Here, we show that the synaptic vesicle protein SV2A is the brain binding site of levetiracetam (LEV), a new antiepileptic drug with a unique activity profile in animal models of seizure and epilepsy. The LEV-binding site is enriched in synaptic vesicles, and photoaffinity labeling of purified synaptic vesicles confirms that it has an apparent molecular mass of Ϸ90 kDa. Brain membranes and purified synaptic vesicles from mice lacking SV2A do not bind a tritiated LEV derivative, indicating that SV2A is necessary for LEV binding. LEV and related compounds bind to SV2A expressed in fibroblasts, indicating that SV2A is sufficient for LEV binding. No binding was observed to the related isoforms SV2B and SV2C. Furthermore, there is a high degree of correlation between binding affinities of a series of LEV derivatives to SV2A in fibroblasts and to the LEV-binding site in brain. Finally, there is a strong correlation between the affinity of a compound for SV2A and its ability to protect against seizures in an audiogenic mouse animal model of epilepsy. These experimental results suggest that SV2A is the binding site of LEV in the brain and that LEV acts by modulating the function of SV2A, supporting previous indications that LEV possesses a mechanism of action distinct from that of other antiepileptic drugs. Further, these results indicate that proteins involved in vesicle exocytosis, and SV2 in particular, are promising targets for the development of new CNS drug therapies.

The biosphere is dominated by microorganisms (32), yet most microbes in nature have not been studied. Traditional methods for culturing microorganisms limit analysis to those that grow under laboratory conditions (14,25). The recent surge of research in molecular microbial ecology provides compelling evidence for the existence of many novel types of microorganisms in the environment in numbers and varieties that dwarf those of the comparatively few microorganisms amenable to laboratory cultivation (7,13,31). Corroboration comes from estimates of DNA complexity and the discovery of many unique 16S rRNA gene sequences from numerous environmental sources (8,10,28). Collectively, the genomes of the total microbiota found in nature, which we termed the metagenome (11), contain vastly more genetic information than is contained in the culturable subset. Given the profound utility and importance of microorganisms to all biological systems, methods are needed to access the wealth of information within the metagenome.Cloning large fragments of DNA isolated directly from microbes in natural environments provides a method to access soil metagenomic DNA. Previously, we investigated the use of the bacterial artificial chromosome (BAC) vector to express Bacillus cereus genomic DNA (20). The advantage of BAC vectors is that they maintain very large DNA inserts (greater than 100 kb) stably in Escherichia coli (23), facilitating the cloning of large fragments of DNA. Our results demonstrated that expression of heterologous DNA from B. cereus in an E. coli BAC system was detectable at a reasonable frequency (20), validating the idea that the low-copy BAC vector (one to two per cell) (23) could be used to express foreign DNA from foreign promoters in E. coli.Here we describe the construction and initial screening of two BAC libraries made with DNA isolated directly from soil. We found detectable levels of several biochemical activities from BAC library clones. Sequence analysis of selected BAC plasmids encoding such activities and of 16S rRNA genes in one of the libraries confirms the novelty of the genomic information cloned in our libraries. The results show that DNA extracted directly from soil is a valuable source of new genetic information and is accessible by using BAC libraries. Our results demonstrate that both traditional and functional genomics of uncultured microorganisms can be carried out by this approach and that screening of metagenome libraries for activities or gene sequences can provide a basis for conducting genomic analyses of uncultured microorganisms. MATERIALS AND METHODSBacterial strains and plasmids. E. coli strain DH10B and the BAC vector pBeloBAC11 were provided by H. Shizuya (15). Bacillus subtilis strain BR151(pPL608) is strain 1E32 (lys-3 metB10 trpC2) from the Bacillus Genetic Stock Center, Ohio State University. -TnphoA was used as described before (20).

