Scientists Discover How TGF-Beta Sends Its Message Even When Attached to the Cell Membrane

For years, scientists thought that TGF-beta, a signaling protein that controls a surprising number of cellular processes, from embryonic development to cancer, could do its job only once it had escaped from a lasso-like “straitjacket.”

But now, thanks to cryogenic electron microscopy (cryo-EM), a powerful technique that allows scientists to create moving, three-dimensional models of molecules at atomic resolution, experts at UC San Francisco have discovered that this protein is much more clever than they thought.

He shakes and wiggles out of his straitjacket, extending a few fingers to activate a nearby receptor despite being locked on the cell’s surface.

The results, published on September 16 in Cellupends decades-old dogma about how TGF-beta works. That could help scientists improve the many therapies aimed at controlling it, including an important new class of cancer therapies called checkpoint inhibitors, which have had less effect than expected.

And at a more basic level, the work suggests an even crazier picture than scientists had imagined, as key players like TGF-Beta morph into unexpected forms to accomplish the impossible in our cells.

“Historically, the field has focused on stabilizing these types of signals to get a high-resolution image, but in doing so, it has ignored how flexibility might be part of their function,” said Yifan Cheng, Ph.D., professor of cellular and molecular pharmacology at UCSF and co-senior author of the paper. “For TGF-Beta, this flexibility plays a critical role, and we think it could explain how other poorly understood signals work, with implications for understanding and treating disease.”

A stationary signal manages to transmit its message

Four years ago, Cheng and co-senior author Stephen Nishimura, MD, discovered that TGF-beta could signal a receptor even when it was bound in its straitjacket, whose scientific name is latency-associated protein (LAP).

This result contradicts decades of scientific research suggesting that TGF-beta must be released by LAP to reach its receptor. If it weren’t released, they thought, basic processes, such as how the body makes new cells without developing tumors, would go haywire.

But when the team engineered a permanent link between TGF-Beta and the straitjacket in mice, the mice survived. TGF-Beta could still do its job even when bound by LAP.

Cheng and Nishimura took a closer look at the situation using cryo-EM, their specialty.

Cryo-EM involves rapidly freezing a mixture of proteins and taking hundreds of thousands of pictures of them to see how they interact. Typically, powerful algorithms align these microscopic snapshots to reveal the most common, and therefore most important, protein arrangements.

But this approach can leave out many possibilities, and previous studies considered only two: either TGF-beta was bound inside the LAP and therefore inert; or it was free to float from cell to cell and unlock its receptor.

Knowing that TGF-beta was able to unlock its receptor when bound to LAP, the UCSF scientists suspected that these proteins might have many more states than just two, which, using typical cryo-EM approaches, would appear fuzzy and be ignored.

“In cryo-EM, people tend to report what they see most clearly, but in our data, we realized there could be meaning in the blurrier parts of the image,” said Nishimura, who is a professor of pathology at UCSF. “So that’s what we focused on.”

Meaning of molecular motion

To get a better view of TGF-Beta moving around in its straitjacket, the scientists methodically stabilized different parts of LAP, TGF-Beta, or both, then used cryo-EM to see how these artificial configurations of the molecules interacted with the TGF-Beta receptor.

In each successive experiment, the blurring observed in the data, known as entropy, shifted to other points on the TGF-Beta, suggesting that they might still be moving despite the straitjacket.

This allowed the TGF-Beta to stick just far enough away from the LAP to be detected by the TGF-Beta receptor. The movement was short-lived. But by systematically constraining the system and taking snapshots of it, Cheng and Nishimura got the clearest picture yet of the signal accomplishing the seemingly impossible.

These findings change the fundamental understanding of TGF-beta and many other signals that govern communication within and between cells. Rather than simply switching from one discrete shape to another, these molecules sometimes do their job through more fluid movements.

“From cell communication to cell surface molecules, TGF-Beta, disease modeling and structural biology, we hope these findings will inspire people to think differently,” Cheng said. “Clearly, the data we extract through cryo-EM holds more exciting discoveries for us.”

Other UCSF authors include Mingliang Jin, Robert I. Seed, Guoqing Cai, Tiffany Shing, Li Wang, Saburo Ito, Anthony Cormier, Stephanie A. Wankowicz, Jillian M. Jespersen, Jody L. Baron, Nicholas D. Carey , Melody G. Campbell, Zanlin Yu, Weihua Wen, Jianlong Lou and James Marks.