Structure and time-dependent behavior of acetylated and non-acetylated forms of a molluscan metallothionein.

Cadmium-induced metallothionein in a mollusc, the oyster Crassostrea virginica, occurs in both blocked and unblocked forms (Roesijadi, G., Kielland, S.L. and Klerks, P. (1989) Arch. Biochem. Biophys. 273, 403-413). The block, which is the sole difference in the structure of the two proteins, was identified as an acetyl group with use of tandem mass spectrometry. The blocked and unblocked proteins carried N-acetylserine and serine, respectively, at the N-terminus and were designated CvNAcMT and CvMT. Only CvNAcMT was detected under basal conditions. Both forms were induced by Cd. Pulse-labeling with [35S]cyteine at specified times during exposure showed that the rate of CvNAcMT synthesis in gills increased rapidly, initially exceeding that of CvMT, then declined to the rate attained by CvMT. Turnover rates for Cd-induced CvMT and CvNAcMT were similar to each other. They appeared to be faster when measured in the absence of Cd in the external medium (k = 0.18 and 0.16/day, respectively), than in its presence (k = 0.03 and 0.06/day, respectively).

Primary sequence of duck metallothionein.

Only one metallothionein appears in domesticated duck upon zinc induction. The complete amino acid sequence has been elucidated. This metallothionein has the same sequence as the chicken metallothionein, as determined by chemical sequencing of overlapping peptides produced by selective proteinase digestion and confirmed by mass spectrometry. The observation that animals of divergent origins share a common gene product presents an example of extreme conservation of a stress-inducible protein.

Primary structure of molluscan metallothioneins deduced from PCR-amplified cDNA and mass spectrometry of purified proteins.

The primary structure of metallothioneins (MT) of a mollusc, the oyster Crassostrea virginica, was determined by molecular cloning and mass spectrometry of purified proteins. The cloning strategy included PCR amplification of the responsible cDNAs from total cDNA using completely degenerate oligonucleotides (derived from the N-terminal amino acid sequence) and oligo(dT)20 as primers. Primer extension off mRNA was used as an independent determination of the nucleotide sequence represented by the degenerate PCR primers. The deduced amino acid sequence was consistent with characteristics of class I MT. Twenty-one cysteine residues, were arranged in nine Cys-X-Cys motifs, five as Cys-Lys-Cys. A single Cys-X-X-Cys motif was also observed. Two MTs that differ only in the presence or absence of an N-acetyl group exist in this organism. Masses of tryptic peptides of purified MTs corresponded with those of peptides predicted from tryptic cleavages of the deduced amino acid sequence. Allowing for known N-terminal modifications, 96% of the deduced sequence was confirmed by mass spectrometry. Comparison (FASTA algorithm) of the primary structure of the oyster MTs with those of other species indicated a higher similarity with vertebrate MTs than with those of other invertebrates.

Protein kinase C and amyloid precursor protein processing in skin fibroblasts from sporadic and familial Alzheimer's disease cases.

Non-amyloidogenic alpha-secretase processing of amyloid precursor protein (APP) is stimulated by protein kinase C (PKC). Levels and activity of PKC are decreased in sporadic Alzheimer's disease skin fibroblasts. We investigated whether alterations in PKC and PKC-mediated APP processing occur also in fibroblasts established from individuals with familial Alzheimer's disease APP KM670/671NL, PS1 M146V and H163Y mutations. These pathogenic mutations are known to alter APP metabolism to increase Abeta. PKC activities, but not levels, were decreased by 50% in soluble fractions from sporadic Alzheimer's disease cases. In contrast, familial Alzheimer's disease fibroblasts showed no significant changes in PKC enzyme activity. Fibroblasts bearing the APP KM670/671NL mutation showed no significant differences in either PKC levels or PKC-mediated soluble APP (APPs) secretion, compared to controls. Fibroblasts bearing PS1 M146V and H163Y mutations showed a 30% increase in soluble PKC levels and a 40% decrease in PKC-mediated APPs secretion. These results indicate that PKC deficits are unlikely to contribute to increased Abeta seen with APP and PS1 mutations, and also that PS1 mutations decrease alpha-secretase derived APPs production independently of altered PKC activity.

Mass spectrometric analysis of proteins.

The past year has seen greatly increased acceptance and application of the analytical capabilities of mass spectrometry by the biochemical community. The technique has been used to provide accurate mass determinations of non-covalently bound protein complexes, rapid mapping of molecular weights of altered peptides in protease digests, sequencing by collisional activation in tandem mass spectrometry, characterization of glycosylation and other modifications, and quantitation of peptides used in clinical diagnostics.

