title: The point mutation UCH-L1 C152A protects primary neurons against cyclopentenone prostaglandin-induced cytotoxicity: implications for post-ischemic neuronal injury.
abstract: Cyclopentenone prostaglandins (CyPGs), such as 15-deoxy-Δ(12,14)-prostaglandin J2 (15dPGJ2), are reactive prostaglandin metabolites exerting a variety of biological effects. CyPGs are produced in ischemic brain and disrupt the ubiquitin-proteasome system (UPS). Ubiquitin-C-terminal hydrolase L1 (UCH-L1) is a brain-specific deubiquitinating enzyme that has been linked to neurodegenerative diseases. Using tandem mass spectrometry (MS) analyses, we found that the C152 site of UCH-L1 is adducted by CyPGs. Mutation of C152 to alanine (C152A) inhibited CyPG modification and conserved recombinant UCH-L1 protein hydrolase activity after 15dPGJ2 treatment. A knock-in (KI) mouse expressing the UCH-L1 C152A mutation was constructed with the bacterial artificial chromosome (BAC) technique. Brain expression and distribution of UCH-L1 in the KI mouse was similar to that of wild type (WT) as determined by western blotting. Primary cortical neurons derived from KI mice were resistant to 15dPGJ2 cytotoxicity compared with neurons from WT mice as detected by the WST-1 cell viability assay and caspase-3 and poly ADP ribose polymerase (PARP) cleavage. This protective effect was accompanied with significantly less ubiquitinated protein accumulation and aggregation as well as less UCH-L1 aggregation in C152A KI primary neurons after 15dPGJ2 treatment. Additionally, 15dPGJ2-induced axonal injury was also significantly attenuated in KI neurons as compared with WT. Taken together, these studies indicate that UCH-L1 function is important in hypoxic neuronal death, and the C152 site of UCH-L1 has a significant role in neuronal survival after hypoxic/ischemic injury.
authors.affiliation: Geriatric Research Educational and Clinical Center, VA Pittsburgh Healthcare System, Pittsburgh, PA, USA.|Department of Neurology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
authors.forename: H
authors.initials: H
authors.lastname: Liu
authors.affiliation: Geriatric Research Educational and Clinical Center, VA Pittsburgh Healthcare System, Pittsburgh, PA, USA.|Department of Neurology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
authors.forename: W
authors.initials: W
authors.lastname: Li
authors.affiliation: Geriatric Research Educational and Clinical Center, VA Pittsburgh Healthcare System, Pittsburgh, PA, USA.|Department of Neurology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
authors.forename: M E
authors.initials: ME
authors.lastname: Rose
authors.affiliation: Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
authors.forename: R W
authors.initials: RW
authors.lastname: Hickey
authors.affiliation: Department of Neurology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
authors.forename: J
authors.initials: J
authors.lastname: Chen
authors.affiliation: Genomics and Proteomics Core Laboratories, University of Pittsburgh, Pittsburgh, PA, USA.
authors.forename: G T
authors.initials: GT
authors.lastname: Uechi
authors.affiliation: Genomics and Proteomics Core Laboratories, University of Pittsburgh, Pittsburgh, PA, USA.
authors.forename: M
authors.initials: M
authors.lastname: Balasubramani
authors.affiliation: Genomics and Proteomics Core Laboratories, University of Pittsburgh, Pittsburgh, PA, USA.|Department of Pharmaceutical Sciences, University of Pittsburgh School of Pharmacy, Pittsburgh, PA, USA.
authors.forename: B W
authors.initials: BW
authors.lastname: Day
authors.affiliation: Department of Neurology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
authors.forename: K V
authors.initials: KV
authors.lastname: Patel
authors.affiliation: Geriatric Research Educational and Clinical Center, VA Pittsburgh Healthcare System, Pittsburgh, PA, USA.|Department of Neurology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
authors.forename: S H
authors.initials: SH
authors.lastname: Graham
mesh_terms: D000818:Animals; D001665:Binding Sites; D002545:Brain Ischemia / Q000201:enzymology / Q000235:genetics* / Q000473:pathology; D003517:Cyclopentanes / Q000633:toxicity*; D005260:Female; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009474:Neurons / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism / Q000502:physiology*; D017354:Point Mutation; D011453:Prostaglandins / Q000633:toxicity*; D051381:Rats; D043222:Ubiquitin Thiolesterase / Q000096:biosynthesis / Q000235:genetics*
chemical_list: D003517:Cyclopentanes; D011453:Prostaglandins; D043222:Ubiquitin Thiolesterase; C013905:cyclopentenone; C496976:Uchl1 protein, mouse