Correlation between leucine rich domain and the stability of LRWD1 protein in human NT2/D1 cells

Yung-Chieh Tsai; Yen-Ni Teng; Jui-Hsiang Hung; Chien-Hsing Wu; Yu-Ting Kuo; Pao-Lin Kuo; Chien-Chih Chiu; Bin Huang

doi:10.1016/j.advms.2014.07.002

Correlation between leucine rich domain and the stability of LRWD1 protein in human NT2/D1 cells

Adv Med Sci. 2014 Sep;59(2):266-72. doi: 10.1016/j.advms.2014.07.002. Epub 2014 Aug 13.

Authors

Yung-Chieh Tsai¹, Yen-Ni Teng², Jui-Hsiang Hung³, Chien-Hsing Wu⁴, Yu-Ting Kuo², Pao-Lin Kuo⁵, Chien-Chih Chiu⁶, Bin Huang⁷

Affiliations

¹ 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.
² Department of Biological Sciences and Technology, National University of Tainan, Taiwan.
³ Department of Biotechnology, Chia Nan University of Pharmacy and Science, Tainan, Taiwan.
⁴ Division of Nephrology, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan.
⁵ Department of Obstetrics & Gynecology, National Cheng Kung University Hospital, Tainan, Taiwan.
⁶ Department of Biotechnology, College of Life Science, Kaohsiung Medical University, Kaohsiung, Taiwan.
⁷ 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.

PMID: 25170821
DOI: 10.1016/j.advms.2014.07.002

Abstract

Purpose: 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.

Materials and 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.

Keywords: Co-immunoprecipitation; LRWD1; Leucine rich domain; Proteomics; Spermatogenesis.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Cell Line, Tumor
Gene Expression Regulation*
Humans
Male
Microtubule Proteins / chemistry
Microtubule Proteins / genetics
Microtubule Proteins / metabolism*
Models, Molecular*
Peptide Fragments / chemistry
Peptide Fragments / genetics
Peptide Fragments / metabolism
Protein Interaction Domains and Motifs
Protein Stability
Protein Structure, Tertiary
Recombinant Proteins / chemistry
Recombinant Proteins / metabolism
Sequence Deletion
Spermatogenesis
Testis / metabolism*

Substances

LRWD1 protein, human
Microtubule Proteins
Peptide Fragments
Recombinant Proteins