Ubiquitin Signaling Group – University of Copenhagen

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Ubiquitin Signaling Group

The Ubiquitin Signaling Group is headed by Prof. Niels Mailand, and Assoc. Prof. Simon Bekker-Jensen (Assistant Group Leader). The work in the lab began in September 2009 and the group has now grown to 18 lab members. 

A key focus is on identification and characterization of ubiquitin-mediated signaling processes in human health and disease, and the lab has expertise in a wide range of methodologies within the areas of cell biology, biochemistry, and advanced microscopy. We work in close collaboration with colleagues in our Center and with leading international experts. We strongly value teamwork and a good social atmosphere in the group.

Staff

General

The posttranslational modification of proteins by ubiquitin is a central regulatory mechanism in most aspects of cell biology. The formation of covalently linked ubiquitin-protein conjugates requires three enzymatic steps, catalyzed by a ubiquitin-activating enzyme (E1) and a ubiquitin-conjugating enzyme (E2), which cooperate to transfer the ubiquitin moiety to a Lysine residue in the target protein with the help of a ubiquitin ligase (E3), the principal specificity determinant in the system. The process of protein ubiquitylation can be highly dynamic and reversible, as evidenced by the existence of numerous deubiquitylating enzymes (DUBs). An estimated 600 potential E3 ubiquitin ligases and some 80-90 DUBs predicted to be catalytically active are encoded by the human genome, illustrating the widespread use of substrate-specific ubiquitylation as a regulatory principle in most aspects of cell biology. Of these, however, only the functions and targets of a minor subset are currently understood in appreciable detail. Reflecting its central regulatory importance, mounting evidence has implicated the dysfunction of ubiquitin-dependent signaling pathways in multiple human diseases.

Generally, ubiquitin serves as a recognition signal to mediate interaction with proteins harboring Ubiquitin Binding Domains (UBDs, termed Ub-receptors). Ubiquitin may be conjugated onto target proteins as a single moiety or as a chain of multiple ubiquitin molecules internally linked via any one of seven lysines (K6, K11, K27, K29, K33, K48, K63) or the N-terminal methione (M1). Thereby, at least eight types of ubiquitin chains may be formed, which are molecularly identical but structurally very distinct and lead to different outcomes through interaction with distinct Ub-receptors:

Projects
The overall goal of the group is to utilize the combined powers of unbiased, proteome- and genome-wide approaches and focused cell-based studies to identify and functionally characterize new ubiquitin-dependent signaling responses operating in the DNA damage response and in innate immunity signaling. Through dedicated collaborations with other groups at The Novo Nordisk Foundation Center for Protein Research (NNF CPR), a wide range of cutting-edge technologies and relevant expertise are available for these studies.

Most of our current research efforts fit broadly into the following complementary themes:

Identification of novel ubiquitin- and SUMO-dependent signaling processes in the DNA damage response
(Simon Bekker-Jensen and Niels Mailand)
The small modifier proteins ubiquitin and SUMO play widespread roles in critical DNA damage signaling and repair pathways. A key aim of the group is to utilize unbiased approaches, primarily cutting-edge proteomics, to uncover novel ubiquitin- and SUMO-driven processes functioning to maintain genome stability. In collaboration with the Department of Proteomics at CPR, we are using state-of-the-art mass spectrometry-based approaches to explore DNA damage-regulated ubiquitylation and SUMOylation on an unbiased, proteome-wide scale - a powerful strategy to discover processes that could not have been easily identified by targeted, hypothesis-driven studies. As proof-of-principle, we have recently published the first global survey of DNA damage-regulated, site-specific ubiquitylation together with other CPR researchers (Povlsen et al., Nature Cell Biology 2012), and we used this insight to characterize an important role of ubiquitin-mediated regulation of the PAF15 protein in DNA damage bypass control (Fig. 1).

 

Fig. 1 

These studies are yielding unprecedented insight into how ubiquitin- and SUMO-mediated signaling is involved in cellular responses to DNA damage, and are uncovering surprising new branches of the DNA damage response, which we are currently investigating. Using a variety of cell biology and biochemistry-based approaches, we are characterizing a range of novel factors modified by DNA damage-regulated ubiquitylation and SUMOylation.

Regulatory control of translesion DNA synthesis (TLS)
(Simon Bekker-Jensen and Niels Mailand)
Recent work from the group has identified two important novel factors, PAF15 and DVC1, which regulate translesion DNA synthesis (TLS) during DNA replication in a ubiquitin-dependent manner (Povlsen et al., Nature Cell Biology 2012; Mosbech et al., Nature Structural and Molecular Biology 2012; Figs. 1, 2). Although it is clear that tight regulatory control of TLS is crucial for avoiding excessive mutagenesis, our current understanding of how this is achieved at the molecular level is limited. We have begun to undertake a dedicated, multipronged strategy to unravel the molecular framework of TLS regulation in human cells, involving a range of complementary, cutting-edge approaches. These include siRNA- and mass spectrometry-based screens for novel TLS factors, as well as detailed studies of a number of prospective new TLS-regulatory human proteins we have identified.

 

 

 

Fig. 2

Novel factors and ubiquitylation targets in the cellular response to DNA double-strand breaks (DSBs)
(Simon Bekker-Jensen and Niels Mailand)
Our previous work has uncovered the existence of a ubiquitin-dependent signaling pathway that plays a key role in orchestrating the dynamic formation of DSB repair foci, large multiprotein structures that form around the damaged chromatin and facilitate repair (reviewed in Bekker-Jensen & Mailand, DNA Repair 2010). Moreover, we have discovered several key ubiquitin ligases for this pathway (Bekker-Jensen & Mailand, DNA Repair 2010; Poulsen et al., Journal of Cell Biology 2012; Bekker-Jensen et al., Nature Cell Biology 2010; Doil et al., Cell 2009; Mailand et al., Cell 2007). We are exploiting our expertise in this area in combination with the technology platform at NNF CPR and novel methodologies to identify, in an unbiased fashion, the proteins that reside in DSB repair foci. We are also utilizing recent technological advancements in proteome-wide mapping of site-specific protein ubiquitylation described above to search for cellular substrates of the key ubiquitin ligases and deubiquitylating enzymes implicated in the ubiquitin-mediated DSB signaling response. Finally, we use proteomics-based interaction studies to define in a comprehensive fashion the ’interactomes’ of the key enzymes in the ubiquitin-dependent DSB response, as well as for all new components of this response identified via the approaches described above.

Functional characterization of the ZZ domain, a novel type of SUMO-binding Zinc finger motif
(Simon Bekker-Jensen and Niels Mailand)
From our studies of the DSB-responsive ubiquitin ligase HERC2, we recently identified a novel type of SUMO-binding domain, the ZZ-type Zinc finger motif (Danielsen et al., Journal of Cell Biology 2012), present in some 20 human proteins. We aim to dissect in detail the SUMO-binding properties of individual ZZ domains as well as their involvements in important cellular processes. We are performing detailed structural analysis of the molecular basis of high-affinity SUMO-binding of the ZZ domains, and we are functionally characterizing the SUMO-binding properties of all individual human ZZ domains. Furthermore, we are assessing the utility of isolated ZZ domains as tools for high-affinity isolation and characterization of SUMOylated cellular proteins.