Pharmacogenomics to predict drug response.

From theory to proof-of-concept, pharmacogenomics promises to improve future general healthcare in a number of ways. By identifying individuals who will respond to a particular drug treatment compared to those who have a low probability of response, pharmacogenomic test development hopes to aid the physician in prescribing the optimal medication for each patient. This approach promises faster relief from symptoms, a lowering of side effect risks and a reduction in healthcare costs. Pharmacogenomic tests used by the pharmaceutical companies themselves can be used to help identify suitable subjects for clinical trials, aid in interpretation of clinical trial results, find new markets for current products and speed up the development of new treatments and therapies. This type of approach should also see fewer compounds failing during later phases of development. The questions we are faced with as we enter the new millennium, however, are if and when the promises of pharmacogenomnics in improving healthcare will be fulfilled. Currently, there are only a handful of pharmacogenomic tests and associated products which are commercially available and it remains to be seen what impact these will have on the market and on healthcare in general.

Inhibition of neurotransmitter release in the lamprey reticulospinal synapse by antibody-mediated disruption of SNAP-25 function.

Exocytosis - syntaxin - synaptobrevin - SNARE synaptic vesicle The lamprey giant reticulospinal synapse can be used to manipulate the molecular machinery of synaptic vesicle exocytosis by presynaptic microinjection. Here we test the effect of disrupting the function of the SNARE protein SNAP-25. Polyclonal SNAP-25 antibodies were shown in an in vitro assay to inhibit the binding between syntaxin and SNAP-25. When microinjected presynaptically, these antibodies produced a potent inhibition of the synaptic response. Ba2+ spikes recorded in the presynaptic axon were not altered, indicating that the effect was not due to a reduced presynaptic Ca2+ entry. Electron microscopic analysis showed that synaptic vesicle clusters had a similar organization in synapses of antibody-injected axons as in control axons, and the number of synaptic vesicles in apparent contact with the presynaptic plasma membrane was also similar. Clathrin-coated pits, which normally occur at the plasma membrane around stimulated synapses, were not detected after injection of SNAP-25 antibodies, consistent with a blockade of vesicle cycling. Thus, SNAP-25 antibodies, which disrupt the interaction with syntaxin, inhibit neurotransmitter release without affecting the number of synaptic vesicles at the plasma membrane. These results provide further support to the view that the formation of SNARE complexes is critical for membrane fusion, but not for the targeting of synaptic vesicles to the presynaptic membrane.

The highly conserved synapse protein SNAP-25 displays sequence variability in the cockroach Leucophaea maderae.

SNAP-25 (synaptosome-associated protein of 25 kD) is attached to the intracellular side of presynaptic membranes where it serves as a target receptor for the vesicle docking machinery prior to release of neurotransmitter. SNAP-25 displays a high degree of sequence conservation between vertebrates and Drosophila melanogaster. To obtain more information about conserved regions of SNAP-25, we have isolated cDNA clones from the cockroach Leucophaea maderae. One clone (Lm1) encoded a full-length SNAP-25 protein and its deduced amino acid sequence is 77% identical to Drosophila SNAP-25. Surprisingly, the cockroach protein is 17 amino acids shorter than Drosophila SNAP-25 at the carboxy terminus. Four other cDNA clones encode parts of SNAP-25 and each clone has distinct characteristics, including amino acid replacements and unique carboxy termini. Thus, the highly conserved protein SNAP-25 displays unexpected sequence variability in the cockroach that may indicate specialized SNAP-25 isoforms.

Differential phosphorylation of syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25) isoforms.

