Institute of Biochemistry, Charité-University Medicine Berlin, Corporate Member of Free University Berlin, Humboldt-University zu Berlin, Berlin Institute of Health, Berlin, Germany.
Faculty of Mathematics and Natural Sciences, University of Wuppertal, Wuppertal, Germany.
Antioxid Redox Signal. 2020 Jan 1;32(1):1-17. doi: 10.1089/ars.2019.7751.
Most mammalian genomes involve several genes encoding for functionally distinct arachidonate lipoxygenase (ALOX isoforms). Proinflammatory leukotrienes are formed the ALOX5 pathway, but 12/15-lipoxygenating ALOX isoforms have been implicated in the biosynthesis of pro-resolving mediators. mutagenesis of the triad determinants abolished the leukotriene synthesizing activity of ALOX5, but the biological consequences of these alterations have not been studied. To fill this gap, we created knock-in mice, which express the 12/15-lipoxygenating Phe359Trp + Ala424Ile + Asn425Met Alox5 triple mutant and characterized its phenotypic alterations. The mouse Alox5 triple mutant functions as arachidonic acid 15-lipoxygenating enzyme, which also forms 12S-hydroxy and 8S-hydroxy arachidonic acid. In contrast to the wild-type enzyme, the triple mutant effectively oxygenates linoleic acid to 13S-hydroxy linoleic acid (13S-HODE), which functions as activating ligand of the type-2 nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ). Knock-in mice expressing the mutant enzyme are viable, fertile, and develop normally. The mice cannot synthesize proinflammatory leukotrienes but show significantly attenuated plasma levels of lipolytic endocannabinoids. When aging, the animals gained significantly more body weight, which may be related to the fivefold higher levels of 13-HODE in the adipose tissue. These data indicate for the first time that mutagenesis of the triad determinants of mouse Alox5 abolished the biosynthetic capacity of the enzyme for proinflammatory leukotrienes and altered the catalytic properties of the protein favoring the formation of 13-HODE. triple mutation of the mouse gene impacts the body weight homeostasis of aging mice augmented formation of the activating PPARγ ligand 13-HODE.
大多数哺乳动物基因组涉及多个编码功能不同的花生四烯酸脂氧合酶（ALOX 同工型）的基因。促炎白三烯是由 ALOX5 途径形成的，但 12/15-脂氧合酶同工型已被牵连到促解决介质的生物合成中。三联体决定因素的突变使 ALOX5 的白三烯合成活性丧失，但这些改变的生物学后果尚未研究。为了填补这一空白，我们创建了表达 12/15-脂氧合酶的苯丙氨酸 359 色氨酸+丙氨酸 424 异亮氨酸+天冬氨酸 425 蛋氨酸 Alox5 三重突变体的基因敲入小鼠，并对其表型改变进行了特征描述。小鼠 Alox5 三重突变体作为花生四烯酸 15-脂氧合酶起作用，也形成 12S-羟基和 8S-羟基花生四烯酸。与野生型酶相比，三重突变体有效地将亚油酸氧化为 13S-羟基亚油酸（13S-HODE），后者作为 2 型核受体过氧化物酶体增殖物激活受体γ（PPARγ）的激活配体发挥作用。表达突变酶的基因敲入小鼠是可行的、有生育能力的，并且发育正常。这些小鼠不能合成促炎白三烯，但脂解内源性大麻素的血浆水平显著降低。随着衰老，动物体重显著增加，这可能与脂肪组织中 13-HODE 的五倍水平升高有关。这些数据首次表明，鼠 Alox5 的三联体决定因素的突变使酶合成促炎白三烯的能力丧失，并改变了蛋白质的催化特性，有利于 13-HODE 的形成。小鼠 基因的三重突变影响衰老小鼠的体重平衡，增加激活 PPARγ 配体 13-HODE 的形成。
