Title
**In situ** prolyl oligopeptidase activity assay in neural cell cultures **In situ** prolyl oligopeptidase activity assay in neural cell cultures
Author
Faculty/Department
Faculty of Pharmaceutical, Biomedical and Veterinary Sciences. Pharmacy
Publication type
article
Publication
Amsterdam ,
Subject
Biology
Pharmacology. Therapy
Source (journal)
Journal of neuroscience methods. - Amsterdam
Volume/pages
204(2012) :1 , p. 104-110
ISSN
0165-0270
ISI
000299977400011
Carrier
E
Target language
English (eng)
Full text (Publishers DOI)
Affiliation
University of Antwerp
Abstract
Prolyl oligopeptidase (PREP, E.C.3.4.21.26) is a cytosolic serine protease that hydrolyzes small (<3 kDa), proline-containing peptides on the carboxyl terminal side of proline residues, and is widely distributed in the brain. High PREP activity, due to aging or neurodegenerative disease, has been hypothesised to lead to an increased breakdown of neuropeptides, resulting in a decline of cognitive functions and an acceleration of neurodegeneration. Recent data have suggested that PREP involvement in neurodegeneration cannot be explained by its extracellular space proteolytic activity alone, but may involve intracellular PREP activities as well. In order to test this, appropriate methods for measuring PREP intracellular activity must first be developed. In the present study, we developed and validated an in situ PREP intracellular activity assay in primary rat cortical neurons, using nitroblue tetrazolium chloride salt (NBT) and a PREP specific substrate (S)-benzyl 2-(2-(4-hydroxynaphthalen-l-ylcarbanoyl)pyrrolidin-l-yl)-2-oxoethylcarbamate (UAMC-00682). This novel in situ PREP activity assay was further validated in neuroblastoma SH-SY5Y cells, under conditions of PREP overexpression and inhibited PREP expression. Using this assay, we demonstrated that PREP inhibitors, Z-Pro-Pro-aldehyde-dimethylacetal, Boc-Asn-Phe-Pro-aldehyde, and (S)-1-((S)-1-(4-phenylbutanoyl)-pyrrolidine-2-carbonyl)pyrrolidine-2-carbonitrile (KYP-2047), were able to inhibit intracellular PREP activity in primary rat cortical neurons. KYP-2047 was the most potent PREP inhibitor in all assay systems tested. The validated assay enables localization and quantification of in situ PREP activity in primary rat cortical neurons and neuroblastoma SH-SY5Y cells, as well allows testing cell permeability and efficiency of novel PREP inhibitors.
E-info
https://repository.uantwerpen.be/docman/iruaauth/3b0b37/0de55557c69.pdf
Full text (open access)
https://repository.uantwerpen.be/docman/irua/4f3d77/2037.pdf
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