The conserved asparagine and arginine are essential for catalysis of mammalian adenylyl cyclase

Mammalian adenylyl cyclases have two homologous cytoplasmic domains (C1 and C2), and both domains are required for the high enzymatic activity. Mutational and genetic analyses of type I and soluble adenylyl cyclases suggest that the C2 domain is catalytically active and the C1 domain is not; the role of the C1 domain is to promote the catalytic activity of the C2 domain. Two amino acid residues, Asn-1025 and Arg-1029 of type II adenylyl cyclase, are conserved among the C2 domains, but not among the C1 domains, of adenylyl cyclases with 12 putative transmembrane helices. Mutations at each amino acid residue alone result in a 30-100-fold reduction in Kcat of adenylyl cyclase. However, the same mutations do not affect the Km for ATP, the half-maximal concentration (EC50) for the C2 domain of type II adenylyl cyclase to associate with the C1 domain of type I adenylyl cyclase and achieve maximal enzyme activity, or the EC50 for forskolin to maximally activate enzyme activity with or without Gsalpha. This indicates that the mutations at these two residues do not cause gross structural alteration. Thus, these two conserved amino acid residues appear to be crucial for catalysis, and their absence from the C1 domains may account for its lack of catalytic activity. Mutations at both amino acid residues together result in a 3,000-fold reduction in Kcat of adenylyl cyclase, suggesting that these two residues have additive effects in catalysis. A second site suppressor of the Asn-1025 to Ser mutant protein has been isolated. This suppressor has 17-fold higher activity than the mutant and has a Pro-1015 to Ser mutation.     

The conserved arginine in rho-GTPase-activating protein is essential for efficient catalysis but not for complex formation with Rho.GDP and aluminum fluoride

The Rho family of small GTP-binding proteins are downregulated by an intrinsic GTPase, which is enhanced by GTPase-activating proteins (GAPs). RhoGAPs contain a single conserved arginine residue that has been proposed to be involved in catalysis. Here, the role of this arginine has been elucidated by mutagenesis followed by determination of catalytic and equilibrium binding constants using single-turnover kinetics, isothermal titration calorimetry, and scintillation proximity assays. The turnover numbers for wild-type, R282A, and R282K RhoGAPs were 5.4, 0.023, and 0.010 s-1, respectively. Thus, the function of this arginine could not be replaced by lysine or alanine. Nevertheless, the R282A mutation had a minimal effect on the binding affinity of RhoGAP for either Rho. GTP or Rho.GMPPNP, which confirms the importance of the arginine residue for catalysis as opposed to formation of the protein-protein complex. The R282A mutant RhoGAP still increased the hydrolysis rate of Rho.GTP by 160-fold, whereas the wild-type enzyme increased it by 38000-fold. We conclude that this arginine contributes half of the total reduction of activation energy of catalysis. In the presence of aluminum fluoride, the R282A mutant RhoGAP binds almost as well as the wild type to Rho.GDP, demonstrating that the conserved arginine is not required for this interaction. The affinity of wild-type RhoGAP for the triphosphate form of Rho is similar to that for Rho.GDP with aluminum fluoride. These last two observations show that this complex is not associated with the free energy changes expected for the transition state, although the Rho.GDP.AlF4-.RhoGAP complex might well be a close structural approximation.     

A conserved aspartate of tRNA pseudouridine synthase is essential for activity and a probable nucleophilic catalyst

tRNA pseudouridine synthase I catalyzes the conversion of uridine to pseudouridine at positions 38, 39, and/or 40 in the anticodon loop of many tRNAs. Pseudouridine synthase I was cloned behind a T7 promoter and expressed in Escherichia coli to about 20% of total soluble proteins. Fluorouracil-substituted tRNA caused a time-dependent inactivation of pseudouridine synthase I and formed a covalent complex with the enzyme that involved the FUMP at position 39. Asp60, conserved in all known and putative pseudouridine synthases, was mutated to amino acids with diverse side chains. All Asp60 mutants bound tRNA but were catalytically inactive and failed to form covalent complexes with fluorouracil-substituted tRNA. We conclude that the conserved Asp60 is essential for pseudouridine synthase activity and propose mechanisms which involve this residue in important catalytic roles.     

