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Research Interests


Ubiquitylation is a key regulator of proliferation and differentiation in all eukaryotes. It is carried out by a cascade of three different classes of enzymes, E1, E2, and E3. In humans alone, there are 38 E2s and more than 600 E3s, making ubiquitylation enzymes one of the most abundant and diverse family of enzymes. Loss of essential ubiquitylation enzymes can lead to rapid cell cycle arrest.

The importance of ubiquitylation for cell cycle control is underscored by the tight links between deregulated ubiquitylation and tumorigenesis. Losing the activity of the E3 Brca1/Bard1, for example, can cause breast and ovarian cancer. The overexpression of the ubiquitin ligase Mdm2, which targets the tumor suppressor p53 for degradation, is observed multiple types of cancers. Small molecules that would correct the activities of rampant ubiquitylation enzymes would be exciting additions to the currently available set of chemotherapeutics.


Our ability to exploit the ubiquitin system as a target for drug discovery has been hampered by our limited knowledge of essential enzymes and their substrates, and particularly, by our lack of understanding mechanisms of ubiquitylation.


Current Research Projects

1. Dissecting the ubiquitin code

 

Ubiquitin is covalently linked to lysine residues in substrate proteins. The transfer of a single ubiquitin moiety (monoubiquitylation) usually results in changes of proteins interactions. The modification of the substrate-linked ubiquitin with further ubiquitin molecules leads to the generation of polymeric ubiquitin chains. These chains can be linked through the N-terminus or through each of the seven lysine residues of ubiquitin, resulting in chains of different structure and function. K48-linked chains, for example, trigger degradation by the 26S proteasome, whereas K63-linked chains recruit binding partners into multimeric protein assemblies. For many other chain topologies, however, very little is known about substrates, enzymes, and functions.

 

We have discovered the K11-linked ubiquitin chain as an essential regulator of cell division in higher eukaryotes. K11-linked ubiquitin chains are assembled during mitosis by the ubiquitin ligase APC/C, a finding we recently confirmed in cells using linkage-specific antibodies. We isolated the responsible E2 enzymes for K11-linkage formation, Ube2C/UbcH10 for chain initiation and Ube2S for chain elongation. We are currently investigating the mechanisms of specific and regulated K11-linked chain formation by these enzymes.

 

2. Ubiquitin-dependent regulation of proliferation and differentiation

 

Substrate specificity of ubiquitylation depends on ~600-1000 so-called E3 enzymes, and for the majority of these enzymes, functions or substrates remain unknown. Ubiquitylation often is reversible; the modification then has to be removed by one out of ~100 deubiquitylating enzymes (DUBs), most of which remain uncharacterized. We have developed a ubiquitin-related siRNA library and a robust siRNA-screening platform to isolate new E3s and DUBs with important roles in proliferation and differentiation. Using this setup, we could identify an important role for ubiquitin in regulating the composition and function of the spliceosome. Loss of this pathway led to incorrect tubulin splicing, inaccurate spindle formation and reduced sensitivity to treatment with the chemotherapeutic taxol.


The identification of substrates of cell-cycle regulated ubiquitylation is difficult. We have developed an in vitro-expression cloning/ubiquitylation strategy to isolate new substrates of the E3 APC/C. These new substrates were spindle assembly factors required for Ran-dependent spindle formation. Their degradation was regulated by binding to importin-beta, which also inhibits their function in spindle formation. We are currently investigating the temporal regulation of the APC/C- and Ran-dependent degradation of spindle assembly factors.

3. Small molecule discovery to modulate the activity of ubiquitylation enzymes

We are combining our biochemical insight and the siRNA-based enzyme discovery to identify targets for the development of small molecules against ubiquitylation enzymes. We are screening for small molecules that could either active or inhibit the activity of these enzymes, providing a proof-of-principle that ubiquitylation enzymes are attractive drug targets.