TDP-43 Dimerizes in Human Cells in Culture |
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Authors: | Yuki Shiina Kunimasa Arima Hiroko Tabunoki Jun-ichi Satoh |
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Institution: | (1) Department of Bioinformatics and Molecular Neuropathology, Meiji Pharmaceutical University, 2-522-1 Noshio Kiyose, Tokyo 204-8588, Japan;(2) Department of Psychiatry, National Center Hospital, National Center of Neurology and Psychiatry, Tokyo 187-8551, Japan; |
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Abstract: | 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. |
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