Abnormalities in Alzheimer's disease fibroblasts bearing the APP670/671 mutation.

Abnormalities in cultured fibroblasts from familial Alzheimer's Disease (FAD) cases uniquely enable the determination of how gene defects alter cell biology in living tissue from affected individuals. The current study focused on measures of calcium regulation and oxidative metabolism in fibroblast lines from controls and FAD individuals with the Swedish APP670/671 mutation. Bombesin-induced elevations in calcium in APP670/671 mutation-bearing lines were reduced by 40% (p < 0.05), a striking contrast to the 100% increase seen in sporadic AD and presenilin-1 (PS1) mutation-bearing cells in previously published studies. The APP670/671 mutation-bearing lines did not exhibit the exaggerated 4-bromo-A23187 releasable pool of calcium following 10 nM bradykinin, the enhanced sensitivity of calcium stores to low concentrations of bradykinin, nor the reduced activity of alpha-ketoglutarate dehydrogenase previously reported in cells from sporadic AD and mutant PS1 FAD. Thus, an altered regulation of internal calcium stores is common to all AD lines, but the calcium pool affected and the polarity of the alteration varies, apparently in association with particular gene mutations. Comparison of signal transduction in cell lines from multiple, genetically characterized AD families will allow testing of the hypothesis that these various pathogenic FAD abnormalities that lead to AD converge at the level of abnormal signal transduction.

Differential regulation of adenylyl cyclase in fibroblasts from sporadic and familial Alzheimer's disease cases with PS1 and APP mutations.

beta-Adrenoceptor- and forskolin-stimulated adenylyl cyclase activities were determined in primary skin fibroblasts established from patients with sporadic Alzheimer's disease (AD) and from individuals with familial APP KM670/671NL, PS1 M146V and PS1 H163Y mutations. Our data showed a significantly decreased beta-adrenoceptor-stimulated adenylyl cyclase activity in fibroblasts from sporadic AD compared with age-matched controls (p < 0.001, Student's unpaired t-test). In contrast, both beta-adrenoceptor- and forskolin-stimulated adenylyl cyclase activities were significantly increased in fibroblasts bearing PS1 M146V and PS1 H163Y mutations compared with controls (p < 0.01 and p < 0.05, respectively). No differences were seen between cell lines with and without the Swedish APP KM670/671NL double mutation. We suggest that various gene mutations associated with AD have different consequences for the regulation of adenylyl cyclase signal transduction in this disorder.

Disrupted beta 1-adrenoceptor-G protein coupling in the temporal cortex of patients with Alzheimer's disease.

The efficacy of beta 1-adrenoceptor-G protein coupling was studied in postmortem temporal cortex synaptic membranes from a series of control and Alzheimer's disease subjects. For the control cases, the non-hydrolysable GTP analogue 5'-guanylylimidodiphosphate (Gpp[NH]p) gave a significant reduction in the affinity of the agonist isoprenaline to displace binding of the radiolabelled antagonist (+/)-4-(3-t-butylamino-2-hydroxypropoxy)[5,7-3H]benzimidazol-2-one ([3H]CGP-12177). This effect was attributed to the conversion of high agonist-affinity sites to a lower-affinity state and was not found for the Alzheimer's disease cases. These data indicate that a disruption of beta 1-adrenoceptor-G protein coupling occurs in the temporal cortex of Alzheimer's disease patients.

A novel pathogenic mutation (Leu262Phe) found in the presenilin 1 gene in early-onset Alzheimer's disease.

Several mutations causing early-onset familial Alzheimer's disease (AD) have been detected in the presenilin 1 (PS-1) gene. Pathogenic mutations have also been described in an homologous gene, presenilin 2 (PS-2). In order to screen for mutations in these genes, cDNA samples from early-onset AD cases were analysed, using single strand conformation polymorphism (SSCP) and direct cDNA sequencing. Two missense mutations in the PS-1 gene were detected, a previously unidentified amino acid substitution Leu262Phe and an earlier reported amino acid substitution Glu318Gly. No disease-related mutations were found in the PS-2 gene.

Primary structure of bovine adenosine deaminase.

Derivatized bovine adenosine deaminase is used in enzyme replacement therapy and as an adjunct to gene therapy against severe combined immunodeficiency syndrome. Although a gene sequence is known for human adenosine deaminase, the structure of the bovine enzyme has not been characterized. Structure studies using mass spectrometry are reported here that evaluate sequence, processing, post-translational modifications and the extent of homology between the human protein and its therapeutic surrogate.

