Assessment of MAO-B occupancy in the brain with PET and [11C]-L-deprenyl-D2: a dose-finding study with a novel MAO-B inhibitor, EVT 301.

Inhibition of monoamine oxidase type B (MAO-B) activity in the brain is a putative strategy for the treatment of Alzheimer's disease (AD). We performed a dose-selection and validation study of a novel, reversible MAO-B inhibitor, EVT 301. Sixteen healthy volunteers received selegiline (10 mg) or EVT 301 (25, 75, or 150 mg) daily for 7-8 days, and four subjects with AD received 75 mg of EVT 301. MAO-B occupancy in the brain was assessed using positron emission tomography (PET) with [11C]-L-deprenyl-D2. EVT 301 was found to dose-dependently occupy MAO-B in the human brain, with occupancy ranging from 58-78% at a dose of 25 mg to 73-90% at a dose of 150 mg. The corresponding occupancy after selegiline was 77-92%. Determination of MAO-B inhibition in blood platelets underestimated the actual brain occupancy achieved with EVT 301. A daily EVT 301 dose of 75 or 150 mg appears suitable for clinical efficacy studies in patients with AD.

A dominant gene for developmental dyslexia on chromosome 3.

Developmental dyslexia is a neurofunctional disorder characterised by an unexpected difficulty in learning to read and write despite adequate intelligence, motivation, and education. Previous studies have suggested mostly quantitative susceptibility loci for dyslexia on chromosomes 1, 2, 6, and 15, but no genes have been identified yet. We studied a large pedigree, ascertained from 140 families considered, segregating pronounced dyslexia in an autosomal dominant fashion. Affected status and the subtype of dyslexia were determined by neuropsychological tests. A genome scan with 320 markers showed a novel dominant locus linked to dyslexia in the pericentromeric region of chromosome 3 with a multipoint lod score of 3.84. Nineteen out of 21 affected pedigree members shared this region identical by descent (corrected p<0.001). Previously implicated genomic regions showed no evidence for linkage. Sequencing of two positional candidate genes, 5HT1F and DRD3, did not support their role in dyslexia. The new locus on chromosome 3 is associated with deficits in all three essential components involved in the reading process, namely phonological awareness, rapid naming, and verbal short term memory.

Revisiting the catalytic CuZ cluster of nitrous oxide (N2O) reductase. Evidence of a bridging inorganic sulfur.

Nitrous-oxide reductases (N2OR) catalyze the two-electron reduction of N(2)O to N(2). The crystal structure of N2ORs from Pseudomonas nautica (Pn) and Paracoccus denitrificans (Pd) were solved at resolutions of 2.4 and 1.6 A, respectively. The Pn N2OR structure revealed that the catalytic CuZ center belongs to a new type of metal cluster in which four copper ions are liganded by seven histidine residues. A bridging oxygen moiety and two other hydroxide ligands were proposed to complete the ligation scheme (Brown, K., Tegoni, M., Prudencio, M., Pereira, A. S., Besson, S., Moura, J. J. G., Moura, I., and Cambillau, C. (2000) Nat. Struct. Biol. 7, 191-195). However, in the CuZ cluster, inorganic sulfur chemical determination and the high resolution structure of Pd N2OR identified a bridging inorganic sulfur instead of an oxygen. This result reconciles the novel CuZ cluster with the hitherto puzzling spectroscopic data.

Two translocations of chromosome 15q associated with dyslexia.

Developmental dyslexia is characterised by difficulties in learning to read. As reading is a complex cognitive process, multiple genes are expected to contribute to the pathogenesis of dyslexia. The genetics of dyslexia has been a target of molecular studies during recent years, but so far no genes have been identified. However, a locus for dyslexia on chromosome 15q21 (DYX1) has been established in previous linkage studies. We have identified two families with balanced translocations involving the 15q21-q22 region. In one family, the translocation segregates with specific dyslexia in three family members. In the other family, the translocation is associated with dyslexia in one family member. We have performed fluorescence in situ hybridisation (FISH) studies to refine the position of the putative dyslexia locus further. Our results indicate that both translocation breakpoints on 15q map within an interval of approximately 6-8 Mb between markers D15S143 and D15S1029, further supporting the presence of a locus for specific dyslexia on 15q21.

