Researcher tracks path of key proteins in health and disease

University of Oregon biologist Joe Thornton’s long-running study of protein mutations and cancer has yielded another breakthrough.

Thornton, of the Institute of Ecology and Evolution, and Mike Harms, a postdoctoral scientist who will join the UO chemistry faculty in September, found that two tiny mutations in a single protein 500 million years ago caused steroid hormones to take on their crucial present-day roles.

These roles include key effects on sexual reproduction and development, regulation of stress and immunity and the growth of breast and prostate cancers, reported the scientists from the UO and three other institutions. The findings will be published in the Proceedings of the National Academy of Sciences.

Biologists have long debated whether evolution proceeds gradually by many mutations of small effect or in jumps due to a few mutations of large effect. The gene-resurrection strategy that Thornton and Harms used allowed them to directly answer this question.

Thornton's group discovered the two key mutations by biochemically resurrecting ancient genes, recapitulating changes in DNA that happened hundreds of millions of years ago, and experimentally determining their effects on molecular properties of proteins that the genes encode.

The researchers focused their "molecular time travel" strategy on a family of proteins called steroid receptors, which mediate the effects of steroid hormones on reproduction, development and physiology. In the new paper, the group traced how the ancient progenitor of the entire family, which recognized only estrogens, evolved into descendant proteins that could recognize other steroid hormones that are important today, such as testosterone, progesterone and the stress hormone cortisol.

"Changes in just two letters of the genetic code long ago, before the dawn of vertebrates, caused a massive shift in the function of one little protein and set in motion the evolution of our present-day hormonal and reproductive systems," Thornton said. "If those two mutations had not happened, our bodies today would have to use different mechanisms to regulate pregnancy, the stress response and the development of male and female characteristics at puberty."

Understanding how the genetic code of a protein determines its functions may allow biochemists to better design drugs and predict the effects of mutations on disease. The new findings show how evolutionary analysis of the proteins' histories can advance this goal, Thornton said.

The group first resurrected a series of steroid receptor proteins as they existed through evolutionary time and used molecular assays to determine their sensitivity to various hormones.

This allowed them to narrow down the historical interval during which the capacity to recognize steroids other than estrogen evolved. They then identified the most important mutations that occurred during that interval by introducing them into the ancestral proteins, thus recapitulating ancient molecular evolution in their UO lab.

They observed that just two of the ancient changes in the protein's gene sequence caused a 70,000-fold shift in preference away from estrogens towards other steroid hormones. The researchers also applied a wide variety of state-of-the-art biophysical techniques to answer evolutionary questions, which allowed them to identify the precise atomic-level mechanisms by which the two genetic mutations radically changed the protein's functions. The techniques included hydrogen-deuterium exchange studies and molecular dynamics analysis of the motions of the protein's thousands of atoms, as well as X-ray crystallography to determine the ancient atomic structure.

Estrogens are key regulators of sexual reproduction and development and represent the top cause of breast cancer. Androgens, such as testosterone, are major players in prostate cancer and the primary regulators of male secondary sexual differentiation. Other steroid hormones regulate the long-term response to stress and the regulation of immunity, metabolism, kidney function and blood pressure.

The work, Thornton said, shows that proteins can evolve by sudden large leaps and that complex new molecular functions can emerge from tiny changes in the genetic code.

- from the UO Office of Strategic Communications