ERC FUNDED PROJECTS

The interest on autophagy, an evolutionarily conserved process in eukaryotes, has enormously increased in the last years, since autophagy is involved in many diseases such as cancer and neurodegenerative disorders. Autophagosome formation is the key process in autophagy. Despite extensive work, the model of autophagosome formation is not yet well established. Some important questions on autophagosome biogenesis remain to be elusive, such as where the bona fide marker protein of autophagosome, LC3, is lipidated, how lipidated LC3 functions in autophagosome formation, and how the proteins for LC3 lipidation and delipidation are involved in autophagosome formation. Although genetic approaches have been useful to identify genes involved in autophagy, they are chronic and thereby the dynamics of phenotypic change cannot be followed, making them not suited for study highly dynamic process such as autophagosome formation. Herein, I propose to develop and use novel chemical probes to address these issues. First, I plan to prepare semi-synthetic caged LC3 proteins and apply them to monitor dynamics of autophagosome formation in the cell in order to address those questions on autophagosome formation. The semi-synthetic LC3 proteins are expected to confer a temporal control and to realize manipulation of protein structure, which renders such studies possible. Second, I intend to develop a versatile approach targeting specific endogenous proteins using a reversible chemically induced dimerization (CID) system, termed as “knock on and off” strategy. I plan to use this approach to elucidate the function of two distinct PI3K complexes in autophagosome formation. On one hand, the establishment of novel approaches will open up a new avenue for studying biological processes. On the other hand, the use of the tool will reveal the mechanism of autophagy.

1 500 000 €

Start date: 2016-09-01, End date: 2021-08-31

Mitochondria are required to convert food into usable energy forms and every cell contains thousands of them. Unlike most other cellular compartments, mitochondria have their own genomes (mtDNA) that encode for 13 of the about 90 proteins present in the respiratory chain. All proteins necessary for mtDNA replication, as well as transcription and translation of mtDNA-encoded genes, are encoded in the nucleus. Mutations in nuclear-encoded proteins required for mtDNA maintenance is an important cause of neurodegeneration and muscle diseases. The common result of these defects is either mtDNA depletion or accumulation of multiple deletions of mtDNA in postmitotic tissues. The long-term goal (or vision) of research in my laboratory is to understand in molecular detail how mtDNA is replicated and how this process is regulated in mammalian cells. To this end we use a protein biochemistry approach, which we combine with in vivo verification in cell lines. My group was in 2004 the first to reconstitute mtDNA replication in vitro and we have continued to develop even more elaborate system ever since. In the current application, the major focus is studies of the mitochondrial D-loop region, a triple-stranded structure in the mitochondrial genome. The D-loop functions as a regulatory hub and we will determine how initiation and termination of mtDNA replication is controlled from this region. We will also determine the physical organization of the mtDNA replication machinery at the replication fork and establish how mtDNA deletions, a classical hallmark of human ageing, are formed.

1 999 985 €

Start date: 2016-11-01, End date: 2021-10-31

At the dawn of the 21st century, our knowledge of the molecular mechanism of mammalian fertilization remains very limited. Different lines of evidence indicate that initial gamete recognition depends on interaction between a few distinct proteins on sperm and ZP3, a major component of the extracellular coat of oocytes, the zona pellucida (ZP). On the other hand, recent findings suggest an alternative mechanism in which cleavage of another ZP subunit, ZP2, regulates binding of gametes by altering the global structure of the ZP. Progress in the field has been hindered by the paucity and heterogeneity of native egg-sperm recognition proteins, so that novel approaches are needed to reconcile all available data into a single consistent model of fertilization. Following our recent determination of the structure of the most conserved domain of sperm receptor ZP3 by X-ray crystallography, we will conclusively establish the basis of mammalian gamete recognition by performing structural studies of homogeneous, biologically active recombinant proteins. First, we will combine crystallographic studies of isolated ZP subunits with electron microscopy analysis of their filaments to build a structural model of the ZP. Second, structures of key egg-sperm recognition protein complexes will be determined. Third, we will investigate how proteolysis of ZP2 triggers overall conformational changes of the ZP upon gamete fusion. Together with functional analysis of mutant proteins, these studies will provide atomic resolution snapshots of the most crucial step in the beginning of a new life, directly visualizing molecular determinants responsible for species-restricted gamete interaction at fertilization. The progressive decrease of births in the Western world and inadequacy of current contraceptive methods in developing countries underscore an urgent need for a modern approach to reproductive welfare. This research will not only shed light on a truly fundamental biological problem, but also constitute a solid foundation for the reproductive medicine of the future.

1 499 282 €

Start date: 2011-01-01, End date: 2015-12-31

The length of telomeric DNA is a critical determinant factor for aging and cancer development. In germ cells, the activation of a telomerase-dependent telomere-lengthening pathway is thought to be important in order to maintain telomeric DNA across generations, but the molecular mechanisms involved in this pathway, i.e: how and when telomerase is activated in germ cells, are largely unknown. A DNA-binding protein complex called shelterin constitutively binds telomeric DNA. However, my recent studies have suggested that a multi-subunit DNA-binding complex, TERB1-TERB2-MAJIN, takes over telomeric DNA from shelterin in mammalian germ cells in order to facilitate homologous recombination. These findings represent a hitherto unknown molecular mechanism at work on the telomeres in germ cells. In this project, I hypothesize that the drastic reformation of telomere-binding complexes in germ cells contributes also to the telomere-lengthening pathway. The aim of this project is to test this hypothesis in order to reveal the mechanism underlying the transgenerational inheritance of telomeric DNA throughout meiosis. This work is divided into three work packages. WP1: to determine the molecular rearrangements that take place at telomeres during meiosis. WP2: to determine how and when telomeres are lengthened during germ cell production. WP3: to determine how meiotic recombination is achieved. The proposed project will reveal molecular mechanisms underlying the transgenerational inheritance of genetic information after meiosis, and this will increase our understanding of the etiology of numerous human diseases caused by meiotic errors, such as congenital birth defects and aneuploidy. Further, because the misregulation of telomerase is a leading cause of cancer development, the identification of telomerase-activating mechanisms in germ cells will have multidiscipline impacts in both cancer and reproductive biology fields and will be useful for developing novel cancer therapies.

1 500 000 €

Start date: 2019-01-01, End date: 2023-12-31