Soluble adenylyl cyclase is essential for proper lysosomal acidification.

Lysosomes, the degradative organelles of the endocytic and autophagic pathways, function at an acidic pH. Lysosomes are acidified by the proton-pumping vacuolar ATPase (V-ATPase), but the molecular processes that set the organelle's pH are not completely understood. In particular, pH-sensitive signaling enzymes that can regulate lysosomal acidification in steady-state physiological conditions have yet to be identified. Soluble adenylyl cyclase (sAC) is a widely expressed source of cAMP that serves as a physiological pH sensor in cells. For example, in proton-secreting epithelial cells, sAC is responsible for pH-dependent translocation of V-ATPase to the luminal surface. Here we show genetically and pharmacologically that sAC is also essential for lysosomal acidification. In the absence of sAC, V-ATPase does not properly localize to lysosomes, lysosomes fail to fully acidify, lysosomal degradative capacity is diminished, and autophagolysosomes accumulate.

pH sensing via bicarbonate-regulated "soluble" adenylyl cyclase (sAC).

Soluble adenylyl cyclase (sAC) is a source of the second messenger cyclic adenosine 3', 5' monophosphate (cAMP). sAC is directly regulated by bicarbonate (HCO(-) 3) ions. In living cells, HCO(-) 3 ions are in nearly instantaneous equilibrium with carbon dioxide (CO2) and pH due to the ubiquitous presence of carbonic anhydrases. Numerous biological processes are regulated by CO2, HCO(-) 3, and/or pH, and in a number of these, sAC has been shown to function as a physiological CO2/HCO3/pH sensor. In this review, we detail the known pH sensing functions of sAC, and we discuss two highly-studied, pH-dependent pathways in which sAC might play a role.

Impaired ß-amyloid secretion in Alzheimer's disease pathogenesis.

A central question in Alzheimer's disease (AD) research is what role ß-amyloid peptide (Aß) plays in synaptic dysfunction. Synaptic activity increases Aß secretion, potentially inhibiting synapses, but also decreases intraneuronal Aß, protecting synapses. We now show that levels of secreted Aß fall with time in culture in neurons of AD-transgenic mice, but not wild-type mice. Moreover, the ability of synaptic activity to elevate secreted Aß and reduce intraneuronal Aß becomes impaired in AD-transgenic but not wild-type neurons with time in culture. We demonstrate that synaptic activity promotes an increase in the Aß-degrading protease neprilysin at the cell surface and a concomitant increase in colocalization with Aß42. Remarkably, AD-transgenic but not wild-type neurons show reduced levels of neprilysin with time in culture. This impaired ability to secrete Aß and reduce intraneuronal Aß has important implications for the pathogenesis and treatment of AD.

Synaptic activity reduces intraneuronal Abeta, promotes APP transport to synapses, and protects against Abeta-related synaptic alterations.

A central question in Alzheimer's disease research is what role synaptic activity plays in the disease process. Synaptic activity has been shown to induce beta-amyloid peptide release into the extracellular space, and extracellular beta-amyloid has been shown to be toxic to synapses. We now provide evidence that the well established synaptotoxicity of extracellular beta-amyloid requires gamma-secretase processing of amyloid precursor protein. Recent evidence supports an important role for intraneuronal beta-amyloid in the pathogenesis of Alzheimer's disease. We show that synaptic activity reduces intraneuronal beta-amyloid and protects against beta-amyloid-related synaptic alterations. We demonstrate that synaptic activity promotes the transport of the amyloid precursor protein to synapses using live cell imaging, and that the protease neprilysin is involved in reduction of intraneuronal beta-amyloid with synaptic activity.

Molecular cloning, characterisation and ligand-bound structure of an azoreductase from Pseudomonas aeruginosa.

The gene PA0785 from Pseudomonas aeruginosa strain PAO1, which is annotated as a probable acyl carrier protein phosphodiesterase (acpD), has been cloned and heterologously overexpressed in Escherichia coli. The purified recombinant enzyme exhibits activity corresponding to that of azoreductase but not acpD. Each recombinant protein molecule has an estimated molecular mass of 23,050 Da and one non-covalently bound FMN as co-factor. This enzyme, now identified as azoreductase 1 from Pseudomonas aeruginosa (paAzoR1), is a flavodoxin-like protein with an apparent molecular mass of 110 kDa as determined by gel-filtration chromatography, indicating that the protein is likely to be tetrameric in solution. The three-dimensional structure of paAzoR1, in complex with the substrate methyl red, was solved at a resolution of 2.18 A by X-ray crystallography. The protein exists as a dimer of dimers in the crystal lattice, with two spatially separated active sites per dimer, and the active site of paAzoR1 was shown to be a well-conserved hydrophobic pocket formed between two monomers. The paAzoR1 enzyme is able to reduce different classes of azo dyes and activate several azo pro-drugs used in the treatment of inflammatory bowel disease (IBD). During azo reduction, FMN serves as a redox centre in the electron-transferring system by mediating the electron transfer from NAD(P)H to the azo substrate. The spectral properties of paAzoR1 demonstrate the hydrophobic interaction between FMN and the active site in the protein. The structure of the ligand-bound protein also highlights the pi-stacking interactions between FMN and the azo substrate.