A core promoter hairpin is essential for subgenomic RNA synthesis in alfalfa mosaic alfamovirus and is conserved in other Bromoviridae

OBJECTIVE: In recent years, there has been debate concerning whether distinct subgroups exist within the eating disorder, not otherwise specified (EDNOS) diagnostic category. One subgroup that has been suggested is binge-eating disorder (BED). While BED has received some research attention, relatively little is known about other possible subgroups within the EDNOS category. The purpose of the present study is to empirically investigate whether distinct subgroups exist within the diagnostic category of EDNOS. METHOD: Participants were 53 EDNOS patients who presented to psychotherapy clinics for treatment of an eating disorder. Information gathered from a clinical assessment, which included a clinical interview and self-report questionnaires, was used in the analyses of the study. RESULTS: Using cluster analytic procedures, two subgroups of patients diagnosed with EDNOS were identified. The two subgroups differed from each other in terms of weight, binging, and body image variables. Specifically, the second subgroup (of 11 patients) appeared to be a distinct subgroup of overweight binge-eating patients, while the first subgroup appeared to be a heterogenous group of EDNOS patients. The overweight binge-eating subgroup was significantly higher in current weight, in reported highest adult weight, in reported higher lowest adult weight, and had more binges per week than the heterogenous EDNOS subgroup. Interestingly, the two subgroups did not differ in terms of self-reported purging and/or compensatory behaviors (e.g., vomiting and laxative use). DISCUSSION: The results of the present study provide preliminary support for a distinct subgroup within the EDNOS diagnostic category. This subgroup resembles BED, with the exception of the presence of purging behaviors. The findings of the present study suggest the need to further investigate the exclusionary criteria of purging/compensatory behaviors for the BED diagnosis.     

Covalent catalysis in nucleotidyl transfer. A KTDG motif essential for enzyme-GMP complex formation by mRNA capping enzyme is conserved at the active sites of RNA and DNA ligases

Vaccinia virus RNA capping enzyme, a heterodimer of 95- and 31-kDa subunits, catalyzes transfer of GMP from GTP to the 5'-diphosphate terminus of RNA via a covalent enzyme-guanylate intermediate. The GMP residue is attached to the 95-kDa subunit through a phosphoamide bond to the epsilon-amino group of a lysine residue. The amino acid sequence of the large subunit includes a lysine-containing motif, Tyr-X-X-X-Lys260-Thr-Asp-Gly, that is conserved in the RNA guanylyltransferases encoded by Shope fibroma virus and Saccharomyces cerevisiae. The KXDG motif is also encountered at the sites of covalent adenylylation of bacteriophage T4 RNA ligase and mammalian DNA ligase I (Thogerson, H. C., Morris, H. R., Rand, K. N., and Gait, M. J. (1985) Eur. J. Biochem. 147, 325-329; Tomkinson, A. E., Totty, N. F., Ginsburg, M., and Lindahl, T. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 400-404). We find that conservative amino acid substitutions at three out of four positions within the KTDG sequence of vaccinia capping enzyme either prevent or strongly inhibit enzyme-guanylate formation. The conserved motif is therefore an essential component of the guanylyltransferase domain. Lys260 is implicated as the active site. Comparison of the sequences of capping enzymes and polynucleotide ligases from diverse sources suggests that KX(D/N)G may be a signature element for covalent catalysis in nucleotidyl transfer.     

Ribonuclease A mutant His119 Asn: the role of histidine in catalysis

BACKGROUND: We wished to investigate central serotonergic function in untreated schizophrenia. METHOD: Thirteen drug-naive, DSM-III-R schizophrenic patients were compared with sex, race, age, weight and menstrual phase matched controls. Plasma prolactin and cortisol responses to a specific serotonergic probe, d-fenfluramine, were measured along with BPRS ratings. RESULTS: Prolactin responses were enhanced in schizophrenic patients compared to controls (P < 0.05) and were correlated positively with BPRS items for depression, anxiety and guilt. Baseline cortisol was also raised in those with schizophrenia (P < 0.001). CONCLUSIONS: Central serotonergic tone may be raised in acute, drug-naive schizophrenia and may be associated with the presence of affective symptomatology.     

