ERC FUNDED PROJECTS

The Endoplasmic Reticulum (ER) membrane in all eukaryotic cells has an intricate protein network that facilitates protein biogene-sis and homeostasis. The molecular complexity and sophisticated regulation of this machinery favours study-ing it in its native microenvironment by novel approaches. Cryo-electron tomography (CET) allows 3D im-aging of membrane-associated complexes in their native surrounding. Computational analysis of many sub-tomograms depicting the same type of macromolecule, a technology I pioneered, provides subnanometer resolution insights into different conformations of native complexes. I propose to leverage CET of cellular and cell-free systems to reveal the molecular details of ER protein bio-genesis and homeostasis. In detail, I will study: (a) The structure of the ER translocon, the dynamic gateway for import of nascent proteins into the ER and their maturation. The largest component is the oligosaccharyl transferase complex. (b) Cotranslational ER import, N-glycosylation, chaperone-mediated stabilization and folding as well as oligomerization of established model substrate such a major histocompatibility complex (MHC) class I and II complexes. (c) The degradation of misfolded ER-residing proteins by the cytosolic 26S proteasome using cytomegalovirus-induced depletion of MHC class I as a model system. (d) The structural changes of the ER-bound translation machinery upon ER stress through IRE1-mediated degradation of mRNA that is specific for ER-targeted proteins. (e) The improved ‘in silico purification’ of different states of native macromolecules by maximum likelihood subtomogram classification and its application to a-d. This project will be the blueprint for a new approach to structural biology of membrane-associated processes. It will contribute to our mechanistic understanding of viral immune evasion and glycosylation disorders as well as numerous diseases involving chronic ER stress including diabetes and neurodegenerative diseases.

2 496 611 €

Start date: 2017-04-01, End date: 2022-03-31

An important question of modern neurobiology is how neurons regulate synaptic function in response to excitation. In particular, the roles of alternative pre-mRNA splicing and mRNA translation regulation in this response are poorly understood. We will study the RNA-binding proteins (RBPs) that control these post-transcriptional changes using a UV crosslinking-based purification method (CLIP) and ultra-high throughput sequencing. Computational analysis of the resulting data will define the sequence and structural features of RNA motifs recognized by each RBP. Splicing microarrays and translation reporter assays will then allow us to examine the regulatory functions of RBPs and RNA motifs. By integrating the biochemical and functional datasets, we will relate the position of RNA motifs to the activity of bound RBPs, and predict the interactions that act as central nodes in the regulatory network. The physiological role of these core RBP-RNA interactions will then be tested using antisense RNAs. Together, these projects will provide insights to the regulatory mechanisms underlying neuronal activity-dependent changes, and provide new opportunities for future treatments of neurodegenerative disorders.

900 000 €

Start date: 2008-09-01, End date: 2013-08-31

Bacterial dehalorespiration is a microbial respiratory process in which halogenated hydrocarbons, from natural or anthropogenic origin, act as terminal electron acceptors. This leads to effective dehalogenation of these compounds, and as such their degradation and detoxification. The bacterial species, their enzymes and other components responsible for this unusual metabolism have only recently been identified. Unlocking the full potential of this process for bioremediation of persistent organohalides, such as polychlorinated biphenyls (PCBs) and tetrachloroethene, requires detailed understanding of the underpinning biochemistry. However, the regulation, mechanism and structure of the reductive dehalogenase (the enzyme responsible for delivering electrons to the halogenated substrates) are poorly understood. This ambitious proposal seeks to study representatives of the distinct reductive dehalogenase classes as well as key elements of the associated regulatory systems. Our group has been at the forefront of studying the biochemistry underpinning transcriptional regulation of dehalorespiration, providing detailed insights in the protein CprK at the atomic level. However, it is now apparent that only a subset of dehalogenases are regulated by CprK homologues with little known about the other regulators. In addition, studies on the reductive dehalogenases have been hampered by the inability to purify sufficient quantities. Using an interdisciplinary, biophysical approach focused around X-ray crystallography, enzymology and molecular biology, combined with novel reductive dehalogenase production methods, we aim to provide a detailed understanding and identification of the structural elements crucial to reductive dehalogenase mechanism and regulation. At the same time, we aim to apply the knowledge gathered and study the feasibility of generating improved dehalorespiratory components for biosensing or bioremediation applications through laboratory assisted evolution.

1 148 522 €

Start date: 2008-09-01, End date: 2013-08-31

Inhibitory interneurons function as modulators of local circuit excitability. Their properties are of fundamental importance for normal brain function therefore understanding how these cells are generated during development may provide insight into neurodevelopmental disorders such as epilepsy and schizophrenia, in which interneuron defects have been implicated. Inhibitory GABAergic interneurons of the cerebral cortex (pallium) are generated from proliferating subpallial precursors during development and migrate extensively to populate the cortex. The aim of this proposal is to identify genetic pathways and signalling systems that underlie cortical interneuron migration and integration into functional neuronal circuits. Distinct interneuron subtypes are generated from the two most prominent neuroepithelial stem cell pools in the subpallium: the medial ganglionic eminence (MGE) and the lateral/caudal ganglionic eminence (LGE/CGE). We will genetically tag and purify interneurons originating from these precursors in order to examine their transcriptomes and identify factors involved in specification and migration. We will use Cre-lox fate mapping in transgenic mice to label specific sub-populations of neural stem cells and their differentiated progeny in the embryonic telencephalon. This will allow us to determine whether subdomains of the MGE or LGE/CGE neuroepithelium generate interneurons with distinct neurochemical phenotypes and/or characteristic migratory properties. Electrical activity and/or neurotransmitter receptor activation can act in concert with genetic programs to promote precursor proliferation, neuronal differentiation as well as neuronal migration. We will use gain-of-function and loss-of-function approaches to examine the role of neurotransmitters and neuropeptides at early stages of interneuron migration to the cortex.

