title: Protease Omi facilitates neurite outgrowth in mouse neuroblastoma N2a cells by cleaving transcription factor E2F1.
abstract: OBJECTIVE
  Omi is an ATP-independent serine protease that is necessary for neuronal function and survival. The aim of this study was to investigate the role of protease Omi in regulating differentiation of mouse neuroblastoma cells and to identify the substrate of Omi involved in this process.
  
  
  METHODS
  Mouse neuroblastoma N2a cells and Omi protease-deficient mnd2 mice were used in this study. To modulate Omi and E2F1 expression, N2a cells were transfected with expression plasmids, shRNA plasmids or siRNA. Protein levels were detected using immunoblot assays. The interaction between Omi and E2F1 was studied using immunoprecipitation, GST pulldown and in vitro cleavage assays. N2a cells were treated with 20 μmol/L retinoic acid (RA) and 1% fetal bovine serum to induce neurite outgrowth, which was measured using Image J software.
  
  
  RESULTS
  E2F1 was significantly increased in Omi knockdown cells and in brain lysates of mnd2 mice, and was decreased in cells overexpressing wild-type Omi, but not inactive Omi S276C. In brain lysates of mnd2 mice, endogenous E2F1 was co-immunoprecipitated with endogenous Omi. In vitro cleavage assay demonstrated that Omi directly cleaved E2F1. Treatment of N2a cells with RA induced marked differentiation and neurite outgrowth accompanied by significantly increased Omi and decreased E2F1 levels, which were suppressed by pretreatment with the specific Omi inhibitor UCF-101. Knockdown of Omi in N2a cells suppressed RA-induced neurite outgrowth, which was partially restored by knockdown of E2F1.
  
  
  CONCLUSIONS
  Protease Omi facilitates neurite outgrowth by cleaving the transcription factor E2F1 in differentiated neuroblastoma cells; E2F1 is a substrate of Omi.
authors.affiliation: Laboratory of Molecular Neuropathology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou 215021, China.
authors.forename: Qi
authors.initials: Q
authors.lastname: Ma
authors.affiliation: Laboratory of Molecular Neuropathology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou 215021, China.
authors.forename: Qing-song
authors.initials: QS
authors.lastname: Hu
authors.affiliation: Laboratory of Molecular Neuropathology, Key Laboratory of Brain Function and Diseases and School of Life Sciences, University of Science and Technology of China, Chinese Academy of Sciences, Hefei 230027, China.
authors.forename: Ran-jie
authors.initials: RJ
authors.lastname: Xu
authors.affiliation: Laboratory of Molecular Neuropathology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou 215021, China.
authors.forename: Xue-chu
authors.initials: XC
authors.lastname: Zhen
authors.affiliation: Laboratory of Molecular Neuropathology, Jiangsu Key Laboratory of Translational Research and Therapy for Neuro-Psycho-Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou 215021, China.
authors.forename: Guang-hui
authors.initials: GH
authors.lastname: Wang
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D050687:E2F1 Transcription Factor / Q000378:metabolism*; D057809:HEK293 Cells; D000074923:High-Temperature Requirement A Serine Peptidase 2; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D024101:Mitochondrial Proteins / Q000378:metabolism*; D016501:Neurites / Q000378:metabolism* / Q000648:ultrastructure; D009447:Neuroblastoma / Q000378:metabolism; D055495:Neurogenesis; D012697:Serine Endopeptidases / Q000378:metabolism*
chemical_list: D050687:E2F1 Transcription Factor; D024101:Mitochondrial Proteins; D012697:Serine Endopeptidases; C000615914:HTRA2 protein, human; D000074923:High-Temperature Requirement A Serine Peptidase 2; C000615915:Htra2 protein, mouse