The synaptic plasma membrane proteins syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25) are central participants in synaptic vesicle trafficking and neurotransmitter release. Together with the synaptic vesicle protein synaptobrevin/vesicle-associated membrane protein (VAMP), they serve as receptors for the general membrane trafficking factors N-ethylmaleimide-sensitive factor (NSF) and soluble NSF attachment protein (alpha-SNAP). Consequently, syntaxin, SNAP-25, and VAMP (and their isoforms in other membrane trafficking pathways) have been termed SNAP receptors (SNAREs). Because protein phosphorylation is a common and important mechanism for regulating a variety of cellular processes, including synaptic transmission, we have investigated the ability of syntaxin and SNAP-25 isoforms to serve as substrates for a variety of serine/threonine protein kinases. Syntaxins 1 A and 4 were phosphorylated by casein kinase II, whereas syntaxin 3 and SNAP-25 were phosphorylated by Ca2+- and calmodulin-dependent protein kinase II and cyclic AMP-dependent protein kinase, respectively. The biochemical consequences of SNARE protein phosphorylation included a reduced interaction between SNAP-25 and phosphorylated syntaxin 4 and an enhanced interaction between phosphorylated syntaxin 1A and the synaptic vesicle protein synaptotagmin I, a potential Ca2+ sensor in triggering synaptic vesicle exocytosis. No other effects on the formation of SNARE complexes (comprised of syntaxin, SNAP-25, and VAMP) or interactions involving n-Sec1 or alpha-SNAP were observed. These findings suggest that although phosphorylation does not directly regulate the assembly of the synaptic SNARE complex, it may serve to modulate SNARE complex function through other proteins, including synaptotagmin I.

Cloning of two loci for synapse protein Snap25 in zebrafish: comparison of paralogous linkage groups suggests loss of one locus in the mammalian lineage.

Synaptosome-associated protein of 25 kDa (Snap25) is an intracellular protein that is defined as a target receptor for synapse vesicles prior to neurotransmitter release. Snap25 is highly conserved, with 61% identity between human and Drosophila melanogaster. Whereas mammals and chicken have a single locus for Snap25, the tetraploid goldfish has at least three loci. We report that the zebrafish has two loci with 91% amino acid identity to each other. The alternative splicing of exon 5 arose before the gene duplication. The expression patterns of the two loci are virtually identical in adult zebrafish. The two zebrafish snap25 loci are located in paralogous linkage groups that seem to correspond to human chromosome 20, which harbors the SNAP locus, and human chromosome 14. Because no additional Snap25 homologue has been reported for any mammal or chicken, snap25.2 may have been lost in the amniote or even tetrapod lineage.

Complex gene organization of synaptic protein SNAP-25 in Drosophila melanogaster.

The evolutionarily conserved protein SNAP-25 (synaptosome-associated protein 25 kDa (kilodaltons)) is a component of the protein complex involved in the docking and/or fusion of synaptic vesicles in nerve terminals. We report here that the SNAP-25 gene (Snap) in the fruit fly Drosophila melanogaster has a complex organization with eight exons spanning more than 120 kb (kilobases). The exon boundaries coincide with those of the chicken SNAP-25 gene (Bark, 1993). Only a single exon 5 has been found in Drosophila, whereas human, rat, chicken, zebrafish and goldfish have two alternatively spliced versions of this exon. In situ hybridization and immunocytochemistry to whole mount embryos show that SNAP-25 mRNA and protein are detected in stage 14 and later developmental stages, and are mainly localized to the ventral nerve cord. Thus, Snap has an evolutionarily conserved and complex gene organization, and its onset of expression in Drosophila melanogaster correlates with a time in neuronal development when synapses begin to be formed and when other synapse-specific genes are switched on.

Molecular genetic aspects of tetraploidy in the common carp Cyprinus carpio.

Genome duplications are believed to have occurred on multiple occasions in vertebrate evolution. Studies of duplicate gene loci in tetraploid animals may reveal important general aspects of gene duplication, an important mode of gene evolution in metazoans. The common carp Cyprinus carpio has twice as many chromosomes as most other cyprinid fishes due to tetraploidization previously estimated to have occurred 50 Myr ago. Our sequence analyses of duplicate carp loci suggest that the tetraploidization took place less than 16 Myr ago. This is further supported by sequence comparisons with the diploid grass carp, which seems to have diverged from the common carp approximately 19 Myr ago. Duplicate loci appear to remain expressed for millions of years and may accumulate mutations leading to drastic amino acid replacements as shown here for somatotropin. Therefore, both loci should always be characterized in molecular studies of tetraploid animals such as goldfish, salmonid fishes, and Xenopus laevis. The long life of duplicate genes may explain the occurrence of numerous large multigene families in higher metazoans.

