Looking for residues involved in the muscle acylphosphatase catalytic mechanism and structural stabilization: role of Asn41, Thr42, and Thr46

Asn41, Thr42, and Thr46 are invariant residues in both muscle and erythrocyte acylphosphatases isolated so far. Horse muscle acylphosphatase solution structure suggests their close spatial relationship to Arg23, the main substrate binding site. The catalytic and structural role of such residues, as well as their influence on muscle acylphosphatase stability, was investigated by preparing several gene mutants (Thr42Ala, Thr46Ala, Asn41Ala, Asn41Ser, and Asn41Gln) by oligonucleotide-directed mutagenesis. The mutated genes were cloned and expressed in Escherichia coli, and the mutant enzymes were purified by affinity chromatography and investigated as compared to the wild-type enzyme. The specific activity and substrate affinity of Thr42 and Thr46 mutants were not significantly affected. On the contrary, Asn41 mutants showed a residual negligible activity (about 0.05-0.15% as compared to wild-type enzyme), though maintaining an unchanged binding capability of both substrate and inorganic phosphate, an enzyme competitive inhibitor. According to the 1H nuclear magnetic resonance spectroscopy and circular dichroism results, all mutants elicited well-constrained native-like secondary and tertiary structures. Thermodynamic parameters, as calculated from circular dichroism data, demonstrated a significantly decreased stability of the Thr42 mutant under increasing temperatures and urea concentrations. The reported results strongly support a direct participation of Asn41 to the enzyme catalytic mechanism, indicating that Asn41 mutants may well represent a useful tool for the investigation of the enzyme physiological function by the negative dominant approach.     

Glu318 and Thr319 mutations of cytochrome P450 1A2 remarkably enhance homolytic O-O cleavage of alkyl hydroperoxides. An optical absorption spectral study

Interactions of putative distal mutants of cytochrome P450 1A2 (P450 1A2) with hydroperoxides were studied with optical absorption spectroscopy. The Soret absorption at 393 nm of the wild-type P450 1A2 decreased by adding H2O2, tert-butyl hydroperoxide (TBHP), and cumyl hydroperoxide (CHP), whereas the absorption decrease was accompanied by the appearance of a new band around 423 nm for Glu318 and Thr319 mutants with similar treatments. Spectral simulation based on Gaussian analyses and visible bands indicate that H2O2- and TBHP-induced intermediates' spectra of the wild type are close to that of Compound I of horseradish peroxidase, whereas TBHP- and CHP-induced intermediates' spectra of the mutants are close to that of Compound II of horseradish peroxidase. Based on kinetic parameters of the spectral changes, it is suggested that: 1) TBHP and CHP mainly form porphyrin+.FeIV = O due to heterolytic O-O scission in the wild type, while forming porphyrinFeIV = O for the mutants owing to homolytic O-O scission; and 2) heterolytic O-O scission of H2O2 is not changed by the mutations. Heterolytic/homolytic cleavage ratios for the P450 1A2 reactions with CHP obtained from product analysis were consistent with those obtained from spectral changes.     

Compensatory neutral mutations and the evolution of RNA

There are many examples of RNA molecules in which the secondary structure has been strongly conserved during evolution, but the base sequence is much less conserved, e.g., transfer RNA, ribosomal RNA, and ribonuclease P. A model of compensatory neutral mutations is used here to describe the evolution of the base sequence in RNA helices. There are two loci (i.e., the two sides of the pair) with four alleles at each locus (corresponding to A, C, G, U). Watson-Crick base pairs (AU, CG, GC, and UA) are each assigned a fitness 1, whilst all other pairs are treated as mismatches and assigned fitness 1-s. A population of N diploid individuals is considered with a mutation rate of u per base. For biologically reasonable parameter values, the frequency of mismatches is always small but the frequency of the four matching pairs can vary over a wide range. Using a diffusion model, the stationary distribution for the frequency x of any of the four matching pairs is calculated. The shape depends on the combination of variables beta = 8Nu2/9s. For small beta, the distribution diverges at the two extremes, x = 0 and x = 1-z, where z is the mean frequency of mismatches. The population typically consists almost entirely of one of the four types of matching pairs, but occasionally makes shifts between the four possible states. The mean rate at which these shifts occur is calculated here. The effect of recombination between the two loci is to decrease the probability density at intermediate x, and to increase the weight at the extremes. The rate of transition between the four states is slowed by recombination (as originally shown by Kimura in a two-allele model with irreversible mutation). A very small recombination rate r approximately u2/s is sufficient to increase the mean time between transitions dramatically. In addition to its application to RNA, this model is also relevant to the 'shifting balance' theory describing the drift of populations between alternative equilibria separated by low fitness valleys. Equilibrium values for the frequencies of the different allele combinations in an infinite population are also calculated. It is shown that for low recombination rates the equilibrium is symmetric, but there is a critical recombination rate above which alternative asymmetric equilibria become stable.     

