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

脂氧合酶的分布、生物学作用和进化的研究。

• 批准号: 405976130
• 负责人: Dr. Dagmar Heydeck
• 金额: --
• 依托单位: Institut für Biochemie (CCM)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/405976130?language=en
• 关键词: Investigations; into; Distribution; Biological; Role

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.

脂肪氧合酶(ALOX)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体和细菌中。到目前为止，在古细菌和病毒中还没有检测到具有功能的ALOX亚型。本项目旨在探索ALOX亚型在不同地区陆地生命(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-亚型是否存在于病毒中？Ii)铜绿假单胞菌的ALOX异构体是铜绿假单胞菌感染的重要致病因子吗？3)在灵长类动物进化过程中，ALOX15同源基因的反应特异性是否有系统性的变化(ALOX15特异性的进化概念，子项目3-5)？IV)体内反应特异性的人源化(敲入小鼠)是否影响ALOX15和ALOX15B在动物炎症和凝血模型中的病理生理作用。虽然ALOX-亚型是水平基因转移的一个主题，但到目前为止还没有在病毒中描述有功能的ALOX-亚型。我们最近在一个模拟病毒中发现了一个假定的ALOX基因，并将测试该基因是否编码一个功能性ALOX亚型。铜绿假单胞菌(PA)表达一个功能性ALOX，但该酶的病理生理作用尚未被探索。我们将测试这一分泌蛋白可能氧化PA患者宿主红细胞膜脂质从而可能有助于发病的假设。反应特异性对于ALOX功能非常重要，我们最近假设ALOX15同源基因的特异性在从花生四烯酸12-脂氧合(低等哺乳动物)到花生四烯酸15-脂氧合(高度发育的灵长类)的进化过程中发生了系统的变化。大多数哺乳动物的ALOX15同源基因都遵循这种情况，但兔酶是唯一已知的例外。120多个哺乳动物ALOX15同源基因的预测氨基酸序列比对表明了这一概念的有效性。对35个低等(异形、偏热)和高等(优热)哺乳动物的ALOX15同源物的反应特异性的表征将把这一进化概念置于更广泛的实验基础上。与12-脂氧合同源物相比，15-脂氧合ALOX15同源物显示出更好的抗炎脂氧素合成能力。因此，表达具有人源化反应特异性(15-脂氧合)的ALOX15/ALOX15B同源基因的小鼠，应该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)来创建表现出12-脂氧合特性的ALOX15和ALOX15B敲入小鼠，并将在两种不同的炎症(佐剂诱导的爪状肿胀，DSS结肠炎)和一种凝血(尾端截肢)模型中测试这些动物。