Research project P7/44 (Research action P7)
Proteins are the main products of genetic information and are the pivotal biological macromolecules that determine the structure and function of all living cell systems. Proteins direct development of organisms, metabolism and responses to environmental stimuli. They interact with ligands of various sizes that can be substrates, inhibitors, effectors, nucleic acids, lipid bilayers or other proteins. Undesired alterations of these interactions can transform a normal cellular process into an aberrant one, resulting in many types of pathologies. Alternatively, the development of small molecules, which can interfere with crucial protein functions in living cells, takes an important part in human therapy and antimicrobial chemotherapy, which is based on the perturbation of the metabolic processes.
In the post-genomic era, we have access to a very large amount of data about the genomes of an increasing number of organisms. By contrast, we are far from being able to integrate all these data in a detailed understanding of the functioning of living cells. This paradox is explained by the fact that the determination of genomic sequences has progressed much faster than our knowledge of the structure-function relationships and interactions between biomolecules. Traditionally, proteins have been studied as isolated entities. However, in the post-genomic era, a new type of protein research has arisen that, whilst continuing research at the atomic and molecular levels, aims at obtaining a more integrated view of protein function within the context of living organisms. Modern protein science therefore studies all levels of organization of life from single protein molecules, to large protein complexes promoting interactions within proteins or with small ligands and nucleic acids up to the study of the dynamic networks in cells and model organisms. For these reasons, an integrated Protein Science project is a challenging and highly interdisciplinary research domain extending from biophysics to chemistry and cell biology.
The overall aim of iPROS project is to promote an integrative protein science approach in order to increase our understanding of protein structure and function. In addition we will train young researchers to be able to lead and to develop top-level research in protein science. On the fundamental level, we will follow four major lines of research: (i) protein folding, (ii) protein engineering and protein-ligand interactions, (iii) protein targets against bacterial resistance and (iv) protein supramolecular assemblage and cellular metabolism. Each of these thematics will constitute a work package. To contribute to the study of these four lines in the most efficient way, collaboration has been established between the Belgian leading teams in Protein Science and complementary disciplines. These groups will share all the available and related technologies in molecular, structural and cellular biology, bacteriology, biophysics, bioinformatics, protein and membrane chemistry, enzymology, and medicinal and theoretical chemistry. The contribution of the foreign partners is particularly precious in the fields of biophysics, structural biology, bacterial cell wall metabolism and prokaryotic cellular regulation and development. The IAP partners use different, complementary approaches in their Protein Science research. Therefore, this network will generate cross-fertilization in the area of protein interaction studies, from which all partners will benefit. As such, a unique and generic instrument is created for studying protein interactions in Belgium. At cruise speed, students, research groups (and companies) in Belgium will benefit from this unique expertise in the field of protein interactions and folding.
Work packages description
Work package 1: Protein Folding (Partners: P1,P3, P6 and INT1)
Understanding the basic aspects of Protein Folding is crucial in describing many biological processes, ranging from transcription to molecular motors and diseases associated with misfolded proteins. Classical in vitro studies of model proteins, including enzymes from extremophiles will be pursued by using rapid-mixing techniques, in conjunction with optical spectroscopy techniques and NMR, to obtain a complete picture of the folding process. We will investigate the mechanisms underlying protein misfolding and amyloid fibril formation, which occur in various neurodegenerative and systemic amyloidoses, using several protein models. Transgene Caenorhabditis elegans strains expressing selected proteins will be created to study fibril formation and toxicity in vivo. We will also analyze how living organisms take advantage of the inherent ability of proteins to form such structures. One particularly well documented example of functional amyloid is that of curlin, whose fibrils are used by Escherichia coli and Salmonella to form biofilm and colonize non-biological surfaces. We will explore how the amyloidogenic subunits are transported and assembled at the outer membrane surface without cytotoxic effects. We will also investigate oxidative protein folding in the periplasm of Caulobacter crescentus, a favourite model to study the regulation of the cell cycle and cellular differentiation.