Walker mutations reveal loose relationship between catalytic and channel-gating activities of purified CFTR (cystic fibrosis transmembrane conductance regulator)

The cystic fibrosis transmembrane conductance regulator (CFTR) functions as an ATPase and as a chloride channel. It has been hypothesized, on the basis of electrophysiological findings, that the catalytic activity of CFTR is tightly coupled to the opening and closing of the channel gate. In the present study, to determine the structural basis for the ATPase activity of CFTR, we assessed the effect of mutations within the "Walker A" consensus motifs on ATP hydrolysis by the purified, intact protein. Mutation of the lysine residue in the "Walker A" motif of either the first nucleotide binding fold (CFTRK464A) or the second nucleotide binding fold (CFTRK1250A) inhibited the ATPase activity of the purified intact CFTR protein significantly, by greater than 50%. This finding suggests that the two nucleotide binding folds of CFTR are functioning cooperatively in catalysis. However, the rate of channel gating was only significantly inhibited in one of these purified mutants, CFTRK1250A, suggesting that ATPase activity may not be tightly coupled to channel gating as previously hypothesized.     

A kinetic and thermodynamic analysis of cleavage site mutations in the hammerhead ribozyme

We have produced three forms of human Fas: full-length Fas, Fas with a C-terminal deletion, and a chimera between extracellular Fas and the intracellular domain of the tumor necrosis factor receptor I p55 subunit. We transfected cell lines with these constructs to compare the relative capacity of antibody agonists and the physiological Fas ligand (FasL) to stimulate death. With two agonistic antibodies, the chimera is 100- to 1000-fold more sensitive to induction of death than the full-length Fas. The C-terminal deletion mutant also shows greatly enhanced death in comparison to the wild-type receptor. In contrast, when FasL is used to trigger the Fas pathway, wild-type Fas and the deletion mutant are similarly sensitive, whereas the chimera is 100-fold less susceptible to ligand-mediated killing than Fas. This demonstrates that antibody agonists and natural ligand can stimulate different signaling pathways and emphasizes the limitations of defining physiologically important signaling pathways solely by antibody agonists.     

A catalytic antibody that accelerates the hydrolysis of carbonate esters. Prediction of the binding-site structure of the substrate

A recurrent craniopharyngioma associated with moyamoya vessels was successfully treated by partial removal of the tumor via the transsphenoidal approach followed by gamma-knife radiosurgery. This 19-year-old man was first treated by partial tumor removal and radiotherapy (54Gy) at the age of 6 years. Growth hormone and human chorionic gonadotropin were given from the ages of 13 to 18 years. At ag 17 years, follow-up magnetic resonance imaging (MRI) revealed regrowth of the tumor. At the age of 19 years, he was readmitted for treatment of the enlarging remnant tumor. Neurological examination revealed bilateral blindness. MRI showed marked suprasellar, sphenoidal and bilateral cavernous sinus extension of the tumor. Angiography revealed stenosis of the right internal carotid artery and the M1 and A1 segments of the right cerebral arteries, as well as occlusion of the C3 segment of the left internal carotid artery. There were vault and ethmoidal moyamoya vessels. The patient underwent tumor removal via the transsphenoidal approach, instead of craniotomy, to avoid injury to the transdural anastomosis. The intrasellar solid tumor was partially removed. The tumor was then irradiated by the gamma knife. MRI 15 months after the treatment showed marked reduction of the tumor. The pathogenesis of the moyamoya phenomenon and the choice of the treatment in this patient are discussed.     

