Research Program
Nanoscale Cell Biology

Objectives
Degradation of intracellular proteins is required for normal maintenance of cellular processes, including cell proliferation, differentiation, and cell death.  The protein modifier ubiquitin is a signal for proteasome-mediated degradation in eukaryotes.  It has long been a challenge to harness the power of the ubiquitin-proteasome system ( UPS ) to target specific substrates for degradation.  We made an attempt to engineer the single chain E3 ligase CHIP (Carboxyl-terminus of Hsp70-Interacting Protein). Our findings have produced exciting technological breakthroughs, including dramatic progress in engineering functional ubiquitin ligases to target specific protein substrates for ubiquitination.  In addition, these engineered ubiquitin ligases can be converted into small molecule-inducible version.   Thus, engineered ubiquitin ligases afford functional nano-molecules for exploring protein functions. We propose herein to create engineered ubiquitin ligases targeting the disease protein huntingtin (Htt) for ubiquitination. If successful, the engineered ubiquitin ligases will represent an unprecedented level of control over protein function in somatic cells, which would have direct impact on biomedical research by introducing functional nano-molecules.

Methods
To map the structural determinants of recombinant E3 ligases, we will apply genetic engineering approach to replace the substrate-binding domain TPR with FKBP.  The resulted FKBP-E3 will be further engineered by varying the distances of the helical linker, in order to obtain optimal nano-molecules mimicking natural E3. 

To elucidate the dynamics of E2-E3 interaction, we will nanovesicle trapping strategy in combination with single-molecule FRET measurements to quantify the E2 dynamics by different versions of recombinant E3. 

To achieve functional E3 ligases targeting disease protein, such as Htt, we will harness specific intrabodies capable of recognizing disease proteins.   In order to target endogenous substrates, such as protein aggregates associated with neurodegeneration, we use specific substrate binding partner to create recombinant ubiquitin ligases capable of targeting diseases proteins.

Summary
The successful engineering of ubiquitin ligases implies a novel approach to achieving specific protein knockout.  In order to target endogenous substrates, such as protein aggregates associated with neurodegeneration, we use specific substrate binding partner to create recombinant ubiquitin ligases capable of targeting diseases proteins.  By facilitating the clearance of disease proteins associated with neurodegene-rative disorders, the engineered ubiquitin ligases show promises in the improvement of pathological symptoms.  The available mouse models of neurodegenerative diseases have enabled the exploration of therapeutic potential of Htt-E3 in delaying the onset or slowing the progress of neurodegeneration.

Goals/Accomplishments

• Define the structural features of ubiquitin ligase using nano-approach
• Engineer ubiquitin ligase to create functional nano-molecules
• Application of engineered ubiquitin ligases in nanomedicine