Maturation and processing of the amyloid precursor protein is regulated by the potassium/sodium hyperpolarization-activated cyclic nucleotide-gated ion channel 2 (HCN2).

The toxic amyloid ß-peptide (Aß) is a key player in Alzheimer Disease (AD) pathogenesis and selective inhibition of the production of this peptide is sought for. Aß is produced by the sequential cleavage of the Aß precursor protein (APP) by ß-secretase (to yield APP-C-terminal fragment ß (APP-CTFß) and soluble APPß (sAPPß)) and γ-secretase (to yield Aß). We reasoned that proteins that associate with γ-secretase are likely to regulate Aß production and to be targets of pharmaceutical interventions and therefore performed a pull-down assay to screen for such proteins in rat brain. Interestingly, one of the purified proteins was potassium/sodium hyperpolarization-activated cyclic nucleotide-gated ion channel 2 (HCN2), which has been shown to be involved in epilepsy. We found that silencing of HCN2 resulted in decreased secreted Aß levels. To further investigate the mechanism behind this reduction, we also determined the levels of full-length APP, sAPP and APP-CTF species after silencing of HCN2. A marked reduction in sAPP and APP-CTF, as well as glycosylated APP levels was detected. Decreased Aß, sAPP and APP-CTF levels were also detected after treatment with the HCN2 inhibitor ZD7288. These results indicate that the effect on Aß levels after HCN2 silencing or inhibition is due to altered APP maturation or processing by ß-secretase rather than a direct effect on γ-secretase. However, HCN2 and γ-secretase were found to be in close proximity, as evident by proximity ligation assay and immunoprecipitation. In summary, our results indicate that silencing or inhibition of HCN2 affects APP processing and thereby could serve as a potential treatment strategy.

Bacillus cereus-type polyhydroxyalkanoate biosynthetic gene cluster contains R-specific enoyl-CoA hydratase gene.

Bacillus cereus and Bacillus megaterium both accumulate polyhydroxyalkanoate (PHA) but their PHA biosynthetic gene (pha) clusters that code for proteins involved in PHA biosynthesis are different. Namely, a gene encoding MaoC-like protein exists in the B. cereus-type pha cluster but not in the B. megaterium-type pha cluster. MaoC-like protein has an R-specific enoyl-CoA hydratase (R-hydratase) activity and is referred to as PhaJ when involved in PHA metabolism. In this study, the pha cluster of B. cereus YB-4 was characterized in terms of PhaJ's function. In an in vitro assay, PhaJ from B. cereus YB-4 (PhaJYB4) exhibited hydration activity toward crotonyl-CoA. In an in vivo assay using Escherichia coli as a host for PHA accumulation, the recombinant strain expressing PhaJYB4 and PHA synthase led to increased PHA accumulation, suggesting that PhaJYB4 functioned as a monomer supplier. The monomer composition of the accumulated PHA reflected the substrate specificity of PhaJYB4, which appeared to prefer short chain-length substrates. The pha cluster from B. cereus YB-4 functioned to accumulate PHA in E. coli; however, it did not function when the phaJYB4 gene was deleted. The B. cereus-type pha cluster represents a new example of a pha cluster that contains the gene encoding PhaJ.

Cloning and heterologous expression of a novel subgroup of class IV polyhydroxyalkanoate synthase genes from the genus Bacillus.

Many microorganisms harbor genes necessary to synthesize biodegradable plastics known as polyhydroxyalkanoates (PHAs). We surveyed a genomic database and discovered a new cluster of class IV PHA synthase genes (phaRC). These genes are different in sequence and operon structure from any previously reported PHA synthase. The newly discovered PhaRC synthase was demonstrated to produce PHAs in recombinant Escherichia coli.

Identification of novel γ-secretase-associated proteins in detergent-resistant membranes from brain.

In Alzheimer disease, oligomeric amyloid ß-peptide (Aß) species lead to synapse loss and neuronal death. γ-Secretase, the transmembrane protease complex that mediates the final catalytic step that liberates Aß from its precursor protein (APP), has a multitude of substrates, and therapeutics aimed at reducing Aß production should ideally be specific for APP cleavage. It has been shown that APP can be processed in lipid rafts, and γ-secretase-associated proteins can affect Aß production. Here, we use a biotinylated inhibitor for affinity purification of γ-secretase and associated proteins and mass spectrometry for identification of the purified proteins, and we identify novel γ-secretase-associated proteins in detergent-resistant membranes from brain. Furthermore, we show by small interfering RNA-mediated knockdown of gene expression that a subset of the γ-secretase-associated proteins, in particular voltage-dependent anion channel 1 (VDAC1) and contactin-associated protein 1 (CNTNAP1), reduced Aß production (Aß40 and Aß42) by around 70%, whereas knockdown of presenilin 1, one of the essential γ-secretase complex components, reduced Aß production by 50%. Importantly, these proteins had a less pronounced effect on Notch processing. We conclude that VDAC1 and CNTNAP1 associate with γ-secretase in detergent-resistant membranes and affect APP processing and suggest that molecules that interfere with this interaction could be of therapeutic use for Alzheimer disease.