Analysis of RNA polymerase by trypsin cleavage. Different structural changes produced by heparin and DNA

Alterations in RNA polymerase structure can be detected using initial trypsin cleavage rates as a conformational probe. Both template (poly[d(A-T) . d(A-T)] and the RNA polymerase inhibitor, heparin, alter the rates at which the subunits of the enzyme are cleaved. However, while the presence of poly[d(A-T) . d(A-T)] slows the cleavage of subunits beta, sigma, and alpha by trypsin, heparin accelerates the cleavage of beta and sigma. Furthermore, the presence of heparin does not prevent the effect of poly[d(A-T) . d(A-T)] on the beta and sigma cleavage rates. Thus, heparin does not eliminate the interaction between DNA and RNA polymerase. That heparin does alter the nature of this interaction is demonstrated by the fact that template decreases the trypsin cleavage rate of subunit alpha in the absence, but not in the presence, of heparin. Like heparin, the addition of RNA to the reaction increases the accessibility of beta and sigma to trypsin. Hence the interaction of heparin with RNA polymerase may mimic the product, rather than the template, interaction.     

Enzymatic specificity and hydrolysis pattern of the catalytic domain of the xylanase Xynl from Rhodothermus marinus

We have investigated the morphology and transfection activity of cationic liposome-DNA complexes (CLDC) under conditions relevant to both in vivo and in vitro studies. Moreover we have attempted to establish structure-function relationships relevant for high transfection activities under both conditions. CLDC were composed of dimethyldioctadecylammonium bromide with either 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or cholesterol (Chol) interacting either with pre-condensed DNA or with uncondensed plasmid DNA. Furthermore for steric stabilization 1% poly(ethylene glycol)-phospholipid conjugate was added to CLDC containing Chol and plasmid DNA. The in vivo studies were carried out in mice following i.v. injection, and the in vitro studies were performed on SK-BR-3 human breast cancer cells in the presence of media with serum. The morphology of the CLDC, monitored by freeze-fracture electron microscopy, was investigated after mixing with mouse serum or the medium where the cells were kept. The substitution of DOPE with Chol, and the addition of N-[omega-methoxypoly(oxyethylene)-alpha-oxycarbonyl-DSPE+ ++ are producing CLDC which are stabilized with respect to time and serum, and are relatively small (100-300 nm). These stabilized complexes show high expression of a marker gene in mouse lungs reaching expression values up to 10 ng luciferase per mg tissue protein, but relatively low expression in SK-BR-3 cells in vitro. Additionally, some of the complexes containing pre-condensed DNA look like 'map-pin' structures showing heads of the size of liposomes and short, stiff and tapering tails. The in vivo transfection activity of these preparations is highest. Similar complexes containing DOPE rather than Chol as helper lipid precipitate in the presence of serum and especially of cell medium and convert into hexagonal lipid (HII) phase. Such complexes, despite their high transfection activity in vitro, show very little transfection activity in vivo. These comparisons may help us to understand the fundamental difference between in vitro and in vivo activity of CLDC: high in vitro transfection activity seems to be associated with hexagonal lipid precipitates whereas high in vivo activity seems to be related with small, stabilized complexes, which in our case also exhibit some protrusions (map-pin structures).     