Critical role of conserved histidine pairs HNXXH and HDXXH in recombinant human phosphodiesterase 4A

Cyclic AMP-Phosphodiesterases (cAMP-PDEs) catalyse the hydrolysis cAMP to AMP and thus serve to modulate the ligand-->adenylate cyclase-->cAMP-->PKA signal transduction pathway. PDEs exist as a multigene family of enzymes that bear significant sequence homology in the catalytic domains. The sequence alignment of these domains has revealed the presence of two histidine motifs: motif I, HNXXH, and motif II, HDXXH. These amino acid sequences are canonical motifs, which act as ligands for divalent metal cations required for catalytic activity. In this paper, we report human monocyte PDE4A to be a zinc-binding protein. Substitution by site-directed mutagenesis of either histidine in motif I by serine, which is not a ligand for metals, results in complete loss of catalytic activity and loss of sensitivity to divalent metal cation activation. However, similar mutations in motif II gave proteins that retained both approximately 50% of initial activity and the ability to respond differentially to Mg2+, Mn2+ and Co2+. Moreover the motif II mutants exhibited both functional group requirements and retained their pKa values. When the inactive mutants were affinity-labelled with 8-BDB-TcAMP and probed with antibody against cAMP or antibody against PDE4A, Western blots were unaltered. These results show that the conserved histidines in motif I are an absolute requirement for catalytic activity, whereas motif II histidines are required only to achieve maximum activity.     

A novel class of evolutionarily conserved genes defined by piwi are essential for stem cell self-renewal

Germ-line stem cells (GSCs) serve as the source for gametogenesis in diverse organisms. We cloned and characterized the Drosophila piwi gene and showed that it is required for the asymmetric division of GSCs to produce and maintain a daughter GSC but is not essential for the further differentiation of the committed daughter cell. Genetic mosaic and RNA in situ analyses suggest that piwi expression in adjacent somatic cells regulates GSC division. piwi encodes a highly basic novel protein, well conserved during evolution. We isolated piwi homologs in Caenorhabditis elegans and humans and also identified Arabidopsis piwi-like genes known to be required for meristem cell maintenance. Decreasing C. elegans piwi expression reduces the proliferation of GSC-equivalent cells. Thus, piwi represents a novel class of genes required for GSC division in diverse organisms.     

A conserved helicase motif of the AddA subunit of the Bacillus subtilis ATP-dependent nuclease (AddAB) is essential for DNA repair and recombination

We examined the behavior of fetal rat chondrocytes cultured on a bioactive glass-ceramic containing apatite and wollastonite (A.W.G.C.). Biomaterial surface topography and profiles were evaluated by bidimensional profilometry and revealed a rough surface for the glass-ceramic compared to the plastic coverslips used as controls. Chondrocyte attachment was evaluated by measuring the number of attached cells after one day of culture and by morphological observations. Chondrocytes attached in great numbers to the material surface by means of focal contacts containing vinculin and beta1-integrin. Fluorescent labeling of actin and vimentin revealed a poor spreading of chondrocytes on the bioactive glass-ceramic compared to the plastic coverslips, where the cells appeared to adhere intimately to the surface and exhibited polygonal arrays of stress fibers. During the following days of culture, chondrocytes proliferated, colonized the surface of the material, and, finally, on day 10, formed nodular structures composed of round cells separated by a dense extracellular matrix. Furthermore, these clusters of round cells were positive for type II collagen and chondroitin sulfate, both hard markers of the chondrocyte phenotype. In addition, protein synthesis, alkaline phosphatase activity, and proteoglycan production were found to increase gradually during the culture period with a pattern similar to that observed on control cultures. These results demonstrate that the bioactive glass-ceramic tested in this study appears to be a suitable substrate for in vitro chondrocyte attachment, differentiation, and matrix production.     

Effects of substitutions of the conserved histidine residues in human gamma-glutamyl transpeptidase

gamma-Glutamyl transpeptidase possesses two histidine residues at positions 383 and 505 which are conserved in all mammalian and bacterial species. In order to elucidate the functions of these residues, we prepared mutants in which these residues were replaced by Ala. Kinetic analysis of the hydrolysis of L-gamma-glutamyl-p-nitroanilide indicated that substitution at His-383 decreased the Vmax value to 14% of that of the wild type, but had no effect on Vmax/K(m). In reactions involving glycylglycine as the acceptor substrate, the Vmax value of this mutant decreased to 38% with little alteration of Vmax/K(m) for L-gamma-glutamyl-p-nitroanilide as a gamma-glutamyl donor, but with a significant reduction of Vmax/K(m) for the acceptor. These results show that this substitution causes impairment of the step in which the free enzyme is regenerated from the gamma-glutamyl enzyme by water or an acceptor substrate. On the other hand, replacement of His-505 resulted in a decrease of the Vmax value for transpeptidation to about 10% of that of the wild type despite no substantial effect on the Vmax value for the hydrolysis reaction. However, this substitution did not affect Vmax/K(m) for the acceptor on transpeptidation. Thus, the formation of a non-productive enzyme-substrate complex with the acceptor substrate would decrease the Vmax value on transpeptidation. These results suggest that His-383 plays an important catalytic role in facilitating the degradation of the gamma-glutamyl-enzyme through hydrolysis or transfer of the gamma-glutamyl moiety to an acceptor. It was also shown that His-505 is important in the formation of a complex of the gamma-glutamyl enzyme with the acceptor substrate even though it plays no critical role in the catalysis. Although the pH-dependence profile and the van't Hoff plot for the ionic group responsible for enzyme activity were consistent with the requirement of a histidine residue, neither of the conserved histidines could be assigned as such an ionic group. This suggests that another histidine residue(s) might play an essential role in the enzyme function.     

