title: Prolyl endopeptidase is revealed following SILAC analysis to be a novel mediator of human microglial and THP-1 cell neurotoxicity.
abstract: Reactive microglial cells may exacerbate the pathology in some neurodegenerative disorders. Supernatants of stimulated human microglial cells, or their surrogate THP-1 cells, are lethal to cultured human neuroblastoma SH-SY5Y cells. To explore this neurotoxicity, we examined the spectrum of proteins generated by THP-1 cells using the technique of stable isotope labeling by amino acids in cell culture (SILAC). Unstimulated cells were grown in medium with light L-[(12)C(6)] arginine while cells stimulated by lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma) were grown in medium with heavy L-[(13)C(6)] arginine. Proteins isolated from the media were digested with trypsin, and relative concentrations of generated peptides determined by mass spectrometry. More than 1,500 proteins or putative proteins were identified. Of these, 174 were increased and 189 decreased by more than twofold in the stimulated cell supernatant. We selected one upregulated protein, prolyl endopeptidase (PEP), for further investigation of its potential contribution to neurotoxicity. We first confirmed its upregulation by comparing its enzymatic activity in stimulated and unstimulated cell supernatants. We then evaluated two specific PEP inhibitors, Boc-Asn-Phe-Pro-aldehyde and Z-Pro-Pro-aldehyde-dimethyl acetal, for their potential to reduce toxicity of stimulated THP-1 cell and human microglia supernatants towards SH-SY5Y cells. We found both to be partially protective in a concentration-dependent manner. Inhibition of PEP may be a therapeutic approach to neurodegenerative disorders including Alzheimer and Parkinson diseases.
authors.affiliation: Unit of Biology and Physical Geography, Barber School of Arts and Sciences, University of British Columbia Okanagan, Kelowna, British Columbia, Canada.
authors.forename: Andis
authors.initials: A
authors.lastname: Klegeris
authors.forename: Jane
authors.initials: J
authors.lastname: Li
authors.forename: Theo K
authors.initials: TK
authors.lastname: Bammler
authors.forename: Jinghua
authors.initials: J
authors.lastname: Jin
authors.forename: David
authors.initials: D
authors.lastname: Zhu
authors.forename: Daniel T
authors.initials: DT
authors.lastname: Kashima
authors.forename: Sheng
authors.initials: S
authors.lastname: Pan
authors.forename: Sadayuki
authors.initials: S
authors.lastname: Hashioka
authors.forename: John
authors.initials: J
authors.lastname: Maguire
authors.forename: Patrick L
authors.initials: PL
authors.lastname: McGeer
authors.forename: Jing
authors.initials: J
authors.lastname: Zhang
mesh_terms: D000704:Analysis of Variance; D001120:Arginine; D002247:Carbon Isotopes; D002460:Cell Line; D019295:Computational Biology / Q000379:methods; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D013058:Mass Spectrometry; D017628:Microglia / Q000187:drug effects / Q000201:enzymology*; D009000:Monocytes / Q000187:drug effects / Q000201:enzymology*; D009447:Neuroblastoma; D009498:Neurotoxins / Q000037:antagonists & inhibitors* / Q000494:pharmacology; D000085822:Prolyl Oligopeptidases; D012697:Serine Endopeptidases / Q000378:metabolism*; D013778:Tetrazolium Salts; D013844:Thiazoles
chemical_list: D002247:Carbon Isotopes; D004791:Enzyme Inhibitors; D008070:Lipopolysaccharides; D009498:Neurotoxins; D013778:Tetrazolium Salts; D013844:Thiazoles; D007371:Interferon-gamma; D001120:Arginine; D007770:L-Lactate Dehydrogenase; D012697:Serine Endopeptidases; C000705287:PREPL protein, human; D000085822:Prolyl Oligopeptidases; C022616:thiazolyl blue