Macrophages are a key type of immune cell that fights disease-causing microorganisms, such as bacteria. They act by 1) directly fighting the infection by forming a hostile environment to kill the pathogen and 2) producing molecules to alert other immune cells to the danger and ultimately create an inflammatory response. As well as adopting this "killing" state, macrophages can also adopt a "repairing" state, in which they initiate events to reduce inflammation, resolve infection and repair tissues damaged during the inflammatory response. The process by which macrophages adopt either state, known as macrophage polarisation, requires communication within a cell, which is referred to as cell signalling. Proteins are large molecules that carry out critical functions in our cells, from chemical reactions (enzymes) to the regulation of transcription. Transcription makes RNA from the hereditary material (DNA) contained within the cell and can be controlled by proteins called transcription factors. RNA is then the code to make new proteins. Every protein is made up by a unique string of smaller building blocks called amino acids. The sequence of amino acids determines the 3-dimensional structure and function of a protein. During cell signalling, protein modification by small chemical groups can increase, decrease or change their function. During phosphorylation, something called a phosphoryl group is added to specific amino acids of the protein. This reaction is carried out by enzymes called kinases. Some kinases only modify specific types of amino acids called serine and threonine amino acids whereas others can also modify the amino acid tyrosine. Disease-causing bacteria, like Salmonella, use their own proteins to interfere with host cell signalling and thereby host immunity. We have recently found that a protein called SteE, delivered from Salmonella into macrophages, binds a host kinase that normally only modifies serine and threonine amino acids. When together with SteE the kinase now modifies a tyrosine amino acid on a new target, which is a transcription factor. Ultimately, this prompts macrophages to inappropriately adopt the "repair" state rather than the "killing" state. This promotes Salmonella survival and long-term persistence inside the host. This project will 1) define the changes in macrophage DNA transcription mediated by SteE during Salmonella-infection and test whether additional host proteins are required to instruct the "Salmonella-friendly" state of macrophages. 2) Investigate host changes in small molecules (metabolites) during Salmonella infection. 3) Study how the 3D arrangement of SteE and the host kinase are altered in order to allow novel substrates to be modified. Collectively, these findings will reveal the mechanism of how the Salmonella protein SteE promotes disease and provide valuable insight into host immune processes. Salmonella is a major human health challenge; causing a wide range of diseases in humans, from self-limiting diarrhoeal disease, to typhoid fever, a life-threatening systemic disease. Our findings will enable us to gain profound understanding on the pathogenesis of a global, disease-causing bacterium. Ultimately, this may promote the development of novel ways to combat bacterial infections, something which is of vast importance with the rise of antibiotic-resistant bacterial strains.

Macrophages are a key type of immune cell that fights disease-causing microorganisms, such as bacteria. They act by 1) directly fighting the infection by forming a hostile environment to kill the pathogen and 2) producing molecules to alert other immune cells to the danger and ultimately create an inflammatory response. As well as adopting this "killing" state, macrophages can also adopt a "repairing" state, in which they initiate events to reduce inflammation, resolve infection and repair tissues damaged during the inflammatory response. The process by which macrophages adopt either state, known as macrophage polarisation, requires communication within a cell, which is referred to as cell signalling. Proteins are large molecules that carry out critical functions in our cells, from chemical reactions (enzymes) to the regulation of transcription. Transcription makes RNA from the hereditary material (DNA) contained within the cell and can be controlled by proteins called transcription factors. RNA is then the code to make new proteins. Every protein is made up by a unique string of smaller building blocks called amino acids. The sequence of amino acids determines the 3-dimensional structure and function of a protein. During cell signalling, protein modification by small chemical groups can increase, decrease or change their function. During phosphorylation, something called a phosphoryl group is added to specific amino acids of the protein. This reaction is carried out by enzymes called kinases. Some kinases only modify specific types of amino acids called serine and threonine amino acids whereas others can also modify the amino acid tyrosine. Disease-causing bacteria, like Salmonella, use their own proteins to interfere with host cell signalling and thereby host immunity. We have recently found that a protein called SteE, delivered from Salmonella into macrophages, binds a host kinase that normally only modifies serine and threonine amino acids. When together with SteE the kinase now modifies a tyrosine amino acid on a new target, which is a transcription factor. Ultimately, this prompts macrophages to inappropriately adopt the "repair" state rather than the "killing" state. This promotes Salmonella survival and long-term persistence inside the host. This project will 1) define the changes in macrophage DNA transcription mediated by SteE during Salmonella-infection and test whether additional host proteins are required to instruct the "Salmonella-friendly" state of macrophages. 2) Investigate host changes in small molecules (metabolites) during Salmonella infection. 3) Study how the 3D arrangement of SteE and the host kinase are altered in order to allow novel substrates to be modified. Collectively, these findings will reveal the mechanism of how the Salmonella protein SteE promotes disease and provide valuable insight into host immune processes. Salmonella is a major human health challenge; causing a wide range of diseases in humans, from self-limiting diarrhoeal disease, to typhoid fever, a life-threatening systemic disease. Our findings will enable us to gain profound understanding on the pathogenesis of a global, disease-causing bacterium. Ultimately, this may promote the development of novel ways to combat bacterial infections, something which is of vast importance with the rise of antibiotic-resistant bacterial strains.