Formation of cytotoxic transthyretin is not dependent on inter-molecular disulphide bridges commonly found within the amyloid form.

Familial amyloidotic polyneuropathy (FAP) is linked to destabilising point mutations in the human plasma protein transthyretin (TTR). Consistent with similar amyloid disorders, low molecular weight TTR oligomers have been shown to exert the major cytotoxic effect. The amyloid structure of TTR contains non-native inter-molecular disulphide linkages via the cysteine at position 10 (Cys10). Moreover, substitution of Cys10 in a mouse model for TTR-amyloidosis abolishes TTR deposits, indicating an important role of Cys10 in FAP pathogenesis. However, the role of disulphide bridges in TTR cytotoxicity has not been elucidated. By probing Cys10Ser TTR variants to the human neuroblastoma SH-SY5Y cell line, we have addressed this question, and our results clearly show that formation of an inter-molecular disulphide bridge is not a pre-requisite for TTR cytotoxicity. This finding suggests that prevention of inter-molecular TTR disulphide bridges as a therapeutic intervention will not impair the cytotoxic potential of TTR.

Akt activity in presenilin 1 wild-type and mutation transfected human SH-SY5Y neuroblastoma cells after serum deprivation and high glucose stress.

The majority of early-onset familial Alzheimer disease cases are caused by mutations in the genes encoding presenilin 1 (PS1) and presenilin 2 (PS2). Presenilin mutations have been hypothesised to cause Alzheimer disease either by altering amyloid precursor protein metabolism or by increasing the vulnerability of neurons to undergo death by apoptosis. We showed previously that PS1 exon 9 deletion (PS1 DeltaE9) and L250S mutations predispose SH-SY5Y neuroblastoma cells to high glucose stress-induced apoptosis and that the anti-apoptotic effect of insulin-like growth factor I (IGF-I) is compromised by these mutations. The present study investigates whether the susceptibility of PS1 mutation transfected SH-SY5Y cells to undergo apoptosis is likely due to a downregulation of Akt/protein kinase B (Akt), a key intermediate in the phosphatidylinositol 3 (PI3)-kinase arm of the IGF-I signaling pathway. We used two methods to determine the regulation of Akt in response to the pro-apoptotic stimuli of serum deprivation and high glucose stress, as well as treatment with IGF-I. We also looked at the phosphorylatiom state of GSK-3beta at Ser9. Using a kinase assay with immunoprecipitated Akt, we detected an increased Akt activity in PS1 L250S cells at 1 hr after the combination of 20 mM glucose plus 10 nM IGF-I, when compared to the other cell types. This effect, however, was transient in that no mutation related differences were seen at either 6- or 24-hr post-treatment. Immunoblotting for Phospho-Akt as a ratio of total Akt, as well as for GSK-3beta phosphorylated at Ser9 revealed no apparent between cell type and treatment differences. This data strongly indicates that PS1 wt and mutant cells show no major differences in the pattern of Akt regulation after exposure to the pro-apoptotic stimuli of either serum deprivation or high glucose stress, or treatment with IGF-I. It is suggested that another component of IGF-I signaling is likely disrupted in these cells to increase their vulnerability to undergo death by apoptosis.

Recognition of trypsin autolysis products by high-performance liquid chromatography and mass spectrometry.

Potential artifactual contributions are assessed in high-pressure liquid chromatograms and fast atom bombardment mass spectra from autolysis of different preparations of the widely used protease trypsin. Both commercially supplied and laboratory-purified samples were examined. Bovine pancreatic trypsin (1 mg/mL) was found to be completely destroyed in 2 h at pH 8.5, degraded to a complex mixture of small peptides which were characterized by their molecular weights. Some identifications were supported by sequencing by tandem mass spectrometry or by mass spectrometric analysis of the mixture resulting from a single Edman degradation. Autolysis of porcine pancreatic trypsin produced a completely different set of peptides. Five sites of hydrolysis at asparagine residues in bovine trypsin were also identified.

Optimization by mass spectrometry of a tryptophan-specific protein cleavage reaction.

Mass spectrometry has been used to define the reaction of BNPS-skatole (3-bromo-3-methyl-2-(o-nitrophenylsulfenyl)indolenine) with selected proteins and peptides, confirming the cleavage of the protein chain at tryptophan residues, characterizing the products, and identifying the side reactions. A short procedure for carrying out the cleavage reaction with minimal side reactions is presented.