Expression and mutagenesis of ZntA, a zinc-transporting P-type ATPase from Escherichia coli.

Cation-transporting P-type ATPases comprise a major membrane protein family, the members of which are found in eukaryotes, eubacteria, and archaea. A phylogenetically old branch of the P-type ATPase family is involved in the transport of heavy-metal ions such as copper, silver, cadmium, and zinc. In humans, two homologous P-type ATPases transport copper. Mutations in the human proteins cause disorders of copper metabolism known as Wilson and Menkes diseases. E. coli possesses two genes for heavy-metal translocating P-type ATPases. We have constructed an expression system for one of them, ZntA, which encodes a 732 amino acid residue protein capable of transporting Zn(2+). A vanadate-sensitive, Zn(2+)-dependent ATPase activity is present in the membrane fraction of our expression strain. In addition to Zn(2+), the heavy-metal ions Cd(2+), Pb(2+), and Ag(+) activate the ATPase. Incubation of membranes from the expression strain with [gamma-(33)P]ATP in the presence of Zn(2+), Cd(2+), or Pb(2+) brings about phosphorylation of two membrane proteins with molecular masses of approximately 90 and 190 kDa, most likely representing the ZntA monomer and dimer, respectively. Although Cu(2+) can stimulate phosphorylation by [gamma-(33)P]ATP, it does not activate the ATPase. Cu(2+) also prevents the Zn(2+) activation of the ATPase when present in 2-fold excess over Zn(2+). Ag(+) and Cu(+) appear not to promote phosphorylation of the enzyme. To study the effects of Wilson disease mutations, we have constructed two site-directed mutants of ZntA, His475Gln and Glu470Ala, the human counterparts of which cause Wilson disease. Both mutants show a reduced metal ion stimulated ATPase activity (about 30-40% of the wild-type activity) and are phosphorylated much less efficiently by [gamma-(33)P]ATP than the wild type. In comparison to the wild type, the Glu470Ala mutant is phosphorylated more strongly by [(33)P]P(i), whereas the His475Gln mutant is phosphorylated more weakly. These results suggest that the mutation His475Gln affects the reaction with ATP and P(i) and stabilizes the enzyme in a dephosphorylated state. The Glu470Ala mutant seems to favor the E2 state. We conclude that His475 and Glu470 play important roles in the transport cycles of both the Wilson disease ATPase and ZntA.

The active site of the bacterial nitric oxide reductase is a dinuclear iron center.

A novel, improved method for purification of nitric oxide reductase (NOR) from membranes of Paracoccus denitrificans has been developed. The purified enzyme is a cytochrome bc complex which, according to protein chemical and hydrodynamic data, contains two subunits in a 1:1 stoichiometry. The purified NorBC complex binds 0.87 g of dodecyl maltoside/g of protein and forms a dimer in solution. Similarly, it is dimeric in two-dimensional crystals. Images of these crystals have been processed at 8 A resolution in projection to the membrane. The NorB subunit is homologous to the main catalytic subunit of cytochrome oxidase and is predicted to contain the active bimetallic center in which two NO molecules are turned over to N2O. Metal analysis and heme composition implies that it binds two B-type hemes and a nonheme iron but no copper. NorC is a membrane-anchored cytochrome c. Fourier transform infrared spectroscopy shows that carbon monoxide dissociates from the reduced heme in light and associates with another metal center which is distinct from the copper site of heme/copper oxidases. Electron paramagnetic resonance spectroscopy reveals that NO binds to the reduced enzyme under turnover conditions giving rise to signals near g = 2 and g = 4. The former represents a typical nitrosyl-ferroheme signal whereas the latter is a fingerprint of a nonheme iron/NO adduct. We conclude that the active site of NOR is a dinuclear iron center.

Early-onset Alzheimer's disease with a presenilin-1 mutation at the site corresponding to the Volga German presenilin-2 mutation.

