Abstract [en]
This project is a blend of synthetic organic chemistry, bioorganic chemistry and inorganic chemistry within a frame of chemical biology, to create new chemical tools for the specific enrichment and detection of nucleobase mono-phosphate modifications (NMP) on host cell proteins in intracellular bacterial infection. During infection, intracellular pathogens like Legionella, Chlamydia, Coxiella and others, take control over the host cell at all levels to thrive and replicate. Secreted bacterial virulence factors with NMP-transfer capability (fic-domains) are essential for establishment of infection. NMP-modifications on host proteins are poorly characterized ? and often found by serendipity - since no methods exist for the systematic enrichment and detection of native NMPs. We have developed synthetic methods for generation of homogeneous NMP-peptide material and employed it in development of mass spectrometry methods and polyclonal antibodies (AB?s) via immunization in rabbit. Our vision was that the AB?s would be the ultimate tool for immunoprecipitation of NMP-proteins from complex samples - however - our AB?s have only performed well in the detection of NMP-proteins, not for specific enrichment. There is an urgent need for new chemical methods to address NMPs on proteins, with a focus on enrichment, to understand bacterial intracellular infection in detail. From use of our AB?s for detection (WB), we know that specific NMP (AMP, GMP) modifications on host cell proteins are abundant in some infections (Chlamydia, Francisella, Coxiella) ? which is far ahead of current state-of-the-art knowledge. We see a clear niche, a specific application and an absolute need for the development of new affinity reagents against protein bound NMPs - which would allow us to answer important biological questions at highest international level. In this work program, which is intended to span 4 years and employ one full postdoc equivalent (2x2 years each, 100% funded) and one PhD student (50% funded) we will create a verified chemical toolbox for the enrichment and detection of NMPs on proteins. The work program consists of four parts (A-D), where (A) covers year 1-2,5; (B) year 2-3; (C) year 3-4 and (D) year 4. In (A) ? which is the basis and starting point of the project - we will design and synthetize metal-based affinity reagents for the four most common NMPs (AMP, GMP, CMP, UMP) bound to protein side chains (Ser, Thr, Tyr) as model peptides. Here, we will make use of the affinity of AMP to Ru(III)-EDTA-type complexes, for GMP to Pt(II)-diamine complexes, and for CMP and UMP to binuclear complexes (Mn, Co, Ru, Pt) to make up for bidentate interactions with the heteroatoms of the nucleobase. In all cases, the ligand of the complex will be connected to a reporter (biotin or fluorophore). In part B, we will quantitatively evaluate the affinity of the complexes from part A on synthetic peptides carrying the NMP-modifications via complementary fluorophores for FRET and improve affinities in an iterative way. In part C, the affinity reagents from A - optimized in B - will be evaluated against protein bound NMPs. We can enzymatically attach all four NMPs to human Rab1, which is the model protein. The different NMPs will be mass encoded with protein tags, thus allowing easy detection via in gel detection though molecular weight. Here, we will start with isolated NMP-Rab1´s and when verified, we will move to complex matrixes, like cell lysates and validate the system, employing our NMP-Rab1?s as spike-ins in pulldown experiments. When optimized though step B and C, we will subject the affinity reagents to application ? Part D ? where we will enrich NMP-modified proteins from cell lines infected with Chlamydia, Legionella and Francisella and identify the proteins as well as the NMP?s with mass spectrometry. Here, Legionella is our internal control, since we know that human Rab1 carries AMP upon infection. With a positive result in hand, the generated