• Taking it Up a Notch

    Simple protein wields tremendous influence in human development and disease

    by Mary Silva

    Raphael Kopan, PhD, started his research career with a rather modest goal, and failed at it. Luckily, he sees that as part and parcel of the scientific process.

    “It’s the inevitable reality of trying to discover how nature works. There’s no manufacturer’s manual,” he says. “You never know where the path will lead.”

    Kopan was studying skin development — specifically, how to make a hair follicle. He admits it was not life-altering science, but thought it could be useful to certain hair-challenged individuals. Besides, he was having fun.

    “It was an interesting project. I worked on it for about a year, tried lots of crazy procedures. Technically they all worked, but none of them resulted in anything remotely usable,” Kopan says. “I was at the point of my career where it looked like there would be no career.”

    But Kopan’s failed efforts with hair follicles had acquainted him with Notch protein, a molecule that appeared to influence the fate of early cell development. His advisor at the time — it was the late 1980s — was the late molecular biologist Harold Weintraub, PhD, who encouraged him to explore the protein further.

    He did, and found that Notch had little to do with hair follicle induction but a lot to do with his future.                   

    “My lucky break was, the piece of Notch I was studying was very small,” Kopan says.

    “Everybody else was studying the whole gene. I was the only guy whose wimpy little Notch needed help in order to work. And the help it needed was to travel into the nucleus.”

    A Breakthrough Break

    Notch protein was first identified in the early 1900s in a form of the Drosophila fruit fly, where mutations in the gene resulted in the insect’s notched wings. Scientists now know that the protein is found in all multicellular animals and is responsible for the development of virtually every organ. Kopan was able to show how Notch influenced development by discovering how it got into the cell nucleus. His discovery changed the way scientists understood cell signaling.

    “The idea people had about how cells translate a signal into the nucleus involved an army of secondary messengers,” he says. “I found the sequence where the Notch molecule was broken. And I was able to show that this break only happened when the molecule encountered its ligand and not otherwise.”

    Kopan’s discovery of Notch’s breakage and subsequent journey into the nucleus was dubbed the “canonical pathway” by his fellow scientists. Notch protein straddles the cell, half in and half out. When the half that remains outside the cell’s membrane encounters a specific binding protein - its ligand - it breaks in two. The half located inside the cell travels to the nucleus, where it begins to issue genetic directives ranging from what the cell becomes to whether it lives or dies.

    When Kopan and his team engineered Notch molecules that could not be broken, this cell signaling did not occur. “This was the strongest bit of evidence that the mechanisms for signals to be transduced required the molecules to fall apart,” he says.

    The Cellular Coin Toss

    Understanding how Notch issues its orders was the first piece of the puzzle.  Further study revealed the protein’s masterful ability to regulate development.

    Notch acts as a biological traffic cop, often helping one cell to adopt a particular fate — fate A — while stopping its neighbors from doing so. The cells signal to each other; the more Notch signal a cell receives, the less likely it is to activate. The cell receiving the least Notch signal moves on to differentiate into fate B. In this way, Notch organizes normal cellular structures and prevents equivalent cells from all adopting the same cellular fate. This can happen “stochastically,” says Kopan - with a sort of controlled randomness - or in a very controlled manner.

    “Notch is like the quarter you carry in your pocket,” Kopan says. “If you have a binary decision to make, you flip a coin: Heads you go, tails you don’t. That’s Notch.”

    Random Explorations Tie Together

    What led to Kopan’s next discovery about Notch was less stochastic and more completely random. 

    Despite having discovered how it worked, Kopan was still puzzled by how Notch broke apart in the cellular membrane. Proteins are typically broken with water, a process called peptide bond hydrolysis or proteolysis, and the cell membrane is made up almost entirely of lipids, mostly fats. How does Notch come apart in that environment?

    The answer came in the mid 1990s, when scientists researching Alzheimer’s disease discovered the presenilin proteins that live in the cell membrane’s fatty environs. Mutations in these proteins are a cause of inherited Alzheimer’s disease. At the same time, scientists in another laboratory discovered that presenilin corrected an egg-laying defect caused by overactive Notch in the C elegans worm. Kopan and his collaborators realized it is the presenilin protein that Notch depends on to break apart, enter the cell nucleus, and mediate its regulation of normal development. 

    This piecing together of what seem to be unrelated bits of exploration is what Kopan loves about science.

    “In basic science, if you aim your arrow, you often miss — it does not get you what you were after. If you don’t aim where your arrow goes, but are mindful where it lands, you can extract deep meaning from most landing spots. Because everything is connected in biology, even this seemingly random process gets you back to the clinic.”

    Impact On Disease

    Now, Kopan is connecting what he has learned about Notch to determine how its presence or absence affects human disease. He and his team are exploring Notch’s function in a variety of diseases and disorders, including kidney disease, immune disorders and cancer.

    He knows what they learn from any species will be useful because Notch works the same way across all multicellular organisms.

    “We are all made of the same building blocks; all metazoans use Notch signaling,” he says. “We work with mutations that arise in an animal, but sooner or later a similar mutation will be in the human population. Or we can generate a mutation identified in humans in our model organisms, to better understand the mechanism of the disease and to see which levers we can push to get a better outcome.”

    Connecting The Dots

    Kopan came to Cincinnati Children’s just one year ago — his first stint in a pediatric setting — and likes working in an environment where both researchers and clinicians focus on the earliest stages of development. He considers all pediatricians developmental biologists.

    “Here, I don’t need to explain why developmental biology is important,” he says. “There is a great flow of information from the human side to us and from us to them, to get to the bottom of various disorders and diseases, and to understand, treat and manage them. We try to accelerate the process by modeling the organism and coming up with intelligent ways to attack it.”

  • Dr. Raphael Kopan
    Raphael Kopan, PhD
  • Notch Protein
    Notch Protein