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  • Suppression of Aβ toxicity by puromycin-sensitive aminopeptidase is independent of its proteolytic activity.

Suppression of Aβ toxicity by puromycin-sensitive aminopeptidase is independent of its proteolytic activity.

Biochimica et biophysica acta (2013-08-06)
Antonina J Kruppa, Stanislav Ott, Dhia S Chandraratna, James A Irving, Richard M Page, Elena Speretta, Tiffany Seto, Luiz Miguel Camargo, Stefan J Marciniak, David A Lomas, Damian C Crowther
ABSTRACT

The accumulation of β-amyloid (Aβ) peptide in the brain is one of the pathological hallmarks of Alzheimer's disease and is thought to be of primary aetiological significance. In an unbiased genetic screen, we identified puromycin-sensitive aminopeptidase (PSA) as a potent suppressor of Aβ toxicity in a Drosophila model system. We established that coexpression of Drosophila PSA (dPSA) in the flies' brains improved their lifespan, protected against locomotor deficits, and reduced brain Aβ levels by clearing the Aβ plaque-like deposits. However, confocal microscopy and subcellular fractionation of amyloid-expressing 7PA2 cells demonstrated that PSA localizes to the cytoplasm. Therefore, PSA and Aβ are unlikely to be in the same cellular compartment; moreover, when we artificially placed them in the same compartment in flies, we could not detect a direct epistatic interaction. The consequent hypothesis that PSA's suppression of Aβ toxicity is indirect was supported by the finding that Aβ is not a proteolytic substrate for PSA in vitro. Furthermore, we showed that the enzymatic activity of PSA is not required for rescuing Aβ toxicity in neuronal SH-SY5Y cells. We investigated whether the stimulation of autophagy by PSA was responsible for these protective effects. However PSA's promotion of autophagosome fusion with lysosomes required proteolytic activity and so its effect on autophagy is not identical to its protection against Aβ toxicity.

MATERIALS
Product Number
Brand
Product Description

Millipore
Protein A, HRP conjugate, 1 mg Protein A, HRP conjugate for Dot Blot, ELISA, Western Blotting & IHC.
Sigma-Aldrich
Puromycin dihydrochloride from Streptomyces alboniger, powder, BioReagent, suitable for cell culture
Sigma-Aldrich
Puromycin dihydrochloride from Streptomyces alboniger, ≥98% (HPLC), powder
Sigma-Aldrich
Monoclonal Anti-β-Actin antibody produced in mouse, clone AC-15, ascites fluid
Sigma-Aldrich
Puromycin dihydrochloride, Ready Made Solution, from Streptomyces alboniger, 10 mg/mL in H2O, suitable for cell culture