We describe a new mutation causing Alzheimer's disease (AD) in presenilin-1 (N135D) that is at the homologous site to the presenilin 2 mutation (N141I) in Volga German kindreds. The phenotype of PS1 N135D is an early-onset (34-38 years) disease. The mutation forms part of, and extends, the alpha-helical array of mutations in transmembrane 2 of the presenilins and leads to the suggestion that disruption of this helical face is the molecular insult that leads to disease.

A further presenilin 1 mutation in the exon 8 cluster in familial Alzheimer's disease.

Recent studies suggest that mutations in the presenilin 1 gene, which encodes a polypeptide predicted to be a multispanning membrane protein, are responsible for the majority of cases of early onset, autosomal dominant Alzheimer's disease. Here we describe a further mutation in the presenilin 1 gene (R269G) in a family with early onset Alzheimer's disease. This mutation is in exon 8 which appears to be a favoured region for pathogenic mutations. In the presenilin protein the region coded for by this exon is likely to comprise a domain located on the membrane surface. We discuss the likely effects of the exon 8 mutations on the structure of the exon and in the pathogenesis of the disease.

Autosomal recessive adult-onset amyotrophic lateral sclerosis associated with homozygosity for Asp90Ala CuZn-superoxide dismutase mutation. A clinical and genealogical study of 36 patients.

We describe 36 patients (six were apparently sporadic cases and 30 were cases from nine families) with amyotrophic lateral sclerosis (ALS) characterized by a distinct phenotype associated with homozygosity for an Asp90Ala mutation in the CuZn-superoxide dismutase gene. The presenting motor manifestation in all patients was paresis in the legs, with slow progression to the upper extremities and finally to the bulbar muscles. The age of ALS onset varied from 20 to 94 years, with a mean of 44 years. Mean survival time was 13 years for the 11 deceased patients. However, this is probably biased and untypical (low) when compared with the disease duration in the surviving patients, and when considering other medical complications in the deceased patients. The rate of progression was highly variable, even within families. All patients showed signs of involvement of both upper and lower motor neurons. Other neurological features included painful muscle spasms and paraesthesiae in the lower extremities. Two-thirds of patients experienced difficulty with micturition. Electrophysiological studies confirmed the slow progression and spatial distribution of clinical symptoms in the peripheral motor system. Furthermore, [corrected] potentials evoked by transcranial magnetic stimulation (MEP) were compared with those evoked by cervical or lumbosacral electrical stimulation and often revealed marked slowing of transmission in central motor pathways. In Sweden and Finland ALS patients homozygous for the Asp90Ala mutation constitute a phenotypically characteristic subset of motor neuron disease.

Subunit III of cytochrome c oxidase influences the conformation of subunits I and II: an infrared study.

The secondary structure of wild-type Paracoccus denitrificans cytochrome c oxidase obtained by decomposition of the infrared amide I band contains 44% alpha-helix, 18% beta-sheet, 14% beta-turns, 18% loops, and 6% nonordered segments. The mutant lacking subunit III presents a small but significant increase (from 18% to 24%) in the percentage of loops and slight differences in the other components. Using band/area ratios and tyrosine side chain absorption as an inner standard, it is shown that in the absence of subunit III the structure of subunits I and II is altered although no changes in their alpha-helix or beta-sheet content are observed. In the bacterial oxidase, thermal infrared studies show a complex denaturation pattern characterized by the presence of a partially denatured intermediate state. Of the seven predicted subunit III alpha-helices, only four are resistant toward the thermal challenge and behave as expected for typical transmembrane helices. The observation that the absence of subunit III influences the conformation of loop regions in the two other subunits suggests that part of the interaction surface between subunit III and the catalytic subunits might be located outside the lipid bilayer.

Forces and factors that contribute to the structural stability of membrane proteins.

