title: Subcellular localization suggests novel functions for prolyl endopeptidase in protein secretion.
abstract: For a long time, prolyl endopeptidase (PEP) was believed to inactivate neuropeptides in the extracellular space. However, reports on the intracellular activity of PEP suggest additional, as yet unidentified, physiological functions for this enzyme. Here, we demonstrate using biochemical methods of subcellular fractionation, immunocytochemical double-labelling procedures and localization of PEP-enhanced green fluorescent protein fusion proteins that PEP is mainly localized to the perinuclear space, and is associated with the microtubulin cytoskeleton in human neuroblastoma and glioma cell lines. Disassembly of the microtubules by nocodazole treatment disrupts both the fibrillar tubulin and PEP labelling. Furthermore, in a two-hybrid screen, PEP was identified as binding partner of tubulin. These findings indicate novel functions for PEP in axonal transport and/or protein secretion. Indeed, a metabolic labelling approach revealed that both PEP inhibition and PEP antisense mRNA expression result in enhanced peptide/protein secretion from human U-343 glioma cells. Because disturbances in intracellular transport and protein secretion mechanisms are associated with a number of ageing-associated neurodegenerative diseases, cell-permeable PEP inhibitors may be useful for the application in a variety of related clinical conditions.
authors.affiliation: Probiodrug AG, Halle/S., Germany.
authors.forename: Ingo
authors.initials: I
authors.lastname: Schulz
authors.forename: Ulrike
authors.initials: U
authors.lastname: Zeitschel
authors.forename: Thomas
authors.initials: T
authors.lastname: Rudolph
authors.forename: David
authors.initials: D
authors.lastname: Ruiz-Carrillo
authors.forename: Jens-Ulrich
authors.initials: JU
authors.lastname: Rahfeld
authors.forename: Bernd
authors.initials: B
authors.lastname: Gerhartz
authors.forename: Volker
authors.initials: V
authors.lastname: Bigl
authors.forename: Hans-Ulrich
authors.initials: HU
authors.lastname: Demuth
authors.forename: Steffen
authors.initials: S
authors.lastname: Rossner
mesh_terms: D000409:Alanine / Q000031:analogs & derivatives / Q000494:pharmacology; D045744:Cell Line, Tumor; D005452:Fluoresceins / Q000494:pharmacology; D005910:Glioma / Q000378:metabolism* / Q000473:pathology; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D007150:Immunohistochemistry; D049408:Luminescent Agents; D009447:Neuroblastoma / Q000378:metabolism* / Q000473:pathology; D000085822:Prolyl Oligopeptidases; D011506:Proteins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013347:Subcellular Fractions / Q000378:metabolism*; D014018:Tissue Distribution; D014404:Tubulin / Q000378:metabolism
chemical_list: C503951:9-fluorenylmethoxycarbonyl-alanyl-pyrrolidine-2-nitrile; D005452:Fluoresceins; D049408:Luminescent Agents; D011506:Proteins; D011993:Recombinant Fusion Proteins; D015842:Serine Proteinase Inhibitors; D014404:Tubulin; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D012697:Serine Endopeptidases; C000705287:PREPL protein, human; D000085822:Prolyl Oligopeptidases; D000409:Alanine