Evolutionary and Mechanistic Studies of the Multifunctional Innate Immune Protein S100A9
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Date
2020-09-24
Authors
Harman, Joseph
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Publisher
University of Oregon
Abstract
Many questions remain regarding the molecular mechanisms by which proteins evolve new properties and functions. How do proteins evolve new functions without perturbing existing ones? How do changes in protein biophysics affect function? How are protein functions maintained, altered, or improved over time?
We used the multifunctional innate immune protein S100A9 (A9) as a model to dissect mechanisms of protein evolution. A9 performs two primary innate immune functions. As a homodimer, it potently stimulates inflammation by interacting with Toll-like receptor 4 (TLR4). As part of a heterocomplex with S100A8 (A8/A9), it is potently antimicrobial. A9 and the A8/A9 complex are further regulated by proteolysis: the A9 homodimer is highly proteolytically susceptible, while the A8/A9 heterocomplex is resistant. The evolutionary origins and mechanisms by which these functions arose are poorly understood, and the mechanism by which A9 activates TLR4 to drive inflammation is unknown.
We took an evolutionary biochemical approach to determine how A9 evolved its innate immune functions. Chapter I comprises an introduction. Chapter II examines the role of pleiotropy in the evolution of A9 multifunctionality. We find that A9s gained proinflammatory activity and lost proteolytic resistance from a weakly proinflammatory, proteolytically resistant ancestral protein. A single ancient substitution had pleiotropic effects on A9 without affecting the A8/A9 complex, revealing a beneficial role for pleiotropy in the evolution of multifunctionality. Chapter III examines the biophysical mechanism by which A9 activates TLR4 to drive inflammation. We show that reverting the ancient substitution identified in chapter II compromises A9 activation of TLR4 by restricting access to a functionally necessary conformation of the protein. These findings highlight how subtle changes to a protein’s conformational energy landscape can have critical impacts on protein evolution. In chapter IV, I outline ongoing work examining how later-diverging A9s have evolved more potent and promiscuous activation of TLR4 and determining how, mechanistically, A9 activates TLR4. This work provides novel insight into how a key innate immune protein evolved multifunctionality and highlights how basic changes in protein biophysics can have profound impacts on biological systems.
This dissertation includes both previously published/unpublished and co-authored material.
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Keywords
ancestral sequence reconstruction, Evolutionary biochemistry, inflammation, innate immunity, S100 proteins, Toll-like receptor 4