Characterization of histidine residues essential for receptor binding and activity of nerve growth factor

The role of the four histidine residues in receptor binding and activity of mouse nerve growth factor (NGF) was investigated using both site-directed mutagenesis and chemical modification with diethyl pyrocarbonate. Replacement of His-75 or His-84 with alanine resulted in decreased biological activity and decreased affinity for p140(trkA); however, with H75A only, a 5-fold increased affinity toward p75(LANR) was observed. The effect of simultaneous replacement of both His-75 and His-84 was neither additive nor synergistic. Slight perturbations in circular dichroism spectra and weakened self-association of the mutants indicated that His-75 and His-84 may be involved in stability, dimerization, and/or folding of NGF. Diethyl pyrocarbonate modification of His-4 and His-8 in the H75A/H84Q double mutant abolished neuritogenesis, binding to both receptors, and phosphorylation of p140(trkA) in PC12 cells. These chemical and mutational results confirm and clarify previous evidence for the involvement of His-75 and His-84 (Dunbar, J. C., Tregear, G. W., and Bradshaw, R. A. (1984) J. Protein Chem. 3, 349-356) or His-4 and His-8 (Shih, A., Laramee, G. R., Schmelzer, C. H., Burton, L. E., and Winslow, J. W. (1994) J. Biol. Chem. 269, 27679-27686) in receptor binding of NGF. At least three and possibly all four histidines, which are located in three spatially distinct regions, contribute to maintenance of functional sites that are essential for receptor binding and activity of NGF.     

Identification of a histidine residue essential for enzymatic activity of group B streptococcal hyaluronate lyase

Parascalene block is a technique of blocking the brachial plexus at the lateral border of the anterior scalene muscle superior to the clavicle. The objective of this study was to define the position of the needle in parascalene block with relationship to the brachial plexus and the dome of the pleura, which is important in determining whether this technique minimizes the incidence of pneumothorax. In the first group, 10 patients scheduled for minor upper extremity surgery agreed to parascalene block, which was performed in the computed tomographic examination room. In the second group, 10 volunteers agreed to have markers placed at the point where a needle would be inserted for parascalene block. The computed tomographic study at the level of the needle insertion or the marker revealed that this level was superior to the dome of the pleura. The distances from the skin to the interscalene groove and the interscalene groove to the first rib at the level of the needle insertion or the marker in both groups were measured to be 17 +/- 4 mm and 15 +/- 3 mm, respectively. This study suggests that the level of the parascalene needle entry is superior to the dome of the pleura. At this level, the incidence of pneumothorax should be minimized.     

Sonic hedgehog signaling is essential for hair development

BACKGROUND: The skin is responsible for forming a variety of epidermal structures that differ amongst vertebrates. In each case the specific structure (for example scale, feather or hair) arises from an epidermal placode as a result of epithelial-mesenchymal interactions with the underlying dermal mesenchyme. Expression of members of the Wnt, Hedgehog and bone morphogenetic protein families (Wnt10b, Sonic hedgehog (Shh) and Bmp2/Bmp4, respectively) in the epidermis correlates with the initiation of hair follicle formation. Further, their expression continues into either the epidermally derived hair matrix which forms the hair itself, or the dermal papilla which is responsible for induction of the hair matrix. To address the role of Shh in the hair follicle, we have examined Shh null mutant mice. RESULTS: We found that follicle development in the Shh mutant embryo arrested after the initial epidermal-dermal interactions that lead to the formation of a dermal papilla anlage and ingrowth of the epidermis. Wnt10b, Bmp2 and Bmp4 continued to be expressed at this time, however. When grafted to nude mice (which lack T cells), Shh mutant skin gave rise to large abnormal follicles containing a small dermal papilla. Although these follicles showed high rates of proliferation and some differentiation of hair matrix cells into hair-shaft-like material, no hair was formed. CONCLUSIONS: Shh signaling is not required for initiating hair follicle development. Shh signaling is essential, however, for controlling ingrowth and morphogenesis of the hair follicle.     

