TDP-43 dimerizes in human cells in culture.
TAR DNA-binding protein-43 (TDP-43) is a 43-kDa nuclear protein involved in regulation of gene expression. Abnormally, phosphorylated, ubiquitinated, and aggregated TDP-43 constitute a principal component of neuronal and glial cytoplasmic and nuclear inclusions in the brains of frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) and amyotrophic lateral sclerosis (ALS), although the molecular mechanism that triggers aggregate formation remains unknown. By Western blot analysis using anti-TDP-43 antibodies, we identified a band with an apparent molecular mass of 86-kDa in HEK293, HeLa, and SK-N-SH cells in culture. It was labeled with both N-terminal-specific and C-terminal-specific TDP-43 antibodies, enriched in the cytosolic fraction, and the expression levels were reduced by TDP-43 siRNA but unaltered by treatment with MG-132 or by expression of ubiqulin-1 or casein kinase-1. By immunoprecipitation analysis, we found the interaction between the endogenous full-length TDP-43 and the exogenous Flag-tagged TDP-43, and identified the N-terminal half of TDP-43 spanning amino acid residues 3-183 as an intermolecular interaction domain. When the tagged 86-kDa tandemly connected dimer of TDP-43 was overexpressed in HEK293, it was sequestered in the cytoplasm and promoted an accumulation of high-molecular-mass TDP-43-immunoreactive proteins. Furthermore, the 86-kDa band was identified in the immunoblot of human brain tissues, including those of ALS. These results suggest that the 86-kDa band represents dimerized TDP-43 expressed constitutively in normal cells under physiological conditions.
Shiina
Yuki
Y
Department of Bioinformatics and Molecular Neuropathology, Meiji Pharmaceutical University, 2-522-1 Noshio Kiyose, Tokyo, 204-8588, Japan.
Arima
Kunimasa
K
Tabunoki
Hiroko
H
Satoh
Jun-ichi
J
D000328:Adult; D000368:Aged; D000818:Animals; D001921:Brain / Q000166:cytology / Q000378:metabolism; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary
D004268:DNA-Binding Proteins