1 250 000 €

Start date: 2008-07-01, End date: 2014-08-31

Transcription, the copying of DNA into RNA, is the first step in the realisation of genetic information. RNA is either directly used by the cell or decoded into proteins during translation. The accuracy of transcription is thus essential for proper functioning of the cell. In all living organisms transcription is performed by multisubunit RNA polymerases, enzymes that are highly conserved in evolution from bacteria to humans. Surprisingly, the mechanisms that ensure accuracy of transcription remain largely unknown. Recently I discovered a novel mechanism of transcriptional proofreading used by bacterial RNA polymerase. I showed that the RNA transcript itself assists RNA polymerase in identifying and correcting mistakes. This discovery led to the hypothesis that this transcript-assisted proofreading is the universal mechanism of transcriptional error correction in all three domains of life. In this proposal we will investigate this hypothesis and search for other mechanisms of transcriptional proofreading used by bacterial, archaeal, and three eukaryotic RNA polymerases. For the first time experimental systems will be built for the simultaneous investigation of transcription elongation complexes formed by bacterial, archaeal and eukaryotic RNA polymerases I, II and III, which will be used to elucidate the mechanisms of error correction used by these RNA polymerases. Using molecular modelling, directed mutagenesis and in vivo screenings we will investigate the impact of these proofreading mechanisms on the total fidelity of transcription in vitro and in vivo. Experimental systems built in this research may be of use for screening of potential antibacterial and antifungal drugs taking advantage of the simultaneous investigation of RNA polymerases from all domains of Life. This research may also have potential applications in drug design by providing new targets for antibiotics.

1 149 831 €

Start date: 2008-11-01, End date: 2013-10-31

Gene expression is tightly regulated to allow rapid responses to cellular stimuli. In eukaryotes, the 3´ polyA tail of mRNAs plays key roles in post-transcriptional control. The Cleavage and Polyadenylation Factor (CPF), Ccr4–Not and Pan2–Pan3 multiprotein complexes add or remove polyA tails to regulate mRNA stability and efficiency of translation. They control expression of genes in the inflammatory response, miRNA-targeted gene silencing and expression of maternal mRNAs in oocyte development. These processes are deregulated in disease, including cancer and neurological disorders. Although the proteins that add and remove polyA tails are known, their mechanisms are poorly understood. My lab recently established methods to reconstitute the polyA machinery. This led to new insights into the link between transcription and polyadenylation, new understanding of the molecular mechanisms of deadenylation, and details of RNA recruitment. In this proposal, my objective is to understand the molecular basis for polyadenylation and deadenylation of specific mRNAs. This is now possible because of our novel methodological and biological advances. We will determine high-resolution structures of the polyA machinery using electron cryo-microscopy (cryo-EM), reconstitute their biochemical activities in vitro and study their in vivo functional roles. We use this integrated approach to study intact multiprotein complexes, not individual subunits or domains. This involves considerable technical challenges and an investment in developing high quality purifications and new structural methods. I will determine how the four enzymatic activities of CPF are coupled, the mechanisms by which Ccr4–Not targets specific RNAs, and the molecular basis for RNA recognition by Pan2–Pan3. Together, this will provide new biological and technological insights, leading to understanding of fundamental processes in gene expression and the role of polyA tails in disease.

2 016 697 €

Start date: 2017-04-01, End date: 2022-03-31

The posttranslational modification of proteins with polyubiquitin regulates virtually all aspects of cell biology. This versatility arises from eight distinct linkage types between individual ubiquitin moieties in polyubiquitin, which co-exist in cells, are independently regulated, and eventually determine the fate of the modified protein. However, ubiquitin chain architecture can be highly complex, and the extent of ‘chain branching’ is unknown. Moreover, ubiquitin also undergoes phosphorylation and acetylation, which can dramatically alter its function. A true appreciation of the complexity in the ubiquitin code can only be achieved when all above aspects are considered, and only then will it be possible to assign cellular readouts to distinct ubiquitination events and differentiate between ubiquitin signals in cells. While the complexity of ubiquitination is daunting, work from many laboratories including my own has exemplified how basic biochemistry, a detailed understanding of mechanism and quantitative mass-spectrometry allows us to study, and eventually understand, the ubiquitin code. In this proposal, new methods and approaches are outlined that will allow a detailed monitoring of polyubiquitin chain architectures from cellular samples (AIM 1), and also lead to an in depth understanding of additional posttranslational modifications, such as ubiquitin phosphorylation and acetylation in cells (AIM 2). Moreover, new research tools for unstudied K6- and K33-linked polyubiquitin will give insights into cellular roles for these linkage types (AIM 3). Our studies will focus on ubiquitination events on mitochondria leading to mitophagy, where unstudied K6-linked chains as well as phospho-ubiquitin are part of complex chain architectures, and mechanisms of signalling are still unclear. Our work will reveal fundamental principles in ubiquitination, and are of high medical relevance due to the links to Parkinson’s disease, infectious disease, and cancer.

1 990 125 €

Start date: 2017-10-01, End date: 2022-09-30