While a considerable amount of literature deals with the structural energetics of water-soluble proteins, relatively little is known about the forces that determine the stability of membrane proteins. Similarly, only a few membrane protein structures are known at atomic resolution, although new structures have recently been described. In this article, we review the current knowledge about the structural features of membrane proteins. We then proceed to summarize the existing literature regarding the thermal stability of bacteriorhodopsin, cytochrome-c oxidase, the band 3 protein, Photosystem II and porins. We conclude that a fundamental difference between soluble and membrane proteins is the high thermal stability of intrabilayer secondary structure elements in membrane proteins. This property manifests itself as incomplete unfolding, and is reflected in the observed low enthalpies of denaturation of most membrane proteins. By contrast, the extramembranous parts of membrane proteins may behave much like soluble proteins. A brief general account of thermodynamics factors that contribute to the stability of water soluble and membrane proteins is presented.

Amyotrophic lateral sclerosis associated with homozygosity for an Asp90Ala mutation in CuZn-superoxide dismutase.

Recent reports have shown heterozygosity for some twenty different mutations in the CuZn-superoxide dismutase (CuZn-SOD) gene in familial amyotrophic lateral sclerosis (FALS), and analysed samples from patients have shown decreased enzymic activity. Here we report homozygosity for an exon 4 mutation, Asp90Ala in fourteen patients among four unrelated ALS families and four apparently sporadic ALS patients from Sweden and Finland. The erythrocyte CuZn-SOD activity is essentially normal. Our findings suggest that this CuZn-SOD mutation causes ALS by a gain of function rather than by loss, and that the Asp90Ala mutation is less detrimental than previously reported mutations.

Forces and factors that contribute to the structural stability of membrane proteins.

While a considerable amount of literature deals with the structural energetics of water-soluble proteins, relatively little is known about the forces that determine the stability of membrane proteins. Similarly, only a few membrane protein structures are known at atomic resolution, although new structures have recently been described. In this article, we review the current knowledge about the structural features of membrane proteins. We then proceed to summarize the existing literature regarding the thermal stability of bacteriorhodopsin, cytochrome-c oxidase, the band 3 protein, Photosystem II and porins. We conclude that a fundamental difference between soluble and membrane proteins is the high thermal stability of intrabilayer secondary structure elements in membrane proteins. This property manifests itself as incomplete unfolding, and is reflected in the observed low enthalpies of denaturation of most membrane proteins. By contrast, the extramembranous parts of membrane proteins may behave much like soluble proteins. A brief general account of thermodynamics factors that contribute to the stability of water soluble and membrane proteins is presented.

Thermodynamic and structural stability of cytochrome c oxidase from Paracoccus denitrificans.

The structural stability of the integral membrane protein cytochrome c oxidase from Paracoccus denitrificans has been measured by high-sensitivity differential scanning calorimetry and Fourier transform infrared spectroscopy. Contrary to the mammalian enzyme or the yeast enzyme, which are composed of 13 subunits, the bacterial enzyme has only three or four subunits, thus providing a unique opportunity to examine the magnitude of the forces that stabilize this enzyme and to establish accurate structural assignments of events observed calorimetrically. In this paper, experiments have been performed with the wild-type enzyme and with a mutant enzyme lacking subunit III. Our results show that subunits I and II form a highly cooperative complex which denatures as a single cooperative unit at 67 degrees C, while subunit III is less stable and denatures 20 degrees C earlier. Reduction of the enzyme causes a large increase in the stability of subunits I and II but has absolutely no effect on subunit III. Despite the lack of a strong interaction between subunit III and the catalytic subunits, the absence of subunit III leads to a turnover-induced loss of electron-transfer activity. The magnitude of the energetic parameters and the infrared spectroscopic experiments indicate that the enzyme does not completely unfold upon thermal denaturation and that significant degrees of structure are preserved. The amount of native alpha-helix structure, which is 45% in the native state, decreases only to 30% after thermal denaturation. Presumably, the residual helical structure existing after thermal denaturation belongs to the intramembranous portions of the protein. The calorimetric behavior of subunit III does not fully conform to that expected for a highly alpha-helical membrane protein. The picture that emerges from these experiments is that, in the temperature-denatured form of the enzyme, most of the extramembranous structural elements are denatured while most of the intramembranous secondary structure is maintained even though native tertiary interactions appear to be disrupted.

Two cysteines, two histidines, and one methionine are ligands of a binuclear purple copper center.

