Investigations into the Distribution, the Biological Role and the Evolution of Lipoxygenases. Functional Charcterization of Genetically Modified Mice with Humanized Reaction Specificity.

• Applicants: Dr. Dagmar Heydeck; Professor Dr. Hartmut Kühn
• Subject Area: Biochemistry ; Developmental Biology
• Term: from 2018 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 405976130

Lipoxygenases (ALOXs) are widely distributed in higher plants and mammals but they also occur sporadically in lower multicellular organisms and in bacteria. No functional ALOX isoforms have been detected so far in archeae and viruses. This project is aimed at exploring the distribution, the biological function and the evolutionary relations of ALOX-isoforms in different areas terrestrial life (viruses, bacteria, mammals). The project should specifically answer the following questions: i) Do ALOX-isoforms occur in viruses? ii) Is the ALOX-isoform of Pseudomonas aeruginosa important as pathogenicity factor for P. aeruginosa infections? iii) Was there a systematic change in the reaction specificity of ALOX15 orthologs during primate evolution (evolutionary concept of ALOX15 specificity, subprojects 3-5)?iv) Does in vivo humanization of the reaction specificity (knock-in mice) impact the pathophysiological role of ALOX15 and ALOX15B in animal inflammation and coagulation models. Although ALOX-isoforms are a subject of horizontal gene transfer no functional ALOX-isoforms have been described so far in viruses. We recently identified a putative ALOX gene in a mimivirus and will test whether this gene encodes for a functional ALOX-isoform.Pseudomonas aeruginosa (PA) expresses a functional ALOX but the patho-physiological role of this enzyme has not been explored. We will test the hypothesis that this secreted protein might oxidize in PA patients the membrane lipids of host erythrocytes and thus, may contribute to pathogenesis.The reaction specificity is important for ALOX functionality and we have recently hypothesized that the specificity of ALOX15 orthologs was systematically altered during evolution from arachidonic acid 12-lipoxygenation (lower mammals) to arachidonic acid 15-lipoxygenating (highly developed primates). Most mammalian ALOX15 orthologs adhere to this scenario but the rabbit enzyme is the only known exception. Predictive amino acid sequence alignments of more than 120 mammalian ALOX15 orthologs suggested the validity of this concept. Characterization of the reaction specificity of 35 ALOX15 orthologs of lower (protheria, metatheria) and higher (eutheria) mammals will put this evolutionary concept on a broader experimental basis.15-lipoxygenating ALOX15 orthologs exhibit an improved synthetic capacity for anti-inflammatory lipoxins when compared 12-lipoxygenating orthologs. Thus, mice expressing ALOX15/ALOX15B orthologs with humanized reaction specificity (15-lipoxygenating), should be protected in experimental inflammation models. We will create ALOX15 and ALOX15B knock-in mice exhibiting 12-lipoxygenating properties by introducing point mutations (Crispr-Cas9 technology) and will test these animals in two different inflammation (adjuvans-induced paw edema, DSS-colitis) and one coagulation (tail tip amputation) model.

• DFG Programme: Research Grants