Erlin-2 is associated with active γ-secretase in brain and affects amyloid ß-peptide production.

The transmembrane protease complex γ-secretase is responsible for the generation of the neurotoxic amyloid ß-peptide (Aß) from its precursor (APP). Aß has a causative role in Alzheimer disease, and thus, γ-secretase is a therapeutic target. However, since there are more than 70 γ-secretase substrates besides APP, selective inhibition of APP processing is required. Recent data indicates the existence of several γ-secretase associated proteins (GSAPs) that affect the selection and processing of substrates. Here, we use a γ-secretase inhibitor for affinity purification of γ-secretase and associated proteins from microsomes and detergent resistant membranes (DRMs) prepared from rat or human brain. By tandem mass spectrometry we identified a novel brain GSAP; erlin-2. This protein was recently reported to reside in DRMs in the ER. A proximity ligation assay, as well as co-immunoprecipitation, confirmed the association of erlin-2 with γ-secretase. We found that a higher proportion of erlin-2 was associated with γ-secretase in DRMs than in soluble membranes. siRNA experiments indicated that reduced levels of erlin-2 resulted in a decreased Aß production, whereas the effect on Notch processing was limited. In summary, we have found a novel brain GSAP, erlin-2, that resides in DRMs and affects Aß production.

Proton myo-inositol cotransporter is a novel γ-secretase associated protein that regulates Aß production without affecting Notch cleavage.

γ-Secretase is a transmembrane protease complex that is responsible for the processing of a multitude of type 1 transmembrane proteins, including the amyloid precursor protein and Notch. γ-Secretase processing of amyloid precursor protein results in the release of the amyloid ß-peptide (Aß), which is involved in the pathogenesis in Alzheimer's disease. Processing of Notch leads to the release of its intracellular domain, which is important for cell development. γ-Secretase associated proteins (GSAPs) could be of importance for substrate selection, and we have previously shown that affinity purification of γ-secretase in combination with mass spectrometry can be used for finding such proteins. In the present study, we used this methodology to screen for novel GSAPs from human brain, and studied their effect on Aß production in a comprehensive gene knockdown approach. Silencing of probable phospholipid-transporting ATPase IIA, brain-derived neurotrophic factor/neurotrophin-3 growth factor receptor precursor and proton myo-inositol cotransporter (SLC2A13) showed a clear reduction of Aß and these proteins were selected for further studies on Aß production and Notch cleavage using small interfering RNA-mediated gene silencing, as well as an overexpression approach. Silencing of these reduced Aß secretion in a small interfering RNA dose-dependent manner. Interestingly, SLC2A13 had a lower effect on Notch processing. Furthermore, overexpression of SLC2A13 increased Aß40 generation. Finally, the interaction between γ-secretase and SLC2A13 was confirmed using immunoprecipitation and a proximity ligation assay. In summary, SLC2A13 was identified as a novel GSAP that regulates Aß production without affecting Notch cleavage. We suggest that SLC2A13 could be a target for Aß lowering therapy aimed at treating Alzheimer's disease.

Human brain proteins showing neuron-specific interactions with γ-secretase.

The transmembrane protease complex γ-secretase is a key enzyme in Alzheimer disease pathogenesis as it liberates the neurotoxic amyloid ß-peptide (Aß); however, the mechanism of regulation of its activity in various cell types and subcellular compartments is largely unknown. Several γ-secretase inhibitors have been developed, but none have been released due to side-effects that appear to arise from reduced processing of Notch, one of many γ-secretase substrates. Hence, it is desirable to specifically inhibit Aß production. In our previous studies, we have identified several γ-secretase-associated proteins (GSAPs) from brain, which affect Aß production without having any major effects on Notch processing. In the present study using detergent-resistant membranes prepared from brain, we have identified four GSAPs that affect Aß production to a greater extent than Notch processing. We evaluated the interaction between GSAPs and γ-secretase in various cell types and their mRNA expression in various human organs. Using an in situ proximity ligation assay, we demonstrated that many GSAPs showed considerably greater interaction with γ-secretase in neurons than in human embryonic kidney cells stably over-expressing APP, and showed that several GSAPs are highly expressed in human brain. This study underscores the importance of studying protein-protein interactions in relevant cell types, and suggests that reducing Aß production by interfering with brain- or neuron-specific γ-secretase/GSAP interactions may reduce the risk of unwanted side-effects associated with treatment of Alzheimer disease.

Determination of endogenous peptides in the porcine brain: possible construction of peptidome, a fact database for endogenous peptides.

Peptides play crucial roles in many physiological events. However, a database for endogenous peptides has not yet been developed, because the peptides are easily degraded by proteolytic enzymes during extraction and purification. In this study, we demonstrated that the data for endogenous peptides could be collected by minimizing the proteolytic degradation. We separated porcine brain peptides into 5250 fractions by 2-dimensional chromatography (first ion-exchange and second reversed-phase high-performance liquid chromatography), and 75 fractions of average peptide contents were analyzed in detail by mass spectrometers and a protein sequencer. Based on the analysis data obtained in this study, more than 10000 peptides were deduced to be detected, and more than 1000 peptides to be identified starting from 2 g of brain tissue. Thus, we deduce that it is possible to construct a database for endogenous peptides starting from a gram level of tissue by using 2-dimensional high-performance liquid chromatography coupled with a mass spectrometer.

