MECHANISM OF MACROMOLECULAR MACHINES
Our goal is to understand complex cell biological processes in the detailed quantitative manner with which chemists understand conventional enzymatic reactions. We then use this information to discover novel regulatory principles and to develop strategies to interfere in cellular processes therapeutically or to engineer cellular metabolism. Currently, the focus of the lab is on developing a quantitative understanding of the Ubiquitin Proteasome System (UPS) in the cell and on applying this information to treat human disease.
Protein Unfolding by ATP-dependent proteases
Cellular protein concentrations are controlled through the rates of synthesis and degradation and the UPS is responsible for most protein degradation in the cytosol and nucleus of eukaryotic cells. Many UPS substrates are regulatory proteins so that the UPS plays a part in effectively all cellular processes1-4. The UPS also removes misfolded and damaged proteins as part of the stress response5. Through these functions the UPS is involved in diseases including neurodegeneration and cancer and thus is an important therapeutic target.
We understand the broad outlines of how the UPS works. At the center of the system is a proteolytic particle called the proteasome. It is a large ATP-dependent molecular machine of almost the size of the ribosome (~2,500 kDa and 40 different subunits). Many proteins are targeted to the proteasome by the covalent attachment of multiple copies of the small protein ubiquitin. The proteasome recognizes the ubiquitin tags and degrades the substrate while recycling the ubiquitin tags. However, fundamental questions remain.
We’re currently investigating two aspects of proteasome function: how the proteasome selects its substrates, and how the proteasome moves along its substrates as it digests them processively. As experimental methods we us techniques ranging from combinatorial protein engineering, quantitative biochemical assays, cell biological methods, genome-scale screens, and single molecule biophysics.
1) A second targeting code
We found that ubiquitin tags by themselves are not sufficient to target folded proteins for proteasomal degradation (Prakash et al. Nat. Struct. Mol. Biol. 11, 830 (2004)). Instead, degradation signals or degrons contain a second part in the form of an intrinsically disordered initiation regions in substrates that can serve as initiation sites for the proteasome. Thus, the proteasome binds to its substrates through the ubiquitin tag and engages them at the initiation site.
We investigate how the initiation site contributes to proteasome targeting. For example, we found that the initiation region has to be long enough (Fishbain et al. Nat. comm. 2, 192. (2011)) and placed at the correct distance from the ubiquitin tag so that the proteasome can bind them both simultaneously (Inobe et al. Nat. Chem. Biol. 7, 161 (2011)). Surprisingly, the proteasome also shows strong preferences for the amino acid sequence of the initiation region in the substrate. This raises the question of how the ubiquitin tag and the initiation region can compensate for each other. For example, can a substrate with a poor initiation region be degraded if it contains a better ubiquitin tag? Can a substrate with a very good initiation region be degraded even without a ubiquitin tag? Ultimately, we would like to understand the dynamics of how the two components interplay to contribute to efficient yet specific proteasomal degradation.
2) Proteasome adaptors
We found that the ubiquitin tag and initiation region can work together in trans so that a ubiquitinated receptor can target a binding partner containing only the initiation region for proteolysis by the proteasome (Prakash et al. Nat. Chem. Biol. 5, 29 (2009)). This is surprising since several regulatory processes in the cell rely on the proteasome to remodel protein complexes by extracting a specific subunit while leaving the rest of the complex intact. We predict that the subunit specificity of the proteasome depends on its selection of the correct initiation site. It is possible that trans-targeting is used physiologically, for example by viruses that want to be sure to deplete a cellular protein by targeting it to the proteasome with an adaptor protein that binds proteasome and substrate simultaneously. Finally, it may be possible to construct artificial adaptors to deplete toxic proteins, to interfere in a cellular process by depleting a regulatory protein, or to redirect metabolic flow by removing a key enzyme in a pathway after a branch point.
3) Partial degradation
Once the proteasome has engaged a substrate it runs along the protein’s polypeptide chain degrading it sequentially and processively from the initiation site (Lee et al. Mol. Cell 7, 627 (2001)) into small peptides of some 10 amino acids in lengths. This prevents the formation of protein fragments with unwanted biological activities. However, there are a few examples where the proteasome stops degradation part way through a polypeptide chain and releases a partially degraded protein. We think that this partial degradation occurs when an internal ubiquitination site sends the proteasome to run into a stop signal encoded in the substrate (Tian et al. Nat. Struct. Mol. Biol. 12, 1045 (2005); Schrader et al. J. Biol. Chem. 286, 39051 (2011)). We are now looking for more natural examples of partial degradation and are using biophysical techniques to understand the mechanism.