Differentiation of Chlamydia spp. by sequence determination and restriction endonuclease cleavage of RNase P RNA genes

The amplification of DNA from Chlamydia trachomatis by PCR with degenerated primers yielded a 345-bp fragment of the putative RNase P RNA gene. From the deduced DNA sequence of this gene in C. trachomatis, a modified primer pair was designed. The primer pair was subsequently used to obtain the corresponding gene products from Chlamydia pneumoniae and Chlamydia psittaci. Sequence comparisons revealed similarities of 76.6% between C. trachomatis and C. pneumoniae, 79.5% between C. trachomatis and C. psittaci, and 84.7% between C. pneumoniae and C. psittaci. Furthermore, the three species were differentiated by fragment length polymorphism analysis after restriction enzyme cleavage of the PCR products. Sequence variations among 14 serotypes of C. trachomatis were confined to one purine base substitution in the putative RNase P RNA gene of lymphogranuloma venereum strains L1 to L3. Complete sequence similarity was found for nine strains of C. pneumoniae of different geographic origins. Taken together, our results indicate a possibility of the general application of this method in clinical bacteriology. Analysis of the secondary structures of the putative RNase P RNA genes from the different Chlamydia species suggested that a novel structural element in the domain of RNase P RNA is involved in base pairing with the 3'-terminal CCA motif of a tRNA precursor. This structure has not previously been found among RNase P RNAs of members of the division Bacteria.     

Three novel SRY mutations in XY gonadal dysgenesis and the enigma of XY gonadal dysgenesis cases without SRY mutations

Mutations in the Y-located testis-determining gene SRY are one cause for XY sex reversal. We have previously identified four SRY mutations in a total of 45 sex-reversed females with XY gonadal dysgenesis (XY GD). In a new sample of 16 XY GD cases, three previously undescribed SRY mutations were identified. Two are point mutations that lead to amino acid substitutions in the HMG domain of SRY, M64R, and F67V. The third SRY mutation is a single base insertion 5' to the HMG box within codon 43, converting this lysine codon to a stop codon (K43X). A total of 33 SRY mutations have so far been described that account for only 10-15% of XY GD females. A further 10-15% of these cases result from deletion of SRY due to aberrant X/Y interchange. The etiology of the remaining 70-80% of XY GD cases is still enigmatic. Possible explanations for these XY sex-reversal cases are discussed.     

Enzymatic activity of poliovirus RNA polymerases with mutations at the tyrosine residue of the conserved YGDD motif: isolation and characterization of polioviruses containing RNA polymerases with FGDD and MGDD sequences

The poliovirus RNA-dependent RNA polymerase (3Dpol) shares a region of homology with all RNA polymerases, centered around the amino acid motif YGDD, which has been postulated to be involved in the catalytic activity of the enzyme. Using oligonucleotide site-directed mutagenesis, we substituted the tyrosine at this motif of the poliovirus RNA-dependent RNA polymerase with cysteine, histidine, isoleucine, methionine, phenylalanine, or serine. The enzymes were expressed in Escherichia coli, and in vitro enzyme activity was tested. The phenylalanine and methionine substitutions resulted in enzymes with activity equal to that of the wild-type enzyme. The cysteine substitution resulted in an enzyme with approximately 50% of the wild-type activity, while the serine substitution resulted in an enzyme with approximately 10% of the wild-type activity; the isoleucine and histidine substitutions resulted in background levels of enzyme activity. To assess the effects of the mutants in viral replication, the mutant polymerase genes were subcloned into the infectious cDNA clone of poliovirus. Transfection of poliovirus cDNA containing the phenylalanine mutation in 3Dpol gave rise to virus in all of the transfection trials, while cDNA containing the methionine mutation resulted in virus in only 3 of 40 transfections. Transfection of cDNAs containing the other substitutions at the tyrosine residue did not result in infectious virus. The recovered viruses demonstrated kinetics of replication similar to those of the wild-type virus, as measured by [3H]uridine incorporation at either 37 or 39 degrees C. RNA sequence analysis of the 3Dpol gene of both viruses demonstrated that the tyrosine-to-phenylalanine or tyrosine-to-methionine mutation was still present. No other differences in the 3Dpol gene between the wild-type and phenylalanine-containing virus were found. The virus containing the methionine mutation also contained two other nucleotide changes from the wild-type 3Dpol sequence; one resulted in a glutamic acid-to-aspartic acid change at amino acid 108 of the polymerase, and the other resulted in a C-to-T base change at nucleotide 6724, which did not result in an amino acid change. To confirm that the second amino acid mutation found in the 3Dpol gene of the methionine-substituted virus allowed for replication ability, a mutation corresponding to the glutamic acid-to-aspartic acid change was made in the polymerase containing the methionine substitution, and this double-mutant polymerase was expressed in E. coli. The double-mutant enzyme was as active as the wild-type enzyme under in vitro assay conditions.(ABSTRACT TRUNCATED AT 400 WORDS)     

