title: In situ prolyl oligopeptidase activity assay in neural cell cultures.
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.
authors.affiliation: Department of Pharmacology, Centre of Excellence for Translational Medicine, University of Tartu, 19 Ravila Str., 51014 Tartu, Estonia. Electronic address: lindaklimaviciusa@yahoo.com.
authors.forename: Linda
authors.initials: L
authors.lastname: Klimaviciusa
authors.affiliation: Department of Pharmacology, Centre of Excellence for Translational Medicine, University of Tartu, 19 Ravila Str., 51014 Tartu, Estonia.
authors.forename: Rajeev Kumar
authors.initials: RK
authors.lastname: Jain
authors.affiliation: Department of Pharmacology, Centre of Excellence for Translational Medicine, University of Tartu, 19 Ravila Str., 51014 Tartu, Estonia.
authors.forename: Külli
authors.initials: K
authors.lastname: Jaako
authors.affiliation: Laboratory of Medical Biochemistry, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1 S6, B 2610 Antwerp, Belgium.
authors.forename: Roos
authors.initials: R
authors.lastname: Van Elzen
authors.affiliation: Interdisciplinary Research Centre, Katholieke Universiteit Leuven-Kortrijk, B-8500,Belgium.
authors.forename: Melanie
authors.initials: M
authors.lastname: Gerard
authors.affiliation: Laboratory of Medicinal Chemistry, Department of Pharamaceutical Sciences, University of Antwerp, Universiteitsplein 1, B-2610 Wilrijk, Antwerp, Belgium.
authors.forename: Pieter
authors.initials: P
authors.lastname: van Der Veken
authors.affiliation: Laboratory of Medical Biochemistry, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1 S6, B 2610 Antwerp, Belgium.
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Lambeir
authors.affiliation: Department of Pharmacology, Centre of Excellence for Translational Medicine, University of Tartu, 19 Ravila Str., 51014 Tartu, Estonia.
authors.forename: Alexander
authors.initials: A
authors.lastname: Zharkovsky
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D001681:Biological Assay / Q000379:methods*; D002478:Cells, Cultured; D004789:Enzyme Activation; D020869:Gene Expression Profiling / Q000379:methods*; D009474:Neurons / Q000201:enzymology*; D000085822:Prolyl Oligopeptidases; D051381:Rats; D017208:Rats, Wistar; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D012697:Serine Endopeptidases; D000085822:Prolyl Oligopeptidases