Current Research and Scholarly Interests
Overview – The lab concentrates on the invention of molecules and techniques that enable better studies of biological processes. In short, we invent tools for biology and we are motivated by approaches that enable entirely new experiments with unprecedented control. These new techniques may also provide a window into mechanisms that cells use to maintain protein homeostasis. Protein quality control is a particular interest at present.
New Approaches for Conditional Control of Protein Function – We have developed an experimental system in which the stability of a specific protein depends on the presence or absence of a cell-permeable molecule. We started with a well-studied protein-ligand pair: the FKBP12 protein and a high-affinity ligand we synthesized called Shield-1. We screened a library of FKBP sequences to identify mutants that are unstable in the absence of Shield-1 and are stabilized by Shield-1. Further characterization of these mutants revealed that the most destabilizing mutants caused a 50-fold to 100-fold reduction in the expression levels of the proteins to which they were fused. Importantly, this instability is transmitted to any fused partner protein, allowing us to regulate the stability of any protein-of-interest using Shield-1.
These reagents are called destabilizing domains (DDs), and we have developed a broad portfolio of these reagents based on several distinct protein-ligand combinations. Most of these reagents are available through Addgene. The systems work well in cultured mammalian cells, a variety of model organisms (e.g., flies, worms, frogs) and in living mice and rats. This technique allows rapid and reversible elimination of a specific protein in a variety of biological contexts. We have also engineered a DD system that functions in the opposite sense. The fusion protein is stable in the absence of the ligand, and administration of the ligand causes the fusion protein to be rapidly degraded. Similarly, we have engineered a conditional stability system where light is used to regulate protein stability. Most of these reagents have been reviewed recently (Rakhit et al., Chemistry & Biology 2014, 21, 1238).
Protein Quality Control in Cells – The DDs can be thought of as model substrates that have the potential to help us understand how cells detect and deal with misfolded or unfolded proteins. The ability to conditionally regulate the folding state of these domains using high-affinity ligands allows us to correlate specific biophysical properties with biological stability. One focus of the lab is understanding how these technologies work in cells. Using purified proteins we have shown that the DD proteins are either unfolded or significantly populate the unfolded state in the absence of the stabilizing ligand (Egeler et al., JBC 2011, 286, 31328). We have also used a focused RNAi strategy to identify proteins involved in the cellular response to the unfolded DDs (Chu et al., JBC 2013, 288, 34575).
More recently, we are using the DDs as conditionally folded proteins to create an acute unfolded protein stress by withdrawing the stabilizing ligand from the cell culture media. This approach allowed us to identify a novel coordinated transcriptional response that mammalian cells trigger when unfolded protein appears (Miyazaki et al., eLIFE 2015). Interestingly, there is little overlap between this new cellular response and the heat shock response, which conventional wisdom holds as the "cytosolic unfolded protein response". Additionally, creating unfolded DD in either the cytosol or nucleus elicits distinct responses, suggesting that mammalian cells maintain different protein quality control surveillance environments in these compartments. Understanding the molecular mechanisms behind these new cellular responses as well as how they help to maintain protein homeostasis is a major focus of the lab.
