title: Identification of proteins that interact with TANK binding kinase 1 and testing for mutations associated with glaucoma.
abstract: OBJECTIVE
  Copy number variations (duplications) of TANK binding kinase 1 (TBK1) have been associated with normal tension glaucoma (NTG), a common cause of blindness worldwide. Mutations in other genes involved in autophagy (TLR4 and OPTN) have been associated with NTG. Here we report searching for additional proteins involved in autophagy that may also have roles in NTG.
  
  
  METHODS
  HEK-293T cells were transfected to produce synthetic TBK1 protein with FLAG and S tags. Proteins that associate with TBK1 were isolated from HEK-293T lysates using tandem affinity purification (TAP) and polyacrylamide gel electrophoresis (PAGE). Isolated proteins were identified with mass spectrometry. A cohort of 148 NTG patients and 77 controls from Iowa were tested for glaucoma-causing mutations in genes that encode identified proteins that interact with TBK1 using high resolution melt (HRM) analysis and DNA sequencing.
  
  
  RESULTS
  TAP studies show that three proteins expressed in HEK-293T cells (NAP1, TANK and TBKBP1) interact with TBK1. Testing cohorts of NTG and normal controls for disease-causing mutations in TANK, identified a total of nine unique variants including three non-synonymous changes, one synonymous changes and five intronic changes. When analyzed alone or as a group, the non-synonymous TBK1 coding sequence changes were not associated with either NTG or primary open angle glaucoma.
  
  
  CONCLUSIONS
  TAP showed that NAP1, TANK and TBKBP1 interact with TBK1 and are good candidates for contributing to NTG. A mutation screen of TANK detected three non-synonymous variants. Although, it remains possible that one or more of these TANK mutations may have a role in NTG, the data in this report do not provide statistical support for an association between TANK variants and NTG.
authors.affiliation: Department of Ophthalmology and Visual Sciences, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA.
authors.forename: Seongjin
authors.initials: S
authors.lastname: Seo
authors.forename: Frances
authors.initials: F
authors.lastname: Solivan-Timpe
authors.forename: Ben R
authors.initials: BR
authors.lastname: Roos
authors.forename: Alan L
authors.initials: AL
authors.lastname: Robin
authors.forename: Edwin M
authors.initials: EM
authors.lastname: Stone
authors.forename: Young H
authors.initials: YH
authors.lastname: Kwon
authors.forename: Wallace L M
authors.initials: WL
authors.lastname: Alward
authors.forename: John H
authors.initials: JH
authors.lastname: Fingert
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D018628:Gene Dosage / Q000235:genetics; D020440:Gene Duplication / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D057066:Low Tension Glaucoma / Q000235:genetics* / Q000378:metabolism; D017354:Point Mutation / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D047992:TNF Receptor-Associated Factor 2 / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C484406:TANK protein, human; C524349:TBKBP1 protein, human; D047992:TNF Receptor-Associated Factor 2; D017346:Protein Serine-Threonine Kinases; C403191:TBK1 protein, human