Identification of a novel complex AßPP:Fe65:PP1 that regulates AßPP Thr668 phosphorylation levels.

The amyloid-ß protein precursor (AßPP) binds several proteins determining metabolism, processing, and the physiological fate of the former. Among these is Fe65, a protein with specific functional significance for AßPP, in particular conferring stability when the latter is dephosphorylated on Thr668. Thus, it follows that phosphatases like protein phosphatase 1 (PP1) are relevant to AßPP processing. Consequently, the identification of AßPP binding proteins, which can be modulated directly or indirectly by PP1, take on added relevance in terms of biological significance. Using the yeast tri-hybrid system and co-immunoprecipitation assays, we describe a novel tri-complex comprising AßPP, Fe65 and PP1. We show that the trimeric complex (AßPP:Fe65:PP1γ) occurs in COS-7 cells, rat hippocampal and cortical primary neurons, and in adult rat hippocampus and cortex. Using overlay assays, we demonstrate that Fe65 is in fact the bridging protein in the complex formed and thus we simultaneously describe another PP1 binding protein. This is singularly important given that PP1 binding proteins determine and confer subcellular localization, as well as substrate specificity, thus regulating the phosphatase activity and subsequent intracellular events. Additionally, we show that this interaction correlates with AßPP Thr668 phosphorylation state, consistent with the role of protein (de)phosphorylation as a key mechanism in regulating cellular events.

PP1 inhibition by Abeta peptide as a potential pathological mechanism in Alzheimer's disease.

Abnormal protein phosphorylation has been associated with several neurodegenerative disorders, including Alzheimer's disease (AD). Abeta is the toxic peptide that results from proteolytic cleavage of the Alzheimer's amyloid precursor protein, a process where protein phosphatases are known to impact. The data presented here demonstrates that protein phosphatase 1 (PP1), an abundant neuronal serine/threonine-specific phosphatase highly enriched in dendritic spines, is specifically inhibited by Abeta peptides both in vitro and ex vivo. Indeed, the pathologically relevant Abeta(1-40) and Abeta(1-42) peptides, as well as Abeta(25-35), specifically inhibit PP1 with low micromolar potency, as compared to inactive controls and other disease related peptides (e.g. the prion related Pr(118-135) and Pr(106-126)). Interestingly, PP1 inhibition is increased by Abeta aggregation, indicating a possible direct neurotoxic effect of the aggregated peptide. PP1 involvement in processes like long-term depression, memory and learning, and synaptic plasticity, prompt us to suggest that PP1 may constitute an important physiological target for Abeta and, therefore, increased Abeta production and/or aggregation may lead to abnormal PP1 activity and likely contribute to the progressive neuropsychiatric AD condition. Thus, PP1 activity and levels constitute potential biomolecular candidates for diagnostics and therapeutics.

Mutation of surface cysteine 374 to alanine in monoamine oxidase A alters substrate turnover and inactivation by cyclopropylamines.

Modification of cysteine (Cys) residues inactivates monoamine oxidases (MAO) yet the crystal structure shows no conserved cysteines in the active site of MAO A (Ma, J. et al. J. Mol. Biol.2004, 338, 103-114). MAO A cysteine 374 was mutated to alanine and the purified enzyme characterized kinetically. The mutant was active but had decreased k(cat)/K(m) values compared to the wild-type enzyme. Cyclopropylamine-containing mechanism-based inactivators similarly showed lower turnover rates. Spectral studies and measurement of free thiols established that 1-phenylcyclopropylamine (1-PCPA) formed an irreversible flavin adduct whereas 2-phenylcyclopropylamine (2-PCPA) and N-cyclo-alpha-methylbenzylamine (N-CalphaMBA) formed adducts that allowed reoxidation of the flavin on denaturation and decreased cysteine in both wild-type and mutant MAO A. In the 1-PCPA and N-CalphaMBA inactivations, the partition ratio was decreased by more than 50% in the mutant. The data suggest that mutation of Cys374 influences MAO A catalysis, which has implications for MAO susceptibility to redox damage. These results are compared with previous work on the equivalent residue in MAO B, namely, cysteine 365.