The Escherichia coli aspartate receptor, a dimer of identical subunits, has two transmembrane regions (TM1, residues 7-30; TM2, residues 189-212) of 24 residues each. To study the relative placement and orientation of the regions, cysteine residues were introduced individually into the center of each: at positions 17,18, and 19 in TM1; and at positions 198, 199, 200, and 201 in TM2. Based on the patterns of disulfide cross-linking observed between subunits in the mutant receptors, there appears to be close contact between the TM1 and TM1' regions at the dimer interface but no such direct interaction between the TM2 and TM2' regions. The cross-linking results are consistent with an a-helical structure extending across the transmembrane region up through at least residue 36, which lies on the periplasmic side of TM1. The ability ofan 18-18' cross-linked dimer to transmit an aspartateinduced transmembrane signal is also supportive of such an extended helix. The changes in relative rates of disulfide cross-linking provide experimental evidence of a conformational change transmitted through the transmembrane domain during signaling. Once formed, disulfides between the transmembrane regions are unusually resistant to reduction by low molecular weight thiols in the presence of denaturants like SDS.These targeted disulfide cross-links can be used to reveal structural and dynamic aspects of protein function. Many transmembrane receptors have now been identified, cloned, and sequenced, but the mechanisms of transmembrane signaling remain obscure. Structurally, transmembrane receptors fall into at least three categories. One class of receptors contains ion channels, which transmit a signal by the passage of ions (1). A second class, which contains seven transmembrane regions, is exemplified by rhodopsin and the ,3-adrenergic receptors, which function by interacting with GTP binding proteins (2). The third class of receptors contains one or two highly hydrophobic transmembrane sequences of the type found in the epidermal growth factor receptor (3), the insulin receptor (4), and the aspartate and serine receptors of chemotaxis (5, 6). Transmembrane receptors containing one or two transmembrane sequences have been postulated to operate by either an association/ dissociation reaction, as suggested for the epidermal growth factor receptor (7), or by a ligand-induced conformational change through an individual subunit, as suggested for the aspartate receptor (8-10).To investigate the properties of the aspartate receptor, a new tool, targeted disulfide cross-linking, was developed (9). Using site-specific mutagenesis, cysteine residues were introduced at various locations throughout the aspartate receptor. The rates of formation of disulfide bonds between monomers revealed properties of the juxtaposition and flexibility of the protein domains. A rate is a probability function, and therefore the rate of cross-linking is related to the proximity of the residues, the flexibility of the protein, and the environment around th...

No abstract

Previous studies on calcitonin gene related peptide (CGRP) have demonstrated that it has the characteristics of an amphiphilic peptide, and from an examination of the sequence, we have proposed that it contains an amphiphilic alpha-helix. We have synthesized two analogues of CGRP which have different lengths of idealized amphiphilic alpha-helical secondary structure. The first model, CGRM-1, has been substituted with residues generating an idealized amphiphilic alpha-helix in the region between residues 8 and 25, equivalent to approximately five turns of an alpha-helix. This peptide is not an agonist in any of our bioassays, but it does bind with low affinity to rCGRP receptors in crude liver membranes. Our second model, CGRM-2, has an idealized amphiphilic alpha-helix between residues 8 and 18, which is equivalent to approximately three turns of an alpha-helix. In an in vitro rat vas deferens assay, this peptide is an agonist with a potency one-fourth that of the native hormone. However, the potency of CGRM-2 in an adenylate cyclase assay is much lower, only 1/140th the potency of CGRP. Both model peptides display amphiphilic characteristics commensurate with their design. We conclude that there is an amphiphilic alpha-helix in rCGRP between residues 8 and 18 and that this helix terminates in the vicinity of residue 18.

No abstract

Synaptic dysfunction is a pathological feature in many neurodegenerative disorders, including Alzheimer’s disease, and synaptic loss correlates closely with cognitive decline. Histone deacetylases (HDACs) are involved in chromatin remodeling and gene expression and have been shown to regulate synaptogenesis and synaptic plasticity, thus providing an attractive drug discovery target for promoting synaptic growth and function. To date, HDAC inhibitor compounds with prosynaptic effects are plagued by known HDAC dose-limiting hematological toxicities, precluding their application to treating chronic neurologic conditions. We have identified a series of novel HDAC inhibitor compounds that selectively inhibit the HDAC–co-repressor of repressor element-1 silencing transcription factor (CoREST) complex while minimizing hematological side effects. HDAC1 and HDAC2 associate with multiple co-repressor complexes including CoREST, which regulates neuronal gene expression. We show that selectively targeting the CoREST co-repressor complex with the representative compound Rodin-A results in increased spine density and synaptic proteins, and improved long-term potentiation in a mouse model at doses that provide a substantial safety margin that would enable chronic treatment. The CoREST-selective HDAC inhibitor Rodin-A thus represents a promising therapeutic strategy in targeting synaptic pathology involved in neurologic disorders.

LEV (levetiracetam), an antiepileptic drug which possesses a unique profile in animal models of seizure and epilepsy, has as its unique binding site in brain, SV2A (synaptic vesicle protein 2A). Previous studies have used a chimaeric and site-specific mutagenesis approach to identify three residues in the putative tenth transmembrane helix of SV2A that, when mutated, alter binding of LEV and related racetam derivatives to SV2A. In the present paper, we report a combined modelling and mutagenesis study that successfully identifies another 11 residues in SV2A that appear to be involved in ligand binding. Sequence analysis and modelling of SV2A suggested residues equivalent to critical functional residues of other MFS (major facilitator superfamily) transporters. Alanine scanning of these and other SV2A residues resulted in the identification of residues affecting racetam binding, including Ile273 which differentiated between racetam analogues, when mutated to alanine. Integrating mutagenesis results with docking analysis led to the construction of a mutant in which six SV2A residues were replaced with corresponding SV2B residues. This mutant showed racetam ligand-binding affinity intermediate to the affinities observed for SV2A and SV2B.