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Rewiring the Regulatory Mechanisms of Signaling Proteins

  • Author(s): Sallee, Nathan
  • Advisor(s): Lim, Wendell
  • et al.
Abstract

Eukaryotic cells must process large amounts of information and respond correctly to stimuli in order to maintain the health of an organism. Much of this information processing is done by networks of signal transduction proteins. Many signaling proteins behave as allosteric systems that have both active and inactive states. These proteins must recognize specific molecular inputs and, in response, modify their catalytic or binding activities to create the appropriate output. One such allosteric switch is the GTPase-binding domain (GBD) of the actin-regulatory protein WASP (Wiskott-Aldrich syndrome protein). Basally, the GBD autoinhibits WASP's actin polymerization activity through an intramolecular interaction with the C helix, but intermolecular binding of the GTPase Cdc42 to the GBD disrupts this autoinhibitory interaction. The inspiration behind this work was to gain an understanding of some of the mechanisms by which the regulation of signaling proteins such as WASP can be rewired. Specifically we were interested in strategies for bypassing the endogenous regulation of these proteins and making them responsive to different inputs that we can control. We took two approaches to accomplish this: (1) engineering novel protein switching components and (2) studying the mechanism by which the pathogenic bacterium EHEC is able to hijack WASP regulation.

In the first case, we found that by overlapping the primary sequences of pairs of protein interaction modules, we could render their two interactions mutually exclusive. We characterized the behaviors of these engineered interaction switches and also incorporated them into naturally-occurring signaling proteins and pathways. These modified components were able to rewire signaling in vitro and in vivo.

In the second, we characterized the EHEC protein EspFU, which potently activates host cell WASP, leading to actin polymerization. We found that EspFU contains a repeated mimic of the autoinhibitory C helix from WASP, and that this motif competitively disrupts WASP autoinhibition. It is also functionally significant that this motif is repeated six times in EspFU, because potent activity seems to be dependent on EspFU's ability to coordinate the simultaneous activation of at least two WASP proteins.

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