Prolyl endopeptidase is revealed following SILAC analysis to be a novel mediator of human microglial and THP-1 cell neurotoxicity.
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.
Klegeris
Andis
A
Unit of Biology and Physical Geography, Barber School of Arts and Sciences, University of British Columbia Okanagan, Kelowna, British Columbia, Canada.
Li
Jane
J
Bammler
Theo K
TK
Jin
Jinghua
J
Zhu
David
D
Kashima
Daniel T
DT
Pan
Sheng
S
Hashioka
Sadayuki
S
Maguire
John
J
McGeer
Patrick L
PL
Zhang
Jing
J
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
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