Researchers at the Nano Life Science Institute (WPI-NanoLSI), Kanazawa University, report in ACS Nano, how proteins in cells can be controllably activated through heating, an effect that can be used to initiate programmed cell death.
Cellular processes are governed by the activity of proteins. Being able to control the functioning of proteins is therefore highly relevant for the development of biotechnological tools. Doing so with high-enough spatial and temporal precision is hugely challenging, however. One approach for tackling this challenge, called thermogenetics, is based on the thermal response of certain proteins, with slight heating or cooling resulting in (de)activation. Now, Cong Quang Vu and Satoshi Arai from Kanazawa University have developed a thermogenetic tool based on polypeptides that enable easy regulation of a protein’s activation temperature and used it to achieve programmed cell death of human-derived cells.
The scientists worked with so-called elastin-like polypeptides (ELPs), biopolymers composed of repeated amino acid building blocks. ELPs are soluble below a certain temperature; above the temperature threshold, they group into coacervate droplets. An ELP’s transition temperature depends on the precise composition of its monomer building block, as well as on its number of monomers. By changing these characteristics, the transition temperature of an ELP can be fine-tuned, which offers the possibility of designing temperature-responsive biomolecular systems that can be thermally activated and deactivated.
The researchers coupled ELPs to a protein called caspase-8 (CASP8), which is found in humans and other mammals. When activated, CASP8 undergoes structural transformations that ultimately lead to the host cell’s death, a process referred to as apoptosis. Vu and Arai aimed to design an ELP with a transition temperature of a few degrees above body temperature, so that only mild heating was required for inducing the solution-to-coacervate-droplet transition. This ELP was then fused to CASP8. Above the transition temperature, the ELP-CASP8 complexes formed coacervate droplets, causing the CASP8 parts to align in a way that triggers activation.
To be able to monitor whether CASP8 activation actually happens, the scientists developed a CASP8 indicator. The indication mechanism involves fluorescent proteins that, as soon as CASP8 gets activated, translocate from outside to inside nucleus in a cell. Measuring the fluorescence intensity of the fluorescent proteins in the nucleus of the cell then made it possible to distinguish between activated and non-activated CASP8.
To check the feasibility of the proposed thermogenetic CASP8 activation scheme, Vu and Arai applied it to cells derived from a human kidney. They found that the heat-responsive CASP8 system indeed led to induced cell death upon increasing temperature. The researchers also performed experiments with local heating, by means of an infrared laser, demonstrating that their thermogenetically controlled apoptosis approach works on the single-cell level too.
Vu and Arai suggest that their thermogenetic tool may also be applied to cellular processes other than apoptosis, paving the way for various biotechnological applications. Quoting the scientists: “By replacing CASP8 with other biomolecules, additional thermogenetic tools can be developed to modulate various cellular functions, such as enzyme activity, protein–protein interactions, and gene expression.”
Background
Apoptosis
Cell death in organisms can result from unintended cellular injury, or from a process initiated by the organism itself. This process is referred to as apoptosis, or programmed cell death. Apoptosis is the mechanism by which humans lose billions of cells every day; it is a highly regulated process and plays an important part in an organism’s development and life cycle.
Apoptosis happens as a chain of biochemical events. It is triggered by the activation of caspases, proteins that can cleave other proteins.
Cong Quang Vu and Satoshi Arai from Kanazawa University have now developed a biotechnological procedure for activating caspase proteins through mild heating, and showed that it can be used to initiate apoptosis in a controlled way.
Funding
This work was supported by a grant-in-aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan (22K20529 to C.Q.V.), the WPI-NanoLSI Transdisciplinary Research Promotion Grant, Kanazawa University to C.Q.V. in the fiscal years 2022−2023 and 2023−2024, the JST FOREST Program (JPMJFR201E to S.A.), and the World Premier International Research Center Initiative (WPI), MEXT.
Contact
Project Planning and Outreach, NanoLSI Administration Office
Nano Life Science Institute, Kanazawa University
Email: nanolsi-office@adm.kanazawa-u.ac.jp
Kakuma-machi, Kanazawa 920-1192, Japan
About Nano Life Science Institute (WPI-NanoLSI), Kanazawa University
Understanding nanoscale mechanisms of life phenomena by exploring “uncharted nano-realms”.
Cells are the basic units of almost all life forms. We are developing nanoprobe technologies that allow direct imaging, analysis, and manipulation of the behavior and dynamics of important macromolecules in living organisms, such as proteins and nucleic acids, at the surface and interior of cells. We aim at acquiring a fundamental understanding of the various life phenomena at the nanoscale.
https://nanolsi.kanazawa-u.ac.jp/en/
About the World Premier International Research Center Initiative (WPI)
The WPI program was launched in 2007 by Japan's Ministry of Education, Culture, Sports, Science and Technology (MEXT) to foster globally visible research centers boasting the highest standards and outstanding research environments. Numbering more than a dozen and operating at institutions throughout the country, these centers are given a high degree of autonomy, allowing them to engage in innovative modes of management and research. The program is administered by the Japan Society for the Promotion of Science (JSPS).
See the latest research news from the centers at the WPI News Portal:
https://www.eurekalert.org/newsportal/WPI
Main WPI program site:
www.jsps.go.jp/english/e-toplevel
About Kanazawa University
As the leading comprehensive university on the Sea of Japan coast, Kanazawa University has contributed greatly to higher education and academic research in Japan since it was founded in 1949. The University has three colleges and 17 schools offering courses in subjects that include medicine, computer engineering, and humanities.
The University is located on the coast of the Sea of Japan in Kanazawa, a city rich in history and culture. The city of Kanazawa has a highly respected intellectual profile since the time of the fiefdom (1598-1867). Kanazawa University is divided into two main campuses: Kakuma and Takaramachi for its approximately 10,200 students, including 600 from overseas.
http://www.kanazawa-u.ac.jp/en/
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