A conserved deamidation site at Asn 2 in the catalytic subunit of mammalian cAMP-dependent protein kinase detected by capillary LC-MS and tandem mass spectrometry

The N-terminal sequence myr-Gly-Asn is conserved among the myristoylated cAPK (protein kinase A) catalytic subunit isozymes Calpha, Cbeta, and Cgamma. By capillary LC-MS and tandem MS, we show that, in approximately one third of the Calpha and Cbeta enzyme populations from cattle, pig, rabbit, and rat striated muscle, Asn 2 is deamidated to Asp 2. This deamidation accounts for the major isoelectric variants of the cAPK C-subunits formerly called CA and CB. Deamidation also includes characteristic isoaspartate isomeric peptides from Calpha and Cbeta. Asn 2 deamidation does not occur during C-subunit preparation and is absent in recombinant myristoylated Calpha (rCalpha) from Escherichia coli. Deamidation appears to be the exclusive pathway for introduction of an acidic residue adjacent to the myristoylated N-terminal glycine, verified by the myristoylation negative phenotype of an rCalpha(Asn 2 Asp) mutant. This is the first report thus far of a naturally occurring myr-Gly-Asp sequence. Asp 2 seems to be required for the well-characterized (auto)phosphorylation of the native enzyme at Ser 10. Our results suggest that the myristoylated N terminus of cAPK is a conserved site for deamidation in vivo. Comparable myr-Gly-Asn sequences are found in several signaling proteins. This may be especially significant in view of the recent knowledge that negative charges close to myristic acid in some proteins contribute to regulating their cellular localization.     

Deficiency of the Fas apoptosis pathway without Fas gene mutations in pediatric patients with autoimmunity/lymphoproliferation

Fas (CD95) is a transmembrane molecule that induces programmed cell death (PCD) of lymphocytes. We examined its function in children with chronic thrombocytopenia, serum autoantibodies, and lymphadenopathy and/or splenomegaly. We found that T-cell lines from six of seven patients with this autoimmune/lymphoproliferative disease (ALD) were relatively resistant to PCD induced by monoclonal antibodies to Fas. By contrast, Fas function was normal in control patients with typical chronic idiopathic thrombocytopenic purpura (ITP) without lymphadenopathy. The defect was not due to decreased Fas expression, nor to over-production of soluble forms of Fas. Moreover, it specifically involved the Fas system because PCD was induced in the normal way by methylprednisolone. Complementary DNA sequencing of the Fas gene did not identify any causal mutation in patients with ALD. This distinguished them from patients with the human autoimmune lymphoproliferative syndrome (ALPS), who carry mutations of the Fas gene. Moreover, patients with ALD did not show the peripheral expansion of CD4/CD8 double-negative T cells that characterizes the ALPS phenotype. Fas signaling involves activation of a sphingomyelinase-catalyzing production of ceramide. We found that ceramide-induced PCD was defective in patients with ALD and not in patients with typical chronic ITP. These data suggest that the ALD patient defect involves the Fas signaling pathway downstream from the sphingomyelinase and that Fas gene mutations and double-negative T-cell expansion are not the only signs of a defective Fas system.     

