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

脂氧合酶广泛存在于高等植物和哺乳动物中，但也零星存在于低等多细胞生物和细菌中。到目前为止，在archeae和病毒中没有检测到功能性ALOX同工型。该项目旨在探索ALOX异构体在不同领域陆地生命（病毒、细菌、哺乳动物）中的分布、生物学功能和进化关系。该项目应具体回答以下问题：i）ALOX同种型是否存在于病毒中？ii）铜绿假单胞菌的ALOX同种型作为铜绿假单胞菌感染的致病因子是否重要？iii）在灵长类进化过程中ALOX 15直系同源物的反应特异性是否存在系统性变化（ALOX 15特异性的进化概念，子项目3-5）？iv）反应特异性的体内人源化（敲入小鼠）是否影响ALOX 15和ALOX 15 B在动物炎症和凝血模型中的病理生理学作用。虽然ALOX同种型是水平基因转移的对象，但迄今为止在病毒中还没有描述功能性ALOX同种型。我们最近发现了一个假定的ALOX基因在mimivirus，并将测试是否该基因编码的功能ALOX-isoform. Pseudomonasaeruginosa（PA）表达的功能ALOX，但这种酶的病理生理作用尚未探讨。我们将检验这一假设，即这种分泌的蛋白质可能在PA患者中氧化宿主红细胞的膜脂质，反应特异性对于ALOX功能性是重要的，我们最近假设ALOX 15直向同源物的特异性在从花生四烯酸12-脂氧合的进化过程中被系统地改变（低等哺乳动物）到花生四烯酸15-脂氧合（高度发达的灵长类动物）。大多数哺乳动物ALOX 15直系同源物坚持这种情况，但兔酶是唯一已知的例外。超过120个哺乳动物ALOX 15直系同源物的预测性氨基酸序列比对表明了这一概念的有效性。35 ALOX 15直系同源物的反应特异性的表征较低（前兽，后兽）和较高（真兽）的哺乳动物将把这个进化的概念在更广泛的实验基础。15-脂氧合ALOX 15直系同源物表现出改善的合成能力，抗炎脂氧素相比，12-脂氧合直系同源物。因此，表达具有人源化反应特异性（15-脂氧合）的ALOX 15/ALOX 15 B直系同源物的小鼠应在实验性炎症模型中受到保护。我们将通过引入点突变（Crispr-Cas9技术）创建表现出12-脂氧合特性的ALOX 15和ALOX 15 B敲入小鼠，并将在两种不同的炎症（佐剂诱导的爪水肿，DSS-结肠炎）和一种凝血（尾尖截肢）模型中测试这些动物。

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.