Molecular chaperone HSP70 prevents formation of inclusion bodies of the 25-kDa C-terminal fragment of TDP-43 by preventing aggregate accumulation
Transactive response DNA/RNA-binding protein 43-kDa (TDP-43) C-terminal fragments, such as a 25-kDa fragment (TDP-25), have been identified as a ubiquitinated and phosphorylated components of inclusion bodies (IBs) in motor neurons from amyotrophic lateral sclerosis patients. Cells contain proteins that function as molecular chaperones and prevent aggregate formation of misfolded and aggregation-prone proteins. Recently, we reported that heat shock protein (HSP)70, an abundant molecular chaperone, binds to TDP-25 in an ATP-dependent manner; however, whether HSP70 can prevent the formation of TDP-25-related IBs remains unknown. Here, we showed that HSP70 prevented TDP-25 aggregation according to green fluorescent protein-tagged TDP-25 (G-TDP-25) colocalization in the cytoplasm with mCherry-tagged HSP70 (HSP70-R). The mobile fraction of HSP70-R in the cytoplasmic IBs associated with G-TDP-25 increased relative to that of G-TDP-25, suggesting that HSP70 strongly bound to G-TDP-25 in the IBs, whereas a portion remained dissociated from the IBs. Importantly, the proportion of G-TDP-25 IBs was significantly decreased by HSP70-R overexpression; however, G-TDP-25 levels in the insoluble fraction remained unchanged by HSP70-R overexpression, suggesting that G-TDP-25 formed aggregated species that cannot be dissolved, even in the presence of strong detergents. These results indicated that HSP70 prevented the accumulation of G-TDP-25 aggregates in cytoplasmic IBs, but was insufficient for G-TDP-25 disassembly and solubilization.
KeywordsProteostasis Protein aggregation HSP FRAP ALS
Amyotrophic lateral sclerosis
Fluorescence recovery after photobleaching
Fused in sarcoma/translated in liposarcoma
Green fluorescent protein
Heat shock cognate
Heat shock protein
Red fluorescent protein
Sodium dodecyl sulfate
Transactive response element DNA/RNA-binding protein 43-kDa
25-kDa C-terminal fragment of TDP-43
A.K. was supported by a Japan Society for Promotion of Science (JSPS) Grant-in-Aid for the Promotion of Joint International Research (Fostering Joint International Research) (16KK0156), a JSPS Grant-in-Aid for Scientific Research (C) (#26440090), a grant-in-aid from The Nakabayashi Trust for ALS Research (Tokyo, Japan), and by a grant-in-aid from the Japan Amyotrophic Lateral Sclerosis Association (JALSA, Tokyo, Japan) for ALS research. M.K. was partially supported by a grant-in-aid from the Nakatani Foundation for Advancement of Measuring Technologies in Biomedical Engineering.