Biochemical and hydrogen-deuterium exchange studies of the single nucleotide polymorphism Y649C in human platelet 12-lipoxygenase linked to a bleeding disorder.
Biochemical and hydrogen-deuterium exchange studies of the single nucleotide polymorphism Y649C in human platelet 12-lipoxygenase linked to a bleeding disorder.
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DOI：
10.1016/j.abb.2022.109472
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发表时间：
2023-01-01
影响因子：
3.9
通讯作者：
中科院分区：
文献类型：
--
作者：
人血小板12-脂氧合酶（h12-LOX）负责形成氧脂素产物，该产物在血小板聚集中起重要作用。h12-LOX的单核苷酸多态性（SNP）与多种疾病有关。在这项研究中，我们研究了h12-LOX SNP的结构、动力学和功能影响，该SNP在埋藏位点产生酪氨酸至半胱氨酸突变（Y 649 C h12-LOX），并且之前被归因于12（S）-羟基二十碳四烯酸水平降低（12 S-HETE）在分离的血小板中产生。在此，体外Michaelis-Menten动力学显示在生理或较低温度下，与WT h12-LOX相比，Y 649 C的催化速率降低。这两种蛋白质表现出相似的熔融温度，金属含量，和寡聚状态。两种蛋白质的脂质体结合也取决于钙的存在、温度和脂质体组成;然而，发现Y 649 C变体在生理温度下与WT相比具有降低的脂质体结合能力。此外，氢-氘交换质谱（HDX-MS）实验揭示了由突变引起的肽迁移率的区域确定的增强。突变的不稳定性增加源于与拱形螺旋相互作用的变化，拱形螺旋排列在距离突变位点≥15 μ m的底物结合位点上。最后，差示扫描量热法证明了降低的蛋白质（解）折叠焓，与HDX结果一致。总之，这些结果表明突变体和WT h12-LOX之间的显著相似性，然而，活性、膜亲和力和蛋白质稳定性的细微变化可能是Y 649 C SNP在血小板生物学中表现出的显著生理变化的原因。
Human platelet 12-lipoxygenase (h12-LOX) is responsible for the formation of oxylipin products that play an important role in platelet aggregation. Single nucleotide polymorphisms (SNPs) of h12-LOX have been implicated in several diseases. In this study, we investigate the structural, dynamical, and functional impact of a h12-LOX SNP that generates a tyrosine-to-cysteine mutation at a buried site (Y649C h12-LOX) and was previously ascribed with reduced levels of 12(S)-hydroxyeicosa-tetraenoic acid (12S-HETE) production in isolated platelets. Herein, in vitro Michaelis-Menten kinetics show reduced catalytic rