Modular engineering of cellular signaling proteins and networks

Abstract

Living cells respond to their environment using networks of signaling molecules that act as sensors, information processors, and actuators. These signaling systems are highly modular at both the molecular and network scales, and much evidence suggests that evolution has harnessed this modularity to rewire and generate new physiological behaviors. Conversely, we are now finding that, following nature's example, signaling modules can be recombined to form synthetic tools for monitoring, interrogating, and controlling the behavior of cells. Here we highlight recent progress in the modular design of synthetic receptors, optogenetic switches, and phospho-regulated proteins and circuits, and discuss the expanding role of combinatorial design in the engineering of cellular signaling proteins and networks.

Figure 1

Hierarchical organization of signaling systems: cells and individual proteins as input/output nodes. At any scale, a signaling system must have three components — it must have sensors to receive INPUT, an information-processing layer that decides what to make of this information, and an OUTPUT function. These components are found in individual signaling molecules, which detect and effect particular upstream and downstream molecular partners. In receptors that span the cell’s membrane, ligand binding to extracellular domains (INPUT) rapidly regulates the activity of intracellular effector domains (OUTPUT). Similarly, posttranslationally-regulated binding motifs link the activities of upstream enzymes that ‘write’ and ‘erase’ the posttranslational mark (INPUT; e.g. kinases and phosphatases) to recruitment of dedicated ‘reader’ domains (OUTPUT). The same classes of components are found in signaling networks and whole cells, but in this case receptor molecules function as INPUT sensors, networks of intracellular proteins function as the information processing layer, and various cellular response modules control OUTPUT.

Figure 2

Engineering new sensor/receptor systems. (a) The chimeric antigen receptor (CAR) was engineered to sense a tumor antigen and induce an immunogenic response against tumor cells expressing that antigen. Modular recombination of the CAR domains with new sensor modules has enhanced specificity of the CAR-T response either through logic gates requiring combinations of specific antigens or licensed by small molecule dimerization of critical signaling domains [,–21]. A second type of engineered receptor based on Notch (synNotch) allows both input (target antigen) and output (gene expression) to be fully customized [24]. CAR and synNotch receptors can be combined synergistically, refining the specificity and scope of the T cell response [25]. (b) Modular optogenetic tools for controlling receptors and signaling proteins. Protein domains from plants that undergo light-induced dimerization, oligomerization, or steric regulation have been used to regulate signaling activities throughout the cell in a modular fashion.

Figure 3

Post-translational signaling: rewiring phosphorylation devices. Novel linkages between signaling modules enable new functionality in signaling proteins and networks. (a) Synthetic scaffolding of the MAPK pathway was sufficient to induce downstream signaling [55] (top left), while co-scaffolding with negative regulatory effectors can tune the pathway response [57]. (b) Combining kinase docking motifs with phospho-regulated nuclear import and export sequences is a successful strategy to create dynamic fluorescent reporters of specific kinase activity [62^••] (top right). Mutation of a phospho-site in a 3-pronged AND-gate for protein degradation generated a 2-pronged degradation AND-gate [69^•].

Figure 4

Combinatorial design for engineering signaling proteins and networks. We present here a conceptual workflow for engineering signaling networks with desired properties. Small libraries of candidate circuits can be semi-rationally designed using a combination of validated signaling and regulatory components together with computational models. These circuits can then be screened and optimized for the proper function. The design of a network for cell polarization [74^••] is provided as an example of this approach.