ERC FUNDED PROJECTS

Specificity in the ubiquitin-proteasome system is largely conferred by ubiquitin E3 ligases (E3s). Cullin-RING ligases (CRLs), constituting ~30% of all E3s in humans, mediate the ubiquitination of ~20% of the proteins degraded by the proteasome. CRLs are divided into seven families based on their cullin constituent. Each cullin binds a RING domain protein, and a vast repertoire of adaptor/substrate receptor modules, collectively creating more than 200 distinct CRLs. All CRLs are regulated by the COP9 signalosome (CSN), an eight-protein isopeptidase that removes the covalently attached activator, NEDD8, from the cullin. Independent of NEDD8 cleavage, CSN forms protective complexes with CRLs, which prevents destructive auto-ubiquitination. The integrity of the CSN-CRL system is crucially important for the normal cell physiology. Based on our previous work on CRL structures (Fischer, et al., Nature 2014; Fischer, et al., Cell 2011) and that of isolated CSN (Lingaraju et al., Nature 2014), We now intend to provide the underlying molecular mechanism of CRL regulation by CSN. Structural insights at atomic resolution, combined with in vitro and in vivo functional studies are designed to reveal (i) how the signalosome deneddylates and maintains the bound ligases in an inactive state, (ii) how the multiple CSN subunits bind to structurally diverse CRLs, and (iii) how CSN is itself subject to regulation by post-translational modifications or additional further factors. The ERC funding would allow my lab to pursue an ambitious interdisciplinary approach combining X-ray crystallography, cryo-electron microscopy, biochemistry and cell biology. This is expected to provide a unique molecular understanding of CSN action. Beyond ubiquitination, insight into this >13- subunit CSN-CRL assembly will allow examining general principles of multi-subunit complex action and reveal how the numerous, often essential, subunits contribute to complex function.

2 200 677 €

Start date: 2016-01-01, End date: 2020-12-31

"The mismatch repair (MMR) system has evolved to correct errors of DNA replication and prevent recombination between non-homologous sequences. Correspondingly, MMR malfunction leads to increased mutation rates and illegitimate recombination, and individuals with inherited MMR gene mutations are predisposed to cancer of the colon and other organs. However, MMR has recently been implicated in processes ranging from DNA damage signaling and drug sensitivity to antibody maturation, some of which contradict and even subvert the classical role of MMR as a guardian of genomic integrity. We suspect that the latter processes are linked to a new, non-canonical MMR (ncMMR) pathway that can be activated outside of the S- and G2 phases of the cell cycle by a variety of lesions and structures. ncMMR lacks strand directionality, involves long stretches of DNA degradation, and our preliminary in vitro evidence suggests that resynthesis of these repair tracts can be mediated not only by high-fidelity, replicative polymerases, but also by error-prone enzymes. In this scenario, ncMMR would actually contribute to mutagenesis. I plan to deploy proteomic, genomic and imaging technologies to identify the components of the ncMMR “mutasome” and to reconstitute the system from purified recombinant components. Furthermore, I wish to study the “action radius” of MMR proteins by characterizing their interactome and analyze its dependence on endogenous states (e.g. cell cycle stages) and exogenous stimuli (e.g. drug treatments) in human and chicken (DT40) cells, and Xenopus laevis egg extracts. I intend to exploit a new system of inducible protein replacement that was recently developed in my laboratory, to stably express MMR, replication, repair and recombination proteins (both wild type and variants). This program should increase our understanding of the pivotal role of MMR in DNA metabolism and its involvement in human disease and cancer."

2 208 084 €

Start date: 2012-04-01, End date: 2016-08-31

The nuclear envelope (NE) harbors key roles in cellular and organismal homeostasis, reflected by a variety of diseases caused by mutations in NE proteins. Despite the fundamental role of the NE in protecting and organizing the genome, still little is known about the molecular mechanisms underlying NE biogenesis, dynamics and functionality. We will address 3 open questions in NE biology, using vertebrate cells as model system. (1) To understand how the functional specification of the NE by transmembrane proteins is generated, we will decipher how membrane proteins are sorted to the inner nuclear membrane (INM). To reach this goal, we will define targeting signals and the mode of NPC passage of INM proteins, and identify the molecular requirements for transport. (2) Based on structural analysis, we will investigate how the molecular organization of LINC complexes, which are formed by interacting pairs of SUN and KASH proteins spanning the NE, determines their role in NE architecture and as tethers of the NE to the cytoskeleton. (3) We will study dynamic changes of the NE that occur at the onset of ’open’ mitosis, when the nuclear compartment is disintegrated to allow for the formation of a cytoplasmic mitotic spindle. NE breakdown (NEBD) presents a dramatic change of cellular architecture and comprises a series of events including disassembly of nuclear pore complexes, the nuclear lamina and retraction of NE membranes into the endoplasmic reticulum. To elucidate the molecular mechanisms controlling and executing these steps of nuclear disassembly, we will characterize the cellular machinery involved in NEBD and unravel the molecular function of identified components. All these questions will be addressed by a blend of in vivo approaches relying on high-end fluorescence imaging linked to computational image analysis, RNAi screening, as well as powerful in vitro systems recapitulating protein transport to the INM or NEBD that we have developed, and biochemical methods.

2 500 000 €

Start date: 2013-04-01, End date: 2018-03-31

The ends of eukaryotic chromosomes, known as telomeres, play crucial roles as guardians of genome stability and tumor suppressors. Telomeric DNA is maintained by the ribonucleoprotein enzyme telomerase. Most normal human somatic cells express only very low levels of telomerase and telomeres shorten with continuous cell division cycles. Ultimately, short telomeres activate a DNA damage response that leads to a permanent cell cycle arrest or apoptosis. Reactivation of telomerase is a key requisite for human cancer cells to attain unlimited proliferation potential. The key questions that need to be tackled in the telomere field are (1) how telomeres protect from DNA repair activities, (2) how recruitment and regulation of telomerase is mediated by telomere structure, (3) how cell cycle arrest occurs upon telomere shortening, and (4) how telomeres regulate their heterochromatic state. In all four areas, important progress is expected in the near future. We recently made the unexpected discovery that telomeres are transcribed into TElomeric Repeat containing RNA (TERRA) and that this RNA is an integral part of telomeric heterochromatin. Our working hypothesis is that the telomere is an RNA-dependent machine, and that several if not most of its crucial functions are regulated by TERRA. In this proposal we will explore TERRA functions, by elucidating its biogenesis, by identifying its protein partners and by genetic manipulation of the expression of TERRA and TERRA binding proteins. This work should provide fundamental insight into how our chromosome ends function. The gained knowledge may also provide novel avenues on how to manipulate telomere function and dysfunction in cancer cells and other diseased tissue.

2 385 047 €

Start date: 2009-04-01, End date: 2014-03-31