹–Val¹⁰⁸²), a portion of which shares identity (35% at Ala³⁹–Ser¹³⁰⁴ of PsDex) with Pro³²–Ala⁷⁵⁵ of CI glucanotransferase (CITase), a GH-66 enzyme that catalyzes the formation of CIs from dextran. This homologous sequence (Val⁸³⁷–Met⁹³² for PsDex and Tyr⁴⁰⁴–Tyr⁴⁹² for CITase), similar to carbohydrate-binding module 35, was not found in other endodextranases (Dexs) devoid of CITase activity. These results support the classification of GH-66 enzymes into three types: (i) Dex showing only dextranolytic activity, (ii) Dex catalyzing hydrolysis with low cyclization activity, and (iii) CITase showing CI-forming activity with low dextranolytic activity. The fact that a C-terminal truncated enzyme (having Ala³⁹–Ser¹³⁰⁴) has 50% wild-type PsDex activity indicates that the C-terminal 392 residues are not involved in hydrolysis. GH-66 enzymes possess four conserved acidic residues (Asp¹⁸⁹, Asp³⁴⁰, Glu⁴¹², and Asp¹²⁵⁴ of PsDex) of catalytic candidates. Their amide mutants decreased activity (1/1, 500 to 1/40, 000 times), and D1254N had 36% activity. A chemical rescue approach was applied to D189A, D340G, and E412Q using α-isomaltotetraosyl fluoride with NaN₃. D340G or E412Q formed a β- or α-isomaltotetraosyl azide, respectively, strongly indicating Asp³⁴⁰ and Glu⁴¹² as a nucleophile and acid/base catalyst, respectively. Interestingly, D189A synthesized small sized dextran from α-isomaltotetraosyl fluoride in the presence of NaN₃.     

Improvement in the oxidative folding of endothelin-1 by a Lys-Arg extension at the amino terminus: Implication of a salt bridge between Arg-1 and Asp8

Summary An amino-terminal extension of endothelin-l by the lys-Arg dipeptide in the prosequence (KR-ET-1) greatly increased the ratio of native-type to non-native-type disulfide isomer (96/4 versus 71/29) during the oxidative folding reaction. This improvement was completely abolished by substituting Asn for Asp at position 8 (D8N-KR-ET-1), whereas most of it was maintained with similar carboxamide analogues replaced at Glu¹⁰ or Asp¹⁸. Structure analyses by circular dichroism spectroscopy revealed that (i) in the carboxylate state, the α-helical content of the native-type isomer of KR-ET-l is higher than that of the native-type isomer of ET-1, while such a variation is not observed in the corresponding non-native-type isomer of KR-ET-l; and (ii) the enhanced α-helicity resulting from the Lys-Arg extension is largely diminished in D8N-KR-ET-l. From these results and our previous findings that the helical structure in KR-ET-l is stabilized by a particular salt bridge between the extended Arg⁻¹ basic moiety and either the Asp⁸ or Glu¹⁰ acidic side chain in Et-1 [Aumelas, A. et al., Biochemistry, 34 (1995) 4546], we conclude that the formation of a specific salt bridge between the side chains of Arg⁻¹ and Asp⁸ in KR-ET-1 is critical for the predominant generation of the native-type disulfide isomer, probably because it stabilizes the helical structure of parental ET-1.     

Characterization of Various Recombinant Antigens from <Emphasis Type="Italic">Echinococcus multilocularis</Emphasis> for Use in the Immunodiagnosis

Using four clones isolated from Echinococcus multilocularis cDNA library with alveolar echinococcosis (AE) patient sera, various antigens were expressed as ThioHis tag-fused protein. Recombinant EmII/3 antigen was produced as the five fragments divided into the N-terminal (#5 and #5s), the central (#6 and #6s) and the C-terminal domain (#7). Immunoblot analysis revealed that the #7 showed significant reactivity whereas those of #5 and #5s were relatively low. The #6 and #6s also showed lower reactivity than that of #7, although the two minor bands of #6 reacted with every serum. These results suggested that an immunodominant region of EmII/3 locate within the C-terminal one third. The #8s recombinant antigen, Ser²³–Glu¹⁷⁶ of actin filament fragmenting protein (AFFP), apparently reacted with the AE patient sera, while the #1 antigen synthesized as a full-length antigen B1 did not show such high reactivity. Thus, #7 and #8s antigens showed significant potential for use in immunodetection of AE. In addition, the specific antibodies against #7 and #8s reacted with specific antigens in crude extract of E. multilocularis cyst, indicating that these antigens retained antigenicity common to native EmII/3 and AFFP, respectively.     

Crystal structure of glutamyl-queuosine tRNAAsp synthetase complexed with L-glutamate: structural elements mediating tRNA-independent activation of glutamate and glutamylation of tRNAAsp anticodon

Glutamyl-queuosine tRNA^Asp synthetase (Glu-Q-RS) from Escherichia coli is a paralog of the catalytic core of glutamyl-tRNA synthetase (GluRS) that catalyzes glutamylation of queuosine in the wobble position of tRNA^Asp. Despite important structural similarities, Glu-Q-RS and GluRS diverge strongly by their functional properties. The only feature common to both enzymes consists in the activation of Glu to form Glu-AMP, the intermediate of transfer RNA (tRNA) aminoacylation. However, both enzymes differ by the mechanism of selection of the cognate amino acid and by the mechanism of its activation. Whereas GluRS selects l-Glu and activates it only in the presence of the cognate tRNA^Glu, Glu-Q-RS forms Glu-AMP in the absence of tRNA. Moreover, while GluRS transfers the activated Glu to the 3′ accepting end of the cognate tRNA^Glu, Glu-Q-RS transfers the activated Glu to Q34 located in the anticodon loop of the noncognate tRNA^Asp. In order to gain insight into the structural elements leading to distinct mechanisms of amino acid activation, we solved the three-dimensional structure of Glu-Q-RS complexed to Glu and compared it to the structure of the GluRS·Glu complex. Comparison of the catalytic site of Glu-Q-RS with that of GluRS, combined with binding experiments of amino acids, shows that a restricted number of residues determine distinct catalytic properties of amino acid recognition and activation by the two enzymes. Furthermore, to explore the structural basis of the distinct aminoacylation properties of the two enzymes and to understand why Glu-Q-RS glutamylates only tRNA^Asp among the tRNAs possessing queuosine in position 34, we performed a tRNA mutational analysis to search for the elements of tRNA^Asp that determine recognition by Glu-Q-RS. The analyses made on tRNA^Asp and tRNA^Asn show that the presence of a C in position 38 is crucial for glutamylation of Q34. The results are discussed in the context of the evolution and adaptation of the tRNA glutamylation system.