Conserved sequence and structural motifs contribute to the DNA binding and cleavage activities of a geminivirus replication protein

Tomato golden mosaic virus (TGMV), a member of the geminivirus family, has a single-stranded DNA genome that replicates through a rolling circle mechanism in nuclei of infected plant cells. TGMV encodes one essential replication protein, AL1, and recruits the rest of the DNA replication apparatus from its host. AL1 is a multifunctional protein that binds double-stranded DNA, catalyzes cleavage and ligation of single-stranded DNA, and forms oligomers. Earlier experiments showed that the region of TGMV AL1 necessary for DNA binding maps to the N-terminal 181 amino acids of the protein and overlaps the DNA cleavage (amino acids 1-120) and oligomerization (amino acids 134-181) domains. In this study, we generated a series of site-directed mutations in conserved sequence and structural motifs in the overlapping DNA binding and cleavage domains and analyzed their impact on AL1 function in vivo and in vitro. Only two of the fifteen mutant proteins were capable of supporting viral DNA synthesis in tobacco protoplasts. In vitro experiments demonstrated that a pair of predicted alpha-helices with highly conserved charged residues are essential for DNA binding and cleavage. Three sequence motifs conserved among geminivirus AL1 proteins and initiator proteins from other rolling circle systems are also required for both activities. We used truncated AL1 proteins fused to a heterologous dimerization domain to show that the DNA binding domain is located between amino acids 1 and 130 and that binding is dependent on protein dimerization. In contrast, AL1 monomers were sufficient for DNA cleavage and ligation. Together, these results established that the conserved motifs in the AL1 N terminus contribute to DNA binding and cleavage with both activities displaying nearly identical amino acid requirements. However, DNA binding was readily distinguished from cleavage and ligation by its dependence on AL1/AL1 interactions.     

Effects of the mutations Ala30 to Pro and Ala53 to Thr on the physical and morphological properties of alpha-synuclein protein implicated in Parkinson''s disease

Inner centromere protein (INCENP) and centromere protein E (CENPE) are two functionally important proteins of the higher eukaryotic centromere. Using a mouse Incenp genomic DNA and a mouse Cenpe cDNA to analyze recombinant inbred mouse sets, as well as interspecific backcross panels, we have mapped these genes to the proximal regions of mouse Chromosomes 19 and 6, respectively. Comparison of Cenpe and human CENPE, which maps to chromosome region 4q24-->q25, has further identified a new region of homology between the two species.     

Point mutations causing Bloom's syndrome abolish ATPase and DNA helicase activities of the BLM protein

Bloom's syndrome (BS) is a rare human genetic disorder characterized by mutations within the BLM gene whose primary effects are excessive chromosome breakage and increased rates of sister chromatid interchange in somatic cells. We report the characterization of a murine protein (mBLM), highly related to the product of the human BLM gene. This protein exhibits an ATP-dependent DNA-helicase activity that unwinds DNA in a 3'-5' direction. Single amino acid substitutions found in BS cells, abolish both ATPase and helicase activities of this protein, indicating that defects in these BLM functions may be primarily responsible for BS establishment. These results provide the first evidence suggesting that the enzymatic activities of the BLM product are implicated in the upholding of genomic integrity.     

Simon effect with and without awareness of the accessory stimulus.

The authors investigated whether a Simon effect could be observed in an accessory-stimulus Simon task when participants were unaware of the task-irrelevant accessory cue. In Experiment 1A a central visual target was accompanied by a suprathreshold visual lateral cue. A regular Simon effect (i.e., faster cue-response corresponding reaction times [RTs]) was found. Experiment 1B demonstrated that this effect cannot be attributed to perceptual grouping of the target and cue. Experiments 2A, 2B, and 2C showed a reverse Simon effect (i.e., faster noncorresponding RTs) when participants were not aware of the cue. In this condition, the Simon effect would occur relative to the reorientation of attention from the cue, which would initially capture attention, toward the target. This conclusion is supported by the results of Experiments 3A and 3B, in which the reorientation of attention was induced by having the target flash after its onset. With suprathreshold cues either a reverse or regular Simon effect was observed by using a 100-ms or ≥200-ms onset flashing interval, respectively, whereas with subthreshold cues a reverse Simon effect was found irrespective of the interval length. (PsycINFO Database Record (c) 2010 APA, all rights reserved)