Scientists create advanced sense-and-respond systems that mimic natural cellular behavior

Protein phosphorylation is a cornerstone of cellular communication, orchestrating rapid and precise responses to environmental signals.

Recent breakthroughs in synthetic biology, led by researchers at Rice University, and published in the journal, Science, are revolutionizing how these pathways can be engineered for therapeutic purposes. By constructing modular phosphorylation circuits in human cells, this research opens the door to advanced sense-and-respond systems that mimic natural cellular behavior.

Phosphorylation is a biochemical process where a phosphate group is added to a protein, altering its function. This modification plays a critical role in cellular activities such as movement, secretion, and gene expression. In multicellular organisms, phosphorylation cascades amplify external signals through interconnected cycles, akin to a series of falling dominoes. However, the inherent complexity of these pathways has historically limited their application in synthetic systems.

The Rice University team addressed this challenge by reimagining phosphorylation cycles as modular units. Each cycle—composed of kinase and phosphatase activities—operates independently, enabling researchers to assemble entirely novel circuits. These synthetic cycles interact seamlessly with native cellular processes without compromising cell viability or growth.

Caleb Bashor, a leading researcher in the study, emphasized the potential of this approach: “It turns out, phosphorylation cycles are not just interconnected but interconnectable. This dramatically expands the design space for synthetic signaling circuits.”

To validate their strategy, the researchers engineered synthetic kinases (synKins) and synthetic substrates (synSubs). By fusing kinase domains with leucine zippers (LZs), they created a system where synKins selectively phosphorylate synSubs.

These components were introduced into human embryonic kidney (HEK) 293T cells, and multicolor flow cytometry was used to measure phosphorylation efficiency. Variants of synKins were optimized to ensure minimal interference with host cell machinery, as evidenced by negligible nonspecific phosphorylation and preserved cell growth rates.

The team’s modular approach also included synthetically targeted phosphatases (synPhos), which reversed phosphorylation in a recruitment-dependent manner. This bidirectional control allowed researchers to fine-tune phosphorylation cycles by altering molecular properties such as binding affinity and catalytic turnover rates. For example, modifying LZ sequences and active site mutations in synKins resulted in a 10- to 20-fold modulation of synSub phosphorylation.

To predict phosphorylation behavior, the researchers developed a thermodynamic model. Single-cell fluorescence data were normalized and analyzed, yielding precise parameters for LZ interaction affinities and catalytic rates. These predictions were validated across 216 unique circuit compositions, demonstrating the model’s accuracy and the modular system’s versatility. This framework also identified conditions for ultrasensitive responses, enabling circuits to amplify weak inputs into significant outputs.

One remarkable achievement was the integration of synthetic phosphorylation circuits with transcriptional reporters. Engineered SH2 domains, which bind to phosphorylated motifs, were used to establish connections between phosphorylation cycles and downstream gene expression. This capability mirrors native signaling networks, where phosphorylation triggers allosteric regulation, protein stabilization, or new protein-protein interactions.

The potential applications of these innovations are vast. Synthetic phosphorylation circuits can be programmed for real-time responses to extracellular signals, such as inflammatory markers or tumor growth factors.

In proof-of-concept experiments, the team engineered circuits capable of detecting inflammatory cytokines and regulating autoimmune flare-ups. These circuits offer a promising solution for mitigating immunotherapy-related toxicity and advancing precision medicine.

Xiaoyu Yang, the study’s lead author, described the groundbreaking nature of the work: “Imagine tiny processors inside cells made of proteins that can decide how to respond to specific signals like inflammation or blood sugar levels.

This work brings us closer to building smart cells that detect disease and release treatments in real time.” The rapid response capability of these circuits—achieving functional changes within seconds to minutes—surpasses traditional synthetic systems that rely on slower transcriptional processes.

Despite its complexity, the design process was guided by principles of modularity and predictability. The team’s efforts represent a significant leap forward in synthetic biology, akin to the advances made in genetic circuit engineering. By establishing a foundational toolkit for phosphorylation-based circuits, this research paves the way for scalable, user-defined cellular therapies.

Rice University’s Synthetic Biology Institute, under the leadership of Caroline Ajo-Franklin, has been instrumental in fostering this innovation. “If in the last 20 years synthetic biologists have learned how to manipulate the way bacteria gradually respond to environmental cues, this work vaults us forward to controlling mammalian cells’ immediate response to change,” said Ajo-Franklin.

The implications of this research extend beyond medicine. Synthetic phosphorylation circuits could revolutionize biosensing technologies, enabling cells to act as dynamic detectors for environmental or physiological changes. Their ability to operate independently of native pathways ensures high specificity and minimal cross-talk, crucial for therapeutic applications.

In addition to its therapeutic potential, this research offers insights into the fundamental principles of cellular communication. Phosphorylation cycles serve as a model for how evolution has optimized signaling networks for speed and efficiency. By replicating these natural designs, synthetic biology gains a powerful tool for understanding cellular behavior and improving biotechnological processes.

The team also explored the scalability of their approach, testing how these circuits could be expanded to incorporate multiple layers of signaling. They demonstrated that synthetic circuits could process complex inputs, such as combinations of environmental signals, to produce highly specific outputs. This multilayered design mimics the sophistication of native signaling networks, providing a template for creating advanced cellular systems.

Another critical aspect of this work is its potential for customization. Researchers can now design circuits tailored to specific applications, such as detecting rare disease markers or responding to environmental changes. This flexibility makes synthetic phosphorylation circuits an invaluable asset for both research and clinical settings.

While the journey to develop these circuits was not without challenges, the collaborative effort among researchers yielded a system that performs on par with natural pathways. Caleb Bashor summarized the breakthrough: “Our synthetic signaling circuits are not only highly tunable but can function alongside cells’ own processes without compromising viability. This was a major hurdle we’re thrilled to have overcome.”

As the field of synthetic biology continues to evolve, the Rice University team’s work stands as a testament to the power of interdisciplinary innovation.

By leveraging the modularity of natural systems, they have unlocked new possibilities for engineering cellular behavior, offering hope for transformative therapies and technologies.

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