Expression of bradykinin B2 receptor in the mouse ovary.

The amounts of [1-5]-bradykinin in ovary extracts were determined using gonadotropin-treated immature female mice. The bradykinin levels in the ovary were high at 2, 6, and 48 hr after injection of human chorionic gonadotropin (hCG) into pregnant mare's serum gonadotropin (PMSG)-treated mice. Northern blot analysis of total RNAs isolated from the PMSG/hCG-treated mouse ovaries indicated that the B(2) receptor mRNA was constitutively expressed. Bradykinin B(2) receptor protein was detected by Western blot analysis of the ovary extracts. In situ hybridization analysis revealed that the B(2) receptor mRNA is expressed in the granulosa cells of all growing follicles of ovaries from both gonadotropin-treated immature and mature female mice. The effect of bradykinin on the expression of the B(2) receptor gene was examined by RT-PCR analysis with the ovary previously cultured in the presence of bradykinin. Bradykinin treatment of immature female, gonadotropin-treated immature female, and mature female mouse ovaries brought about no apparent changes in the B(2) receptor mRNA level. The present data indicate that the level of B(2) receptor expression in the ovary is fairly constant, and that the biological effect elicited by bradykinin in this organ may be dependent upon concentrations of the ligand produced by operation of the kinin-kallikrein system.

Identification of two novel synaptic γ-secretase associated proteins that affect amyloid ß-peptide levels without altering Notch processing.

Synaptic degeneration is one of the earliest hallmarks of Alzheimer disease (AD) and results in loss of cognitive function. One of the causative agents for the synaptic degeneration is the amyloid ß-peptide (Aß), which is formed from its precursor protein by two sequential cleavages mediated by ß- and γ-secretase. We have earlier shown that γ-secretase activity is enriched in synaptic compartments, suggesting that the synaptotoxic Aß is produced locally. Proteins that interact with γ-secretase at the synapse and regulate the production of Aß can therefore be potential therapeutic targets. We used a recently developed affinity purification approach to identify γ-secretase associated proteins (GSAPs) in synaptic membranes and synaptic vesicles prepared from rat brain. Liquid chromatography-tandem mass spectrometry analysis of the affinity purified samples revealed the known γ-secretase components presenilin-1, nicastrin and Aph-1b along with a number of novel potential GSAPs. To investigate the effect of these GSAPs on APP processing, we performed siRNA experiments to knock down the expression of the GSAPs and measured the Aß levels. Silencing of NADH dehydrogenase [ubiquinone] iron-sulfur protein 7 (NDUFS7) resulted in a decrease in Aß levels whereas silencing of tubulin polymerization promoting protein (TPPP) resulted in an increase in Aß levels. Treatment with γ-secretase inhibitors often results in Notch-related side effects and therefore we also studied the effect of the siRNAs on Notch processing. Interestingly, silencing of TPPP or NDUFS7 did not affect cleavage of Notch. We also studied the expression of TPPP and NDUFS7 in control and AD brain and found NDUFS7 to be highly expressed in vulnerable neurons such as pyramidal neurons in the hippocampus, whereas TPPP was found to accumulate in intraneuronal granules and fibrous structures in hippocampus from AD cases. In summary, we here report on two proteins, TPPP and NDUFS7, which interact with γ-secretase and alter the Aß levels without affecting Notch cleavage.

Mechanism of inhibition of DNA gyrase by ES-1273, a novel DNA gyrase inhibitor.

We investigated the mode of action of ES-1273, a novel DNA gyrase inhibitor obtained by optimization of ES-0615, which was found by screening our chemical library using anucleate cell blue assay. ES-1273 exhibited the same antibacterial activity against S. aureus strains with amino acid change(s) conferring quinolone- and coumarin-resistance as that against a susceptible strain. In addition, ES-1273 inhibited DNA gyrase supercoiling activity, but not ATPase activity of the GyrB subunit of DNA gyrase. Moreover, ES-1273 did not induce cleavable complex. These findings demonstrate that the mechanism by which ES-1273 inhibits DNA gyrase is different from that of the quinolones or the coumarins. Preincubation of DNA gyrase and substrate DNA prevented inhibition of DNA gyrase supercoiling activity by ES-1273. ES-1273 antagonized quinolone-induced cleavage. In electrophoretic mobility shift assay, no band representing DNA gyrase-DNA complex was observed in the presence of ES-1273. Taken together, these results indicate that ES-1273 prevents DNA from binding to DNA gyrase. Furthermore, our results from surface plasmon resonance experiments strongly suggest that ES-1273 interacts with DNA. Therefore, the interaction between ES-1273 and DNA prevents DNA from binding to DNA gyrase, resulting in inhibition of DNA gyrase supercoiling. Interestingly, we also found that ES-1273 inhibits topoisomerase IV and human topoisomerase IIalpha, but not human topoisomerase I. These findings indicate that ES-1273 is a type II topoisomerase specific inhibitor.