ERC FUNDED PROJECTS

Eukaryotic Argonaute proteins are known for their central role in RNA interference pathways. Yet, the evolutionary origin of Argonaute proteins lies in prokaryotes, where other proteins essential for RNA interference are absent. Therefore, prokaryotic Argonaute proteins (pAgos) must have distinct ancestral functions. Although a handful of closely related pAgos interfere with exogenous DNA invaders such as plasmids, pAgos are extremely diverse in terms of sequence conservation and domain architecture. In addition, many pAgos genetically associate with various putative enzyme domains, which suggests that they are functionally interdependent. As such, different pAgos must rely on distinct mechanism and are expected to fulfil a range of different roles. Therefore, the function of most pAgos remains completely unknown. The COMPASS project will map the function of unexplored pAgo systems, in which pAgos associate with auxiliary proteins. I hypothesize that in these systems, pAgo binds exogenous DNA sequences in a guide-dependent manner. This can result in the recruitment and/or activation of the auxiliary proteins. As pAgo-associated auxiliary proteins are homologous to proteins involved in DNA recombination, NAD+ turnover, or protein deacetylation, these pAgo systems are expected to fulfil completely novel roles ranging from stimulating horizontal gene transfer to triggering programmed cell death. The uncharacterized roles of these pAgo systems and the mechanisms underlying their functionality will be elucidated by a multidisciplinary approach combining microbiology, protein biochemistry, and X-ray crystallography techniques. Not only will the results facilitate a deeper understanding of the evolutionary diversification of pAgos, it will also enable the repurposing of programmable pAgo systems for the development of genetic tools that facilitate guide sequence-directed DNA recombination and high-sensitivity detection of target DNA sequences.

1 496 463 €

Start date: 2020-12-01, End date: 2025-11-30

The primary cilium is a microtubule-based organelle that organizes a variety of cellular signaling pathways. Its importance for human health is illustrated by a large collection of cilium-based diseases, the ciliopathies, caused by mutations that alter cilium formation, structure, and function. Importantly, the mammalian Hedgehog signaling pathway is critically dependent on the primary cilium, dysregulation of which contributes to severe developmental defects and a variety of cancers. The ultimate goal of this work program is to enhance our understanding of the molecular mechanisms of ciliary signaling in health and disease. This is accomplished through the development of advanced technologies that provide a currently unattainable level of spatiotemporal control over ciliary proteins in mammalian cells. Unraveling the mechanisms by which the ciliary compartment orchestrates signal transduction is challenging, because ciliary and cytoplasmic roles of proteins involved in signal transduction are tightly connected, difficult to resolve and, importantly, context-specific. Here, an innovative chemical biology program is presented that provides a powerful toolbox to overcome these challenges, allowing the intraciliary manipulation, and therefore study, of ciliary proteins. Combining chemical probes, synthetic ciliary targeting approaches, and a modular enzymatic tagging strategy, this program provides unprecedented opportunities to probe, visualize, inhibit or degrade proteins at specific times and selectively within the ciliary compartment. Specifically, these tools will be used to decipher the relationships between tubulin acetylation state, intraflagellar transport, and Hedgehog signal transduction in wild-type and ciliopathy-mutant cells. These innovative work packages synergistically provide enhanced fundamental understanding of ciliary signaling, and pave the way for novel therapeutic approaches to combat cilium-based diseases.

1 408 776 €

Start date: 2021-01-01, End date: 2025-12-31

Cellular homeostasis is controlled by the RAS-MAPK pathway. This pathway is dysregulated in human diseases, especially cancer, in which more than 50% of cases carry aberrations that hyperactivate RAS-MAPK signaling. In this context, KRAS mutations are the most frequent oncogenic drivers. Therapeutic suppression of pathogenic KRAS-RAF-MAPK signaling to achieve disease control in cancer patients still represents a challenging target. KRAS dimers and multimers at the membrane (collectively referred, together with adaptors and effectors, as to “KRAS signalosome”) influence the activation of KRAS signaling. I provided the first biological evidence that dimerization is required for the function of oncogenic KRAS (Ambrogio et al, Cell, 2018). Indeed, one fascinating and still largely unexplored aspect of KRAS biology is the functional impact of KRAS complexes at the membrane for signaling and drug sensitivity. No inhibitors of oncogenic KRAS clustering have been identified so far. Interestingly, wild-type KRAS antagonizes oncogenic KRAS, resulting in reduced oncogenic signaling. The overarching goal of this proposal is the characterization in vitro and in vivo of the “KRAS signalosome” in terms of functional dynamics and related actionable vulnerabilities. My strong background in KRAS biology provides me with the expertise to propose an ambitious, yet feasible plan to understand the tumor suppressor effect of wild-type KRAS protomers in mutant KRAS-driven complexes by identifying and validating membrane interactors differentially recruited by wild-type and mutant KRAS (Work package 1). In parallel, I will study the relevance of RAF kinases localization at the membrane as key feature to sustain oncogenic MAPK activity in vivo (Work package 2). Finally, I will screen new compounds to interfere with RAFs function at the cell membrane and will determine the therapeutic impact of disrupting mutant KRAS signalosome using mouse models in vivo (Work package 3).

1 996 853 €

Start date: 2021-08-01, End date: 2026-07-31

Life on Earth is sustained by plants. Growth and development in the plant kingdom is mediated by the controlled distribution of sugars and the hormone auxin, but we still know surprisingly little about the molecular details of this essential part of fundamental plant metabolism. MUM-GROW will elucidate the molecular mechanism of sugar and auxin transmembrane transport in plants. It moves the frontiers of the field by shifting the focus to molecular studies in vitro allowing structural and biochemical experiments to be performed. Correct plant growth and development is completely dependent on sugar uptake in growth zones (the meristem), and made possible in all plants by sugar transporters called SUCs and STPs. Growth polarity is created by an asymmetrical gradient of auxin mediated by auxin transporters called PINs. Despite extensive research, the molecular mechanisms of SUC, STP and PIN transport remains unknown. If we knew the molecular determinants of their function, it would allow us to predict, augment and possibly modify plant responses to a changing environment. I will address this using a complementary set of methods founded in structural biology to determine the 3-dimensional structures of key players in these transmembrane transport systems. This will be combined with biochemical characterization to address important mechanistic questions and elucidate their molecular mechanism. Understanding the mechanisms that govern plasticity in growth is essential for determining resilience of whole ecosystems. This proposal will lead to a breakthrough in our understanding of sugar and auxin homeostasis, a fundamental part of basic plant metabolism. It has tremendous potential for the societal challenge to secure sufficient food for our global population in a sustainable balance between environmental impact and resource exploitation. Furthermore, this proposal will uncover general molecular principles of transmembrane uptake and export pertaining to all organisms.

1 999 910 €

Start date: 2021-06-01, End date: 2026-05-31