phenobarbital-sodium and Alzheimer-Disease

We recently reported that glutamate carboxypeptidase II (GCPII) has a new physiological function degrading amyloid-β (Aβ), distinct from its own hydrolysis activity in N-acetyl-L-aspartyl-L-glutamate (NAAG); however, its underlying mechanism remains undiscovered. Using site-directed mutagenesis and S1 pocket-specific chemical inhibitor (compound 2), which was developed for the present study based on in sillico computational modeling, we discovered that the Aβ degradation occurs through S1 pocket but not through S1' pocket responsible for NAAG hydrolysis. Treatment with compound 2 prevented GCPII from Aβ degradation without any impairment in NAAG hydrolysis. Likewise, 2-PMPA (specific GCPII inhibitor developed targeting S1' pocket) completely blocked the NAAG hydrolysis without any effect on Aβ degradation. Pre-incubation with NAAG and Aβ did not affect Aβ degradation and NAAG hydrolysis, respectively. These data suggest that GCPII has two distinctive binding sites for two different substrates and that Aβ degradation occurs through binding to S1 pocket of GCPII.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Binding Sites; Cell Line, Tumor; Dipeptides; Enzyme Inhibitors; Glutamate Carboxypeptidase II; Glutamic Acid; Humans; Mice; Mice, Transgenic; Molecular Docking Simulation; Mutagenesis, Site-Directed; Organophosphorus Compounds; Proteolysis

Proteolytic processing of amyloid peptides (Aβs) is one important mechanism that controls the brain Aβ level. Although several Aβ-degrading enzymes were identified, evidence has suggested the presence of other peptidases. Here, we report a novel function of glutamate carboxypeptidase II (GCPII) in Aβ degradation in brain, which is a peptidase involved in N-acetylaspartylglutamate cleavage, folate metabolism, and prostate tumorigenesis. Maldi-Tof/MS analysis showed that recombinant human GCPII cleaved the Aβ1-40 and Aβ1-42 monomers at their C-termini, producing smaller fragments, and Aβ1-14 that lacked aggregation property and cellular toxicity. GCPII also degrades soluble oligomers and fibrils and can reduce the endogenous plaque size in brain sections obtained from amyloid precursor protein (APP) Swedish/presinilin (PS)-1ΔE9 transgenic mice. Overexpression of GCPII in either HEK293-APP Swedish cells or primary neurons and glial cells reduced the levels of secreted or exogenously supplemented Aβs and reduced Aβ-induced neurotoxicity, suggesting the biological significance of GCPII-mediated Aβ cleavage. Moreover, treatment of 8-mo-old transgenic mice for 1 mo with 2-(phosphonomethyl)-pentanedioic acid (10 mg/kg, intraperitoneally), a specific GCPII inhibitor, increased cerebral Aβ content. These results suggest an important physiological role for GCPII in Aβ clearance in brain and provide the evidence that dysregulation of GCPII is involved in Alzheimer's disease pathology.

Topics: Alzheimer Disease; Amyloidogenic Proteins; Animals; Brain; Cells, Cultured; Enzyme Activation; Enzyme Inhibitors; Glutamate Carboxypeptidase II; HEK293 Cells; Humans; Mice; Mice, Transgenic; Organophosphorus Compounds; Up-Regulation