ERC FUNDED PROJECTS

"DNA damage is a fact of life. Lesions hamper genome function, induce mutations causing cancer and trigger senescence or cell death contributing to aging. Therefore cells are equipped with a sophisticated defence machinery: DNA Damage Response (DDR) including different repair pathways. Nucleotide excision repair (NER) is versatile repair process, eliminating helix-distorting lesions, e.g. bulky adducts and sun-induced lesions. Very cytotoxic transcription-blocking lesions are removed by a dedicated sub-pathway, transcription-coupled (TC-)NER. The impact of NER is highlighted by 4 disorders: xeroderma pigmentosum (XP), Cockayne syndrome (CS), trichothiodystrophy and UV-sensitive syndrome (UVSS). XP patients are cancer-prone due to global-genome (GG-)NER defects, whereas CS patients, impaired in TC-NER, display progeroid features, which are thought to derive from endogenous oxidative DNA lesions hampering transcription. Consistent with this, CS cells are sensitive to oxidative agents, whereas TC-NER-deficient UVSS patients are not sensitive to oxidative agents and do not display aging features. This implies lesion-specific TC-NER, arguing for distinct operational TC-repair machineries. The relative importance of DDR pathways varies with the type of damage, cell type and stage of development determining onset of cancer and aging pathologies. The challenging ambition of this proposal is to gain in depth insight into the role of NER in protection against cancer and aging by an integral multi-disciplinary approach which includes new mouse models for novel TC-NER genes, live cell and tissue NER kinetic analyses, advanced proteomics and analysis of NER-related chromatin dynamics to dissect cross-talk with other pathways. The strength of this project is the comprehensive strategy, availability of unique tools (e.g. collection of bona fide NER mutant mice), operational top notch technical platforms for all proposed approaches and proven competence and expertise."

2 500 000 €

Start date: 2014-01-01, End date: 2018-12-31

My goal is to elucidate how structurally closely-related proteins are selectively partitioned in distinct membrane domains that allow localization, clustering and segregation of specific cellular activities. Although many of the mechanisms that govern membrane organization are increasingly well understood, such as lipid ‘rafts’ or protein-anchoring to the cortical cytoskeleton, these mechanisms are not sufficiently specific to account for the partitioning of closely homologous proteins in separate membrane domains. I believe that the observed highly selective membrane partitioning can only be explained by the combined action of protein-protein and protein-lipid interactions and thermodynamic properties of the membrane (length, charge, degree of hydrophobicity). I aim to gain a full understanding of how homologous proteins partition in distinct functional membrane domains by studying SNARE proteins as a model system. Different SNAREs partition in different domains with different degrees of overlap in the plasma membrane where they catalyze the final membrane fusion steps of various exocytotic pathways. I will employ quantitative super-resolution microscopy to study the effects of selective (biochemical and genetic) perturbations on SNARE partitioning in both precisely controllable artificial membranes and in PC12 cells. This will allow me to elucidate the mechanisms, contributions and interplay of individual membrane clustering mechanisms in SNARE domain organization. I then plan to demonstrate that the mechanisms of SNARE partitioning explain the membrane organization of other (homologous) proteins as well. My ultimate ambitious goal is to generate a complete model of how proteins are organized in biological membranes. I anticipate that my findings will uncover new and general mechanisms of membrane organization and, since membranes are involved in almost all cellular processes, my work may have impact on virtually all areas of the health and life sciences.

1 500 000 €

Start date: 2013-12-01, End date: 2018-11-30

Bile acids play a pivotal role in energy supply as they facilitate the solubilization and absorption of fat in the intestine. Furthermore, bile acids are recently identified as important signalling molecules regulating glucose metabolism, inflammation and energy expenditure. Targeting bile acid signalling is, therefore, appealing to treat metabolic diseases such as diabetes and atherosclerosis. These disorders are potentially affecting >1 billion individuals worldwide and current options to treat them remain insufficient. I postulate that the hepatic bile acid uptake transporter NTCP (gene name SLC10A1) provides an excellent novel target to improve human health as it determines the duration of bile acid signalling by controlling how fast bile acids are removed from serum after a meal. In this proposal I will elucidate the contribution of bile acid dynamics to energy homeostasis and metabolism and identify the molecular mechanisms that regulate NTCP. My aim is to generate novel strategies to reduce hepatic bile acid uptake to prolong bile-acid signalling and increase energy expenditure, improve glucose handling and reduce atherosclerosis. My key objectives are: 1. to determine the consequence of NTCP modulation on systemic bile acid dynamics, glucose and energy metabolism in animal models. To this end, I will perform careful metabolic analysis of a unique Slc10a1 knockout model in combination with diet-induced and genetic models for atherosclerosis and diabetes. 2. to identify novel means to inhibit NTCP-mediated bile acid uptake. To this end, I will make use of a FRET-based bile acid sensor that I recently developed to characterize the molecular regulation of hepatic bile acid uptake and to identify FDA-approved drugs that inhibit NTCP-mediated bile acid uptake. This will establish my new research line on serum bile acid dynamics and ultimately provide new ways to treat metabolic diseases related to disturbed bile acid, lipid, glucose and energy homeostasis.

1 489 320 €

Start date: 2013-12-01, End date: 2018-11-30

The persistence of a transcriptionally competent but latent HIV infected memory CD4+T cell reservoir, despite the effectiveness of Highly Active Antiretroviral therapy (HAART) against active virus, presents the main impediment to HIV eradication. A novel concept in HIV eradication is to activate latent virus to subsequently eliminate with HAART. Much effort has gone into identification of protein complexes that regulate HIV LTR activity. Strategies have mainly relied on candidate approaches. However, due to technical limitations, comprehensive unbiased identification of host proteins associated with and necessary for silencing of the latent HIV LTR has not been possible. Trxn-PURGE proposes a novel multidisciplinary approach combining current knowledge of HIV transcription and new insights into eradication strategies with state of the art high though-put approaches, mycology, virology, genetics and conventional biochemistry to identify novel players in maintenance and activation of HIV transcriptional latency. We will: 1. Use a novel unbiased strategy to identify the in vivo latent LTR-bound protein complex directly from infected T cells. 2. Conduct a cell-based high-throughput Haploid genetic screen to identify novel factors essential for maintenance of HIV latency. 3. Having identified three putative activators from a limited library, we will perform a large-scale screen with unbiased library of fungal supernatants to identify molecules capable of activation of latent HIV. These parallel approaches will identify novel molecular targets and molecules in activation of HIV transcriptional latency, which we will functionally and mechanistically characterize alone and in synergy with known compounds implicated in latent LTR activation in both 4. T cell lines and 5. primary human CD4+T cells harboring latent HIV. By unravelling its molecular mechanisms, Trxn-PURGE will set the stage for the development of a clinical combinatorial therapy to activate latent HIV.

1 499 942 €

Start date: 2014-02-01, End date: 2019-01-31