Projects / Programmes
Design of phosphorylation-regulated signaling pathways in mammalian cells
| Code |
Science |
Field |
Subfield |
| 4.06.00 |
Biotechnical sciences |
Biotechnology |
|
| Code |
Science |
Field |
| 3.04 |
Medical and Health Sciences |
Medical biotechnology |
Synthetic biology, protein logic circuits, protein kinases, protein phosphatases, phosphorylation cascade
Researchers (1)
| no. |
Code |
Name and surname |
Research area |
Role |
Period |
No. of publicationsNo. of publications |
| 1. |
35277 |
PhD Tina Fink |
Biochemistry and molecular biology |
Head |
2020 - 2022 |
25 |
Organisations (1)
| no. |
Code |
Research organisation |
City |
Registration number |
No. of publicationsNo. of publications |
| 1. |
0104 |
National Institute of Chemistry |
Ljubljana |
5051592000 |
21,023 |
Abstract
Response to external and internal signals is an essential feature of all cells, enabling fast and appropriate response to the environmental conditions and/or intracellular signals. We aim to endow cells with new processing and sensing logic that is required for innovative therapies, such as e.g. in CAR T- cell cancer immunotherapy for biotechnological production or sensing. So far, designed cell circuits were based on transcriptional regulation, based on the modular DNA recognition and transcriptional effector domains. Transcriptional regulation is however inherently slower than pathways based on protein interaction and modification. Recently we presented a fast designed signaling pathway, based on proteolysis and coiled-coil interactions (Fink et al., Nat.Chem.Biol. 2019). Fast signal processing in cells is mainly performed by phosphorylation cascades catalyzed by protein kinases and phosphatases, which remains a challenge. Here we propose to design a novel phospho-regulated signaling pathway in mammalian cells which will combine two major advantages: (i) fast signal processing and (ii) reversibility. The underlying idea of the proposed project is to regulate the activity of the target proteins by introducing the phospho-peptide binding domain and the specific kinase substrate peptide into the target protein in such a way that the targeted protein is inactivated and regains its activity in the presence of the selected kinase, which has been confirmed by our preliminary results. Modular design of the proposed system will allow the construction of phosphorylation cascades, logic functions and positive/negative feedback loops, which will be used to design synthetic immunotherapy-relevant phosphorylation signaling cascade in mammalian cells. This strategic goal of the project will be implemented by the following aims that will design, test and demonstrate the function of the designed phosphorylation-regulated signaling pathway in mammalian cells based on the following Aims: (1) Selection of appropriate kinases and their corresponding phospho-peptide binding domains and substrate peptides; (2) Testing different modular architectures of the effector protein; (3) Implementation of phosphorylation-based logic gates in mammalian cells; (4) Development of an immunotherapy-relevant synthetic phosphorylation-based signaling cascade in mammalian cells. The project will lead to the development of a synthetic phosphor-regulated signaling cascade in mammalian cells specifically relevant for cell-based immunotherapy. Therefore, the proposed system could represent an important advance in synthetic biology and could energize further advances of this scientific field.
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