bioRxiv Science⌕ Search

Biology subjects

Kitaguchi, T.

Publications and source records attributed to Kitaguchi, T..

2 recordsLinked to original sources

Photothermal Dye-based Subcellular-sized Heat Spot Enabling the Modulation of Local Cellular Activities

Thermal engineering at microscale such as the control and measurement of temperature is a key technology in basic biological research and biomaterials development, which remains challenge yet. Here, we engineered the polymeric nanoparticle, in which a fluorescent temperature sensory dye and a photothermal dye were embedded in its polymer matrices, termed nanoHT. When a near infrared laser at 808 nm is illuminated to the particle, it enables to create the subcellular-sized heat spot in a live cell, where fluorescence thermometry allows the read out of the temperature increment concurrently at individual heat spots. Owing to the controlled local heating, we found that the cell death of HeLa cells was induced at the certain temperature at rate of a few seconds. It should be also noted that the cell death was triggered from the very local heat spot at subcellular level. Furthermore, nanoHT was applied for the induction of muscle contraction of the C2C12 myotube by heat. We successfully showed that the heat-induced contraction took place at the limited area of a single myotube according to the alteration of protein-protein interactions related to the contraction event. These studies demonstrated that even a single heat spot provided by a photothermal material could be very effective in altering cellular functions, paving the way for novel photothermal therapies.

bioengineering↗

The molecular basis of ubiquitin-specific protease 8 autoinhibition by the WW-like domain

Ubiquitin-specific protease 8 (USP8) is a deubiquitinating enzyme involved in multiple membrane trafficking pathways. The enzyme activity is inhibited by binding to 14-3-3 proteins, and mutations of the 14-3-3 binding motif in USP8 are related to Cushings disease. However, the molecular basis of USP8 enzyme activity regulation remains unclear. Here, we identified amino acids 645-684 of USP8 as an autoinhibitory region, which our pull-down and single-molecule FRET assay results suggested interacts with the catalytic USP domain. In silico modelling indicated that the region forms a WW-like domain structure, plugs the catalytic cleft, and narrows the entrance to the ubiquitin-binding pocket. Furthermore, 14-3-3 was found to inhibit USP8 enzyme activity partly by enhancing the interaction between the WW-like and USP domains. These findings provide the molecular basis of USP8 autoinhibition via the WW-like domain. Moreover, they suggest that the release of autoinhibition may underlie Cushings disease caused by USP8 mutations.

biochemistry↗