title: Correlation between leucine rich domain and the stability of LRWD1 protein in human NT2/D1 cells.
abstract: OBJECTIVE
  LRWD1 is a protein that contains LRR and WDs domains and is important in regulating spermatogenesis. However, the roles of LRR or WDs domains in the expression of LRWD1 remain unclear.
  
  
  METHODS
  The NT2/D1 cells separately transfected with full length of LRWD1 gene (LRWD(WT)) or genes with deleted sequences in the LRR domain (LRWD1(ΔLRR)), WD1 domain (LRWD1(ΔWD1)), WD2 domain (LRWD1(ΔWD2)), WD3 domain (LRWD1(ΔWD3)) and entire three WD domains (LRWD1(Δ3×WD)) were applied to investigate the expression levels of LRWD1 protein by either Western blot or flow cytometry. The associated proteins in these mutated LRWD1 proteins were identified by mass spectrometry.
  
  
  RESULTS
  Deletion of the LRR domain significantly decreased the expression of LRWD1 protein. With the treatment of MG132, the LRR domain may functions in preventing LRWD1 protein from proteasome-mediated degradation. In the co-immunoprecipitation analysis, protein receptor of tumor necrosis factor 2 (TNFR2) was specifically observed to be associated with LRR-deficient LRWD1 protein.
  
  
  CONCLUSIONS
  The LRR domain is significantly correlated to the stability of LRWD1 protein. Determining if the stability is modulated by TNFR2 is worthy of further study.
authors.affiliation: Department of Obstetrics and Gynecology, Chi-Mei Medical Center, Tainan, Taiwan; Department of Medicine, Taipei Medical University, Taipei, Taiwan; Department of Sport Management, Chia Nan University of Pharmacy and Science, Tainan, Taiwan.
authors.forename: Yung-Chieh
authors.initials: YC
authors.lastname: Tsai
authors.affiliation: Department of Biological Sciences and Technology, National University of Tainan, Taiwan.
authors.forename: Yen-Ni
authors.initials: YN
authors.lastname: Teng
authors.affiliation: Department of Biotechnology, Chia Nan University of Pharmacy and Science, Tainan, Taiwan.
authors.forename: Jui-Hsiang
authors.initials: JH
authors.lastname: Hung
authors.affiliation: Division of Nephrology, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan.
authors.forename: Chien-Hsing
authors.initials: CH
authors.lastname: Wu
authors.affiliation: Department of Biological Sciences and Technology, National University of Tainan, Taiwan.
authors.forename: Yu-Ting
authors.initials: YT
authors.lastname: Kuo
authors.affiliation: Department of Obstetrics & Gynecology, National Cheng Kung University Hospital, Tainan, Taiwan.
authors.forename: Pao-Lin
authors.initials: PL
authors.lastname: Kuo
authors.affiliation: Department of Biotechnology, College of Life Science, Kaohsiung Medical University, Kaohsiung, Taiwan.
authors.forename: Chien-Chih
authors.initials: CC
authors.lastname: Chiu
authors.affiliation: Department of Biomedical Science and Environmental Biology, College of Life Science, Kaohsiung Medical University, Kaohsiung, Taiwan; Department of Biological Science, National Sun Yat-sen University, Kaohsiung, Taiwan. Electronic address: huangpin2@kmu.edu.tw.
authors.forename: Bin
authors.initials: B
authors.lastname: Huang
mesh_terms: D045744:Cell Line, Tumor; D005786:Gene Expression Regulation; D006801:Humans; D008297:Male; D008868:Microtubule Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D013091:Spermatogenesis; D013737:Testis / Q000378:metabolism*
chemical_list: C000595278:LRWD1 protein, human; D008868:Microtubule Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins
keywords: Co-immunoprecipitation; LRWD1; Leucine rich domain; Proteomics; Spermatogenesis