Cytochrome c oxidase contains a copper center, CuA, which is involved in electron transfer from cytochrome c to the oxygen-reducing active site. This center is distinct from types 1, 2, and 3 copper sites and related only to a purple copper center in nitrous oxide reductase. At present it is not clear whether this site is mononuclear or is comprised of two copper atoms in a mixed valence (Cu(I)-Cu(II)) configuration. Here we use a model of CuA, engineered into a structurally related but initially copperless protein, to study the structure of this copper center. The results from biochemical analysis, site-directed mutagenesis, and electrospray mass spectrometry support the binuclear model. Two cysteines, two histidines, and one methionine are the major ligands of two coppers. Substitution of these residues results in either a complete loss of color or dramatic changes in the absorbance spectrum. In contrast, substitution of the invariant glutamate residue, which is located between the copper-binding cysteines, leads to a minor perturbation of the optical spectrum.

Reduction of CuA induces a conformational change in cytochrome c oxidase from Paracoccus denitrificans.

Cytochrome c oxidase (cytochrome aa3) from Paracoccus denitrificans contains a tightly bound manganese(II) ion, which responds to reduction of the enzyme by a change in its EPR signal (Seelig et al. (1981) Biochim. Biophys. Acta 636, 162-167). In this paper, the nature of this phenomenon is studied and the bound manganese is used as a reporter group to monitor a redox-linked conformational change in the protein. A reductive titration of the cyanide-inhibited enzyme shows that the change in the manganese EPR signal is associated with reduction of CuA. The change appears to reflect a rearrangement in the rhombic octahedral coordination environment of the central Mn2+ atom and is indicative of a redox-linked conformational transition in the enzyme. The manganese is likely to reside at the interface of subunits I and II, near the periplasmic side of the membrane. One of its ligands may be provided by the transmembrane segment X of subunit I, which has been suggested to contribute ligands to cytochrome a and CuB as well. Another manganese ligand is a water oxygen, as indicated by broadening of the manganese EPR signal in the presence of H2(17)O.

Subunit III of cytochrome c oxidase is not involved in proton translocation: a site-directed mutagenesis study.

Subunit III (COIII) is one of the three core subunits of the aa3-type cytochrome c oxidase. COIII does not contain any of the redox centres and can be removed from the purified enzyme but has a function during biosynthesis of the enzyme. Dicyclohexyl carbodiimide (DCCD) modifies a conserved glutamic acid residue in COIII and abolishes the proton translocation activity of the enzyme. In this study, the invariant carboxylic acids E98 (the DCCD-binding glutamic acid) and D259 of COIII were changed by site-directed mutagenesis to study their role in proton pumping. Spectroscopy and activity measurements show that a structurally normal enzyme, which is active in electron transfer, is formed in the presence of the mutagenized COIII. Experiments with bacterial spheroplasts indicate that the mutant oxidases are fully competent in proton translocation. In the absence of the COIII gene, only a fraction of the oxidase is assembled into an enzyme with low but significant activity. This residual activity is also coupled to proton translocation. We conclude that, in contrast to numerous earlier suggestions, COIII is not an essential element of the proton pump.

Bacillus subtilis expresses two kinds of haem-A-containing terminal oxidases.

The expression of two different aa3-type cytochrome oxidases is demonstrated in Bacillus subtilis. One of them (denoted caa3-605), was predicted by DNA-sequencing of Bacillus cytochrome oxidase genes, but has not been found previously. It contains covalently bound haem C in subunit II and is very similar to the enzyme previously described in the thermophilic bacterium PS3. The other oxidase (denoted aa3-600) deviates from most known oxidases of aa3 type, and is probably identical with the oxidase described by de Vrij et al. [de Vrij, W., Azzi, A. & Konings, W. N. (1983) Eur. J. Biochem. 131, 97-103]. It shows no immunological cross-reactivity to the PS3 enzyme and differs from this spectroscopically; it contains no CuA and does not oxidise cytochrome c despite of its haem-A chromophores. It catalyses oxidation of quinols, which is proposed to be its physiological function.