Parkinson's Disease (PD) is the second most common neurodegenerative disorder worldwide and no treatment is currently available to halt the onset and/or progression of PD pharmacologically. The kinase-activating G2019S mutations in Leucine-Rich Repeat Kinase 2 (LRRK2) is one of the most common genetic causes of PD and has motivated work to develop LRRK2-targeted therapies, including LRRK2 kinase inhibitors. However, current LRRK2 kinase inhibitors, albeit in clinical trials, promote LRRK2 and microtubule association, underlying potential undesirable side effects. Alternative LRRK2-targeting strategy, known as LRRK2 Proteolysis Targeting Chimera (PROTAC) is therefore proposed. LRRK2 PROTACs are heterobifunctional small molecules that consist of a ligand that binds LRRK2, conjugated to a ligand that binds an E3 ubiquitin ligase via a linker. By recruiting a E3 ubiquitin ligase in close proximity to a LRRK2 protein, LRRK2 PROTACs can induce the ubiquitination and subsequent degradation of LRRK2 through the ubiquitin-proteasome pathway. By designing and synthesizing a few sets of LRRK2 PROTAC compounds and screening them with degradation assays on multiple cell lines, we identified potent LRRK2 PROTACs that degraded LRRK2 at nanomolar range concentrations. The goal of this fellowship is to further improve the potencies of these LRRK2 PROTACs through structural modification and qualify them as chemical probes and potential lead compounds for the treatment of PD by detailed in vitro and in vivo characterization and PD-related biology studies.

Modern life generates enormous amounts of plastic waste: 359 million tons of plastics are produced annually worldwide, of which 90% is produced from fossil fuels and 79% accumulates in landfill or in the natural environment. Collectively all these plastics create an environmental hazard. Furthermore, we are losing valuable materials that could be recycled. As Nature did not encounter plastics for most of its evolutionary history, plastic-degrading enzymes with a metabolic role did not exist. However, recent research into communities of bacteria from oceans and wastewater has shown that over the last 50 years some bacteria have evolved enzymes that can exploit this new nutrient. These plastic degrading enzymes, some of which are known as PETases (as they degrade polyesters, PETs), are not very efficient but represent an exciting starting point to discover and engineer more effective enzymes. Furthermore, international 'metagenome' efforts have been capturing vast amounts of bacterial genomic data from these natural environments, which are now available as resources like MGnify at the European Bioinformatics Institute (EBI). In this project we will use bioinformatics to harvest enzymes from these massive metagenomic databases, by classifying them into functional and structural classes with useful 'promiscuous' chemical activities. We will use state-of-the-art artificial intelligence (AI) and machine learning (ML) tools to do this, proven to classify families of proteins with high functional similarity. Putative novel plastic-degrading enzymes identified by this approach will be further analysed by ML tools which screen for predicted solubility. We will also perform chemical studies to assess improvements in enzyme activity, compared to the existing, inefficient, PETases. Any putative plastic-degrading enzymes will then provide a starting point for directed evolution experiments where we select new variants of the enzymes with improved properties. To best explore evolution of plastic degrading ability we will use our unique ultrahigh-throughput assay for particle breakdown, with a throughput of over 10 million clones per day. We can thus directly assess the ability of enzymes to chemically act on plastic particles (rather than substrates that only mimic plastics). This will revolutionise the field of enzymatic plastic degradation, because so far only marginal improvements have been possible using proxy substrates. In addition to efficient screening, the analysis of the output sequences of screening will be fed back into our bioinformatic analyses and target selection. We will also structurally characterise these enzymes to discover how changes in their functional sites have improved their ability to bind and digest plastics. This data will provide detailed insights on how protein sites can diverge and evolve better plastic degrading properties, thus improving our in silico selection protocol. We have performed pilot work on PETases and will build on this and extend to other plastic degrading enzymes (plastizymes). This close integration of 'dry' data science and 'wet' experimental work results in powerful cycles of in silico analysis, experimental tests and refinement of analysis tools that are more powerful than current small scale protein engineering campaigns. The project thus addresses one of the most important (and also most difficult) environmental challenges, but more generally, also provides a paradigm to demonstrate how an interdisciplinary approach can accelerate evolution in cases where no effective natural enzyme is available. If successful, this paradigm would form the basis not just for the 'rules of life' (as mentioned in the call text), but for 'rules beyond life' (as it exists now), targeted to address the future needs of our society.

United States