Periodic Reporting for period 1 - Biosensor Design (Engineering Chemotactic Biosensors for a Diverse Spectrum of Metastatic Markers)
Periodo di rendicontazione: 2020-02-01 al 2022-01-31
The directional movement of cells in response to environmental signals (chemotaxis) is essential in biology. Immune cells use chemotaxis to traffic towards disease sites, however, metastatic cells often remain undetected. The design of chemotactic biosensors for metastatic markers can manipulate the immune system's spectrum of actions. Studies have identified soluble secreted factors that promote the initial escape of tumour cells, their intravasation into circulation, and extravasation into metastatic niches. The EU-funded Biosensor Design project aims to build and validate a general computation-based platform for rational biosensor design. The immediate objective is to create biosensors that will elicit migration of engineered cytotoxic immune cells towards vulnerable metastatic cells.
We developed a computational strategy for designing highly sensitive receptor biosensors of chemokines. We used the structurally well-characterized chemokine receptor, CXCR4 as a template, which we have investigated by engineering variants that alter the quaternary structure of the dimeric and can be tuned for downstream biased signaling functions. We then developed a computational strategy that allowed us to carve ligand binding sites and engineer potent and efficacious signaling into peptide-sensing GPCRs, such as chemokine receptors, from homology models without the need for solved active state structures, which are still in short supply. From our method, we generated several Conformationally Adaptive Peptide Biosensors (CaPSens) that have enhanced chemotactic responses, which we demonstrated in primary T cells. Our designs extend membrane protein engineering and synthetic biology, providing insight into mechanisms of signal transduction, and act as useful tools in engineered cells for redirecting and promoting lymphocyte trafficking. Further methodological developments were established to engineer receptor loops for selective ligand-binding and coupling extracellular ligand-sensing domains to intracellular G-protein coupling within a single-domain GPCR.
The project has produced several novel chemotactic biosensor designs based on limited homologous structural information and confronting the design challenge of the inherent flexibility of receptor:peptide signaling systems. Typical peptide-sensing biosensors rely on rigid bidning domains and fluorescent or transcriptional reporters that offer little route to direct cellular function. Our CaPSens go beyond the state-of-the-art by accounting for the high conformational flexibility inherent to both the peptide ligand and the receptor binding pocket. These biosensors are a major first step towards engineering biosensors that can elicit migration and other cellular processes in response to flexible peptide ligands. Our flexible receptor:peptide design pipeline, along with our design strategies to engineer receptor loops with ligand-specific selectivity, and allosteric rewiring of hybridized GPCRs fold into our development of a holistic biosensor design platform. Our engineered CaPSens have direct applications in immune cell homing and synthetic biology, while our design methods set the stage for generation of immunotherapy tools that can circumvent existing barriers to cancer treatment.