Identification of three aspartic acid residues essential for catalysis by the RusA holliday junction resolvase

RusA is a Holliday junction resolvase encoded by the cryptic prophage DLP12 of Escherichia coli K-12 that can be activated to promote homologous recombination and DNA repair in resolution-deficient mutants lacking the RuvABC proteins. Database searches with the 120 amino acid residue RusA sequence identified 11 homologues from diverse species, including one from the extreme thermophile Aquifex aeolicus, which suggests that RusA may be of ancient bacterial ancestry. A multiple alignment of these sequences revealed seven conserved or invariant acidic residues in the C-terminal half of the E. coli protein. By making site-directed mutations at these positions and analysing the ability of the mutant proteins to promote DNA repair in vivo and to resolve junctions in vitro, we identified three aspartic acid residues (D70, D72 and D91) that are essential for catalysis and that provide the first insight into the active-site mechanism of junction resolution by RusA. Substitution of any one of these three residues with asparagine reduces resolution activity >80-fold. The mutant proteins retain the ability to bind junction DNA regardless of the DNA sequence or of the mobility of the crossover. They interfere with the function of the RuvABC proteins in vivo, when expressed from a multicopy plasmid, an effect that is reproducible in vitro and that reflects the fact that the RusA proteins have a higher affinity for junction DNA in the presence of Mg2+ than do the RuvA and RuvC proteins. The D70N protein has a greater affinity for junctions in Mg2+ than does the wild-type, which indicates that the negatively charged carboxyl group of the aspartate residue plays a critical role at the active site of RusA. Electrostatic repulsions between D70, D72 and D91 may help to form a classical Mg2+-binding pocket.     

Dystonin is essential for maintaining neuronal cytoskeleton organization

The mouse neurological mutant dystonia musculorum (dt) suffers from a hereditary sensory neuropathy. We have previously described the cloning and characterization of the dt gene, which we named dystonin (Dst). We had shown that dystonin is a neural isoform of bullous pemphigoid antigen 1 (Bpag1) with an N-terminal actin-binding domain. It has been shown previously that dystonin is a cytoskeletal linker protein, forming a bridge between F-actin and intermediate filaments. Here, we have used two different antibody preparations against dystonin and detected a high-molecular-weight protein in immunoblot analysis of spinal cord extracts. We also show that this high-molecular-weight protein was not detectable in the nervous system of all dt alleles tested. Immunohistochemical analysis revealed that dystonin was present in different compartments of neurons--cell bodies, dendrites, and axons, regions which are rich in the three elements of the cytoskeleton (F-actin, neurofilaments, and microtubules). Ultrastructural analysis of dt dorsal root axons revealed disorganization of the neurofilament network and surprisingly also of the microtubule network. In this context it is of interest that we observed altered levels of the microtubule-associated proteins MAP2 and tau in spinal cord neurons of different dt alleles. Finally, dt dorsal root ganglion neurons formed neurites in culture, but the cytoskeleton was disorganized within these neurites. Our results demonstrate that dystonin is essential for maintaining neuronal cytoskeleton integrity but is not required for establishing neuronal morphology.     

MacMARCKS is not essential for phagocytosis in macrophages

MacMARCKS (also known as myristoylated alanine-rich protein kinase C substrate (MARCKS)-related protein) is a member of the MARCKS family of protein kinase C substrates. MacMARCKS contains within it a basic effector domain that contains the serine residues that are phosphorylated by protein kinase C, as well as a calcium/calmodulin and actin-binding site. Two previous reports demonstrated that a macrophage cell line expressing a mutant form of MacMARCKS that lacks the effector domain is defective in phagocytosis and cell adhesion (Zhu, Z., Bao, Z., and Li, J. (1995) J. Biol. Chem. 270, 17652-17655; Li, J., Zhu, Z., and Bao, Z. (1996) J. Biol. Chem. 271, 12985-12990). We report here that macrophages from MacMARCKS null mice phagocytose and spread normally. Thus, although MacMARCKS is recruited to phagosomes, it is not absolutely required for phagocytosis.