A new cellular control switch has been built from scratch. It displays a bioactive peptide only when it is presented a distinct trigger, not whenever it encounters a cell signaling molecule that happens to present a vague fit. Developed by bioengineers based at the University of Washington (UW) and the University of California, San Francisco (UCSF), the switch could be used to program living cells to perform novel biotechnological and therapeutic functions.

The switch has a cage-like structure, and it works something like a jack-in-the-box. Its internal mechanism, however, isn’t cranked up and released at random times. The cage consists of six helical protein substructures, one of which—a sort of latch—is displaced by a molecular key. Only then is an active site revealed or a signaling peptide sprung.

And the peptide isn’t clowning around. Depending on its structure, it may switch different cell functions on or off. That is, it may modify gene expression, redirect cellular traffic, degrade specific proteins, or control protein-binding interactions.

The switch system relies on Latching Orthogonal Cage–Key pRotein (LOCKR) technology, details of which appeared July 24 in a pair of Nature papers. The first paper (“De novo design of bioactive protein switches”) describes the considerations behind the switch system’s design:

“First, programming free-energy differences between two states is more straightforward in a system that is governed by inter- and intramolecular competition at the same site than by allosteric activation at distant sites because many of the residue-level interactions can be similar (if not identical). Second, a stable protein framework with an extended binding surface that is available for the competing interactions is more programmable and less likely to engage in off-target interactions than a framework that becomes ordered only upon binding.”

In this paper, the UW and UCSF scientists reported that they have demonstrated the power and generality of LOCKR by caging three distinct functions: binding of a pro-apoptotic peptide, degron-mediated protein degradation, and protein localization via a nuclear export sequence.

“LOCKR brings the programmability of DNA switching technology to proteins, with the added advantages of tunability and flexibility over rewired natural protein systems, and ready interfacing with biological machinery over DNA nanotechnology,” the article’s authors concluded. “More generally, the domain of sophisticated environmentally sensitive and switchable function no longer belongs exclusively to naturally occurring proteins.”

In the second paper (“Modular and tunable biological feedback control using a de novo protein switch”), the UW and UCSF scientists described how they leveraged the plug-and-play nature of degronLOCKR to implement feedback control of endogenous signaling pathways and synthetic gene circuits.

“We first generate synthetic negative and positive feedback in the yeast mating pathway by fusing degronLOCKR to endogenous signaling molecules,” wrote the article’s authors. “We next evaluate feedback control mediated by degronLOCKR on a synthetic gene circuit, to quantify the feedback capabilities and operational range of the feedback control circuit.”

“The modularity of degronLOCKR extends to mammalian cells, which opens the door to a wide range of applications in the design of live cell therapeutics and in biotechnology,” the authors continued. “For example, degronLOCKR feedback control could improve therapies based on chimeric antigen receptor T cells by regulating the activity of synthetic receptors and internal signaling dynamics, or limit the production of toxic intermediates in metabolic pathways.”

The senior authors of the articles are UW’s David Baker, PhD, head of the Institue for Protein Design and professor of biochemistry and UCSF’s Hana El-Samad, PhD, professor of biochemistry and biophysics. “The ability to control cells with designer proteins ushers in a new era of biology,” said El-Samad. “In the same way that integrated circuits enabled the explosion of the computer chip industry, these versatile and dynamic biological switches could soon unlock precise control over the behavior of living cells and, ultimately, our health.”

Once assembled by a cell, a LOCKR switch measures just eight nanometers on its longest side. Having no counterpart in the natural world, LOCKR stands apart from every tool of the biotech trade, including recent technologies like optogenetics and CRISPR. While its predecessors were discovered in nature and then retooled for use in labs, industry, or medicine, LOCKR is among the first biotechnology tools entirely conceived of and built by humans.

“Right now, every cell is responding to its environment,” said Robert A. Langan, a researcher at the UW’s Institute for Protein Design and one of the lead authors of the first article. “Cells receive stimuli, then have to figure out what to do about it. They use natural systems to tune gene expression or degrade proteins, for example.”

Langan and his colleagues set out to create a new way to interface with these cellular systems. They used computational protein design to create self-assembling proteins that present bioactive peptides only upon addition of specific molecular keys.

With a version of LOCKR installed in yeast, the team was able to show that the genetically engineered fungus could be made to degrade a specific cellular protein at a time of the researchers’ choosing. By redesigning the switch, they also demonstrated the same effect in lab-grown human cells.

To stay healthy, cells must tightly control their biochemical processes. Abnormal activity in just one gene, or buildup of the wrong protein, can upset a cell’s equilibrium. This could lead to cell death or even cancer. LOCKR gives scientists a new way to interact with living cells. It could thereby enable a new wave of therapies for diseases as diverse as cancer, autoimmune disorders, and more.

“LOCKR opens a whole new realm of possibility for programming cells,” said Andrew H. Ng, a graduate student at UCSF and the lead author of the second paper. “We are now limited more by our imagination and creativity rather than the proteins that nature has evolved.”

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