Path of key proteins in health and disease began with two ancient mutations

EUGENE, Ore. — (June 25, 2013) — Two tiny mutations in a single protein 500 million years ago caused steroid hormones to take on their crucial present-day roles, including key effects on sexual reproduction and development, regulation of stress and immunity, and the growth of breast and prostate cancers, report scientists from the University of Oregon and three other institutions.

Michael HarmsThe findings — placed online this week ahead of regular publication in the Proceedings of the National Academy of Sciences — are the newest in a long-running series of work under the direction of biologist Joe Thornton of the UO's Institute of Ecology and Evolution.

Thornton's group, including lead authors Michael Harms and Geeta Eick, both UO postdoctoral research associates, 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.

(Links to recently related reports from Thornton's lab are provided below.)

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."

Evolutionary 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‘s group used allowed them to directly answer this question.



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.

"The two mutations changed only a few atoms in the protein, but they radically rewired the network of interactions between the receptor and the hormone," Harms said. "Combining evolutionary history with protein biophysics allowed us to see precisely how the protein's architecture amplified the effects of the mutations into a massive change in function."

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. Thornton said the new findings show how evolutionary analysis of the proteins' histories can advance this goal. Before the research in Thornton's lab, it was not known how the various steroid receptors distinguish estrogens from other hormones.  

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. Along with the two key changes in the receptor, he said, additional mutations, whose precise effects are not yet known, were necessary for the full effects of hormone signaling on the body to evolve.

“Evolutionary biologists at the UO are conducting research that enhances our fundamental understanding of human development, health and disease,” said Kimberly Andrews Espy, vice president for research and innovation and dean of the UO graduate school. “Drs. Thornton and Harm are at the cutting edge of fundamental scientific discoveries that in time have the potential to improve the health and well-being of people through development of better targeted drugs and more effective treatments.”

Co-authors on the study with Harms, Eick, and Thornton were Devrishi Goswami and Patrick R. Griffin of the Scripps Research Institute in Jupiter, Fla., and Jennifer K. Colucci and Eric A. Ortlund of the Emory University School of Medicine in Atlanta, Ga.  



The National Institutes of Health (grants RO1-GM081592 and F32-GM090650), National Science Foundation (grants IOB-0546906 and DEB-0516530) and the Howard Hughes Medical Institute supported the research.

Thornton also is a professor in the Department of Human Genetics and Ecology and Evolution at the University of Chicago, and Harms will join the faculty of the UO Department of Chemistry and Biochemistry as an assistant professor in September.

About the University of Oregon
The University of Oregon is among the 108 institutions chosen from 4,633 U.S. universities for top-tier designation of "Very High Research Activity" in the 2010 Carnegie Classification of Institutions of Higher Education. The UO also is one of two Pacific Northwest members of the Association of American Universities.

Media Contact: Jim Barlow, director of science and research communications, 541-346-3481, jebarlow@uoregon.edu

Sources: Joseph W. Thornton, professor, University of Oregon, University of Chicago, 541-914-2588, joet@uoregon.edu, and Michael J. Harms, postdoctoral research scientist in the UO Institute of Ecology and Evolution and, effective September 2013, assistant professor of chemistry and biochemistry, UO, 541-346-1537, harms@uoregon.edu.

Additional Links:

Related reports on Thornton's research:
• 2012 — Evolution of complexity reconstructed using 'molecular time travel': http://uonews.uoregon.edu/archive/news-release/2012/1/evolution-complexi...
• 2010 — Evolutionary tinkering produced complex proteins with diverse functions: http://uonews.uoregon.edu/archive/news-release/2010/10/evolutionary-tink...
• 2009 — Ratchet-like genetic mutations make evolution irreversible: http://uonews.uoregon.edu/archive/news-release/2009/9/ratchet-genetic-mu...  

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