Evolutionary conservation of synaptosome-associated protein 25 kDa (SNAP-25) shown by Drosophila and Torpedo cDNA clones.

The neuron-specific proteins SNAP-25 (synaptosome-associated protein 25 kDa), synaptobrevin and syntaxin, are localized to presynaptic terminals in mammals and have been found to associate with proteins involved in vesicle docking and membrane fusion. We describe here SNAP-25 cDNA clones from the fruit fly Drosophila melanogaster and the ray Torpedo marmorata. In situ hybridization showed that SNAP-25 mRNA is exclusively found in brain and ganglia in Drosophila with a pattern suggesting expression in most neurons. The Drosophila and Torpedo proteins show 61 and 81% amino acid identity to mouse SNAP-25, a degree of conservation similar to that previously reported for synaptobrevin. None of the SNAP-25 sequences has a membrane-spanning region, but all contain a cluster of cysteine residues that can be palmitoylated for membrane attachment. SNAP-25 displays sequence similarity to syntaxin A and B. These data show that SNAP-25 and synaptobrevin, which are both implicated in vesicle docking and/or membrane fusion, have both been highly conserved during evolution. This supports the existence of a basic molecular machinery for synaptic vesicle docking in vertebrate and invertebrate synapses.

Multiple loci for synapse protein SNAP-25 in the tetraploid goldfish.

The common goldfish Carassius auratus is tetraploid and has 100 chromosomes. We describe here goldfish cDNA clones for SNAP-25, a 200-amino-acid synaptosome-associated protein that has remained highly conserved during evolution. SNAP-25 occurs as a single-copy gene in mouse, chicken, and Drosophila melanogaster. Sequences of six distinct goldfish cDNA clones and Southern hybridizations show that the goldfish has three, or possibly four, SNAP-25 loci rather than two as expected. A gene duplication early in actinopterygian fish evolution gave rise to the loci SnapA and SnapB. The proteins SNAP-A and SNAP-B are 94% and 91% identical to the mouse protein but are only 91% identical to each other. SNAP-B has a larger number of unique amino acid replacements than SNAP-A and also has more dramatic replacements. The tetraploidization resulted in two SnapB loci whose divergence from each other is consistent with a tetraploidization event 15-20 million years ago. The presence of duplicate SnapA loci has not yet been possible to confirm, possibly because they are still very similar to each other. Two of the SnapA cDNA clones and one SnapB cDNA clone have frameshift mutations. As these aberrant alleles otherwise display high sequence identity to the functional alleles, they probably became nonfunctional recently. The findings of allelic variability and aberrant alleles emphasize the importance of characterizing multiple DNA clones in tetraploid species.

Amino acid sequence of a snake venom toxin that binds to the muscarinic acetylcholine receptor.

The green mamba, Dendroaspis angusticeps, has two protein toxins that bind to the muscarinic acetylcholine receptor. The sequence of muscarinic toxin 2 was determined with an automatic gas phase sequencer. The C-terminal residue is Asp as determined by hydrazinolysis and amino acid analysis. Toxin 2 has 65 amino acid residues and a formula weight of 7040. It is homologous to a large number of other snake venom toxins as short alpha-neurotoxins, cardiotoxins/cytotoxins and angusticeps-type toxins of mamba venoms. The sequence is confirmed in the accompanying article (Ducancel, F., Rowan, E.G., Cassart, E., Harvey, A. L., Menez, A. and Boulain, J.-C. Toxicon 29, 516-520, 1991).