Disulfide bonds in native and recombinant fish growth hormones.

Disulfide linkages were characterized for the first time in a fish growth hormone. Trypsin digestion of chum salmon growth hormone, followed by mass spectrometry established that Cys-49 is linked to Cys-161, while Cys-178 is linked to Cys-186. This is analogous to the big loop, little loop pattern found in human growth hormone. Ninety-three percent of the primary structure of a recombinant rainbow trout growth hormone whose cDNA codes for the same amino acid sequence as chum salmon growth hormone was confirmed by mass spectrometric peptide mapping.

Gamma-aminobutyric acid-B (GABAB) binding sites in postmortem suicide brains.

Gamma-aminobutyric acid-B (GABAB) binding sites labelled with [3H]GABA were determined in postmortem frontal cortex samples of 20 control subjects and 16 suicides. The suicide group was further subdivided according to the method of suicide and the existence of depressive symptoms prior to death. No significant differences in GABAB binding were found either between overall suicide and control groups or between the control group and the other subgroups (violent suicide, nonviolent suicide, nondepressed and depressed suicide victims). A significant increase in GABAB binding was observed in those individuals dying from carbon monoxide poisoning. It is concluded that although GABAB binding sites are not altered in our suicide group, a presynaptic dysfunction might account for the increased GABAB binding found in the carbon monoxide subgroup.

Characterization of muscarinic acetylcholine receptors in cultured adult skin fibroblasts: effects of the Swedish Alzheimer's disease APP 670/671 mutation on binding levels.

We have characterised the muscarinic receptor subtypes found in human skin fibroblasts and compared binding levels in cell lines from members of the Alzheimer's disease family with the Swedish amyloid precursor protein (APP) 670/671 mutation. Binding studies with [3H] quinuclidinyl benzilate ([3H]QNB) and the M2/M4 selective antagonist [3H] (+/-)-5,11-dihydro-11-([(2-[(di-propylamino)methyl]-1- piperidinyl]ethyl)amino]carbonyl)-6H-pyrido(2,3-b)(1,4)benzodiazepine-6- one ([3H]AF-DX 384) revealed the presence of a single population of muscarinic receptors on lysed fibroblast membranes. [3H]QNB binding was displaced by a number of selective muscarinic ligands with a rank order of potency: atropine > himbacine > methoctramine > (+/-)-p-fluoro-hexahydro-sila-difenidol hydrochloride > pirenzepine > muscarinic-toxin-3. APP 670/671 mutation carrying cell lines showed 25-35% lower levels of muscarinic receptors labelled with [3H]QNB, [3H]N-methyl scopolamine and [3H]AF-DX 384, compared to controls. This difference was not statistically significant due to large individual variation. It is concluded that muscarinic receptors on adult skin fibroblasts are predominantly of the M2 subtype. Since these cells do not possess M1 and M3 receptor subtypes, they are unlikely to provide a good model for studying muscarinic receptor regulation of APP processing.

Characterization of enzymatic processes by rapid mix-quench mass spectrometry: the case of dTDP-glucose 4,6-dehydratase.

The single-turnover kinetic mechanism for the reaction catalyzed by dTDP-glucose 4,6-dehydratase (4,6-dehydratase) has been determined by rapid mix-chemical quench mass spectrometry. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was employed to analyze quenched samples. The results were compatible with the postulated reaction mechanism, in which NAD(+) initially oxidizes glucosyl C4 of dTDP-glucose to NADH and dTDP-4-ketoglucose. Next, water is eliminated between C5 and C6 of dTDP-4-ketoglucose to form dTDP-4-ketoglucose-5,6-ene. Hydride transfer from NADH to C6 of dTDP-4-ketoglucose-5,6-ene regenerates NAD(+) and produces the product dTDP-4-keto-6-deoxyglucose. The single-turnover reaction was quenched at various times on the millisecond scale with a mixture of 6 M guanidine hydrochloride and sodium borohydride, which stopped the reaction and reductively stabilized the intermediates and product. Quantitative MALDI-TOF MS analysis of the quenched samples allowed the simultaneous observation of the disappearance of substrate, transient appearance and disappearance of dTDP-hexopyranose-5,6-ene (the reductively stabilized dTDP-4-ketoglucose-5,6-ene), and the appearance of product. Kinetic modeling of the process allowed rate constants for most of the steps of the reaction of dTDP-glucose-d(7) to be evaluated. The transient formation and reaction of dTDP-4-ketoglucose could not be observed, because this intermediate did not accumulate to detectable concentrations.