Sommer Lab

Ubiquitin chain formation - generating the ubiquitin code

Protein ubiquitylation represents a highly versatile and dynamic post-translational modification that comes in distinct flavors collectively called the ‘ubiquitin code’. Proteins can be mono-ubiquitylated at a single site, typically a lysine residue, or at several positions. Importantly, the lysines in previously attached ubiquitin (Ub) serve as acceptors for additional Ub molecules giving rise to poly-Ub chains. These different Ub signals target the clients into distinct pathways. In principle, the attachment of Ub to a substrate requires the activity of appointed pairs of so-called Ub conjugating (E2-) enzymes and Ub ligases (E3 enzymes). However, at present we lack detailed knowledge on how a specific Ub signal is generated and how it its decoded by the cell. Acomprehensive characterization of these processes is a pre-requisite to understand the contribution of the diverse Ub signals to the biology of a cell and to identify specific entry points for therapeutic intervention.

Distinct steps during substrate ubiquitylation.
Polyubiquitylation of proteins is initiated by the attachment of single ubiquitin (Ub) moieties to target sites, typically lysine residues, in the client (“priming”). These entities then serve as primers for the conjugation of additional Ub molecules (“elongation”). Poly-Ub chains linked via different lysines within the Ub molecules serve distinct functions in the cell. Most prominently lysine 48-linked Ub chains target client proteins to proteasomal degradation. Priming and elongation during poly-Ub synthesis request different propensities of the involved enzymes. Remarkably, the Doa10 Ub ligase employs distinct E2 enzymes, Ubc6 and Ubc7, for these reactions. Moreover, individual E2 enzymes are stimulated by their co-factors in different ways. Priming of substrates and early steps in chain elongation by Ubc7 relies on its activation by a Ub ligase, whereas the formation of longer Ub chains requires positioning to the growing Ub chain by Cue1. 

© Dr. Annika Weber

Ongoing research:

Ub ligases of the so-called RING-finger type facilitate the transfer of Ub by arranging substrates and E2 enzymes in a defined spatial orientation. We investigate the processivity of selected E2/E3 pairs using fluorescence-basedin vitroassays to capture the dynamics as well as the products of these reactions. We combine these approaches with structural analysis via NMR and X-ray crystallography and verify our results in yeast and mammalian cell culture model systems. 

We are currently investigating howatypical Ub structures like branched Ub chains are generated and aim to identify their function in the cell. Furthermore, we study in detail how different E2 enzymes are activated by their cognate RING Ub ligases for distinct steps in Ub chain formation. We also want to uncover the factors, which define the enzymatic propensities of individual E2 enzymes. Finally, we are measuring the cellular abundance of unusual Ub conjugates like serine and threonine ubiquitylation and want to determine the physiological impact of these modifications.

Former research:

Our work revealed that Ub conjugation of the yeast E2 enzyme Ubc7 at the ER membrane relies on tight binding to a membrane-bound co-factor Cue1 ¹. Cue1 does not only recruit Ubc7 to membrane-bound Ub ligases but also binds to Ub chains and thereby stimulates the activity of the E2 enzyme ². Subsequent studies indicate that Cue1 positions Ubc7 at the tip of the Ub chain and facilitates the attachment of further Ub molecules ³. Results from our work also suggest that the poly-ubiquitylation of proteins represents a two-step process: the attachment of the first Ub entity onto an acceptor site within the target and the mounting of additional Ub molecules to already conjugated Ubs. In the case of the Doa10 Ub ligase these reactions are catalyzed by distinct E2 enzymes ⁴. Ubc6 decorates Doa10 client proteins with mono-Ub, which then serve as primers for the addition of lysine 48-linked poly-Ub by Ubc7. Remarkably, Ubc6 conjugates Ub not only to lysines but also to hydroxylated amino acids like serines and threonines, which substantially expands the target range of the Doa10 Ub ligase. 

1.        Biederer, T., Volkwein, C. & Sommer, T. Role of Cue1p in ubiquitination and degradation at the ER surface. Science278,1806–1809 (1997).

2.        Bagola, K. et al.Ubiquitin Binding by a CUE Domain Regulates Ubiquitin Chain Formation by ERAD E3 Ligases.Mol. Cell50,528–539 (2013).

3.        von Delbrück, M. et al.The CUE Domain of Cue1 Aligns Growing Ubiquitin Chains with Ubc7 for Rapid Elongation. Mol. Cell62,918–928 (2016).

4.        Weber, A. et al.Sequential Poly-ubiquitylation by Specialized Conjugating Enzymes Expands the Versatility of a Quality Control Ubiquitin Ligase. Mol. Cell63,827–839 (2016).