Work package 2: Protein engineering and protein-ligand interactions (Partners: P1, P2, P3, P4)
WP2 will combine biological, structural, chemical and combinatorial approaches to explore protein functions and molecular interactions. The first specific aim of WP2 will focus on improving existing proteins and enzymes to create properties such as affinity for a ligand, lasting effects and/or greater selectivity. In addition, we will develop and characterize hybrid proteins which are able to interact with different polysaccharides or display allosteric regulation. The second scope of WP2 will be dedicated to protein-ligand interactions such as metal ion-protein and lipid-protein interactions. We would focus on zinc/cadmium transporters of the Arabidopsis halleri P-type ATPase. Compared to their prokaryotic homologues, many plant metal transporters have acquired additional cytoplasmic domains which might be involved in metal binding, metal sensing and/or regulation of the protein activity. We propose to evaluate their metal-binding affinity and their potential structural changes upon metal ion binding. Furthermore, the signal transduction mechanism initiated by the presence of penicillin in culture media will be unravelled by deciphering the interactions between lipid and transmembrane domain of BlaR.
Work package 3: Protein targets against bacterial resistance (Partners: P1, P2, P3, P5, P6 and INT2)
WP3 aims at studying proteins identified as targets for combating bacteria and drug resistance, and developing chemical strategies and “lead compound” discovery approaches. The first main topic deals with proteins involved in the peptidoglycan (PG) biosynthesis. The C55-PP phosphatases catalyze the C55-PP dephosphorylation leading to the active form of the lipid carrier required for the translocation of the glycan units across the cytoplasmic membrane. Polymerization of the PG macromolecule is carried out by class A Penicillin-Binding Proteins (PBPs) via their transglycosylase (TGase) and transpeptidase (TPase) activities. A major bacterial resistance problem concerns resistance to -lactam antibiotics via the emergence of modified targets (resistant PBPs, rPBPs) and synthesis of -lactam destroying enzymes (-lactamases). Within the frame of IAP P6/19, tremendous work has already been done for the synthesis of antibiotics directed toward TGases, rPBPs and -lactamases with several potential lead compounds in the pipeline. In addition, new approaches will be envisaged, including synthesis of new molecular scaffolds, screening of peptide libraries and fragment based screening via X-ray crystallography.
Work package 4: Protein supramolecular assemblage and cellular metabolism (Partners : P1, P2, P4, P6, INT2 and INT3)
Proteins do not act as isolated entities. In a cellular context, they are strongly affected by permanent and transient interactions with other biomolecules. Moreover, their expression and activity is intensively regulated through connections in protein networks. Therefore, WP4 is devoted to gain insights in the assembly and mode of action of protein machineries and signaling networks that determine the cellular state. The main topics of WP4 are (1) the analysis of the interactions and the structural changes of the main E. coli divisome proteins in the membrane in order to have a dynamic picture of this multiprotein complex for determining the interactions which are essential for cell division and (2) the understanding of the signalling events and molecular mechanisms resulting from adaptation of organisms towards environmental stress leading to resistance, adaptation or transitions to different cell states (biofilms, sporulation, antibiotic production)
Work package 5: Networking and Dissemination (All partners)
The coordinator will be responsible for the general organisation of the network. To help the coordinator, a general scientific steering committee will be established, consisting of the WP leaders and one senior scientist from each partner. The steering committee will hold regular meetings. WP leaders will coordinate their own work package and will be responsible for the work that will be carried out. A subcommittee will assure the dissemination of the results by a website, newsletters, seminars and international meetings.
Work package 6: Training (All partners)
Particular attention will be given to the training of PhD students and early stage researchers. A training program will be organized to improve their career perspectives by broadening their scientific skills. They are also aimed to support experienced researchers in complementing or acquiring new skills and competencies. Besides the lectures already available in the different Graduate schools, the IAP consortium will organize original activities in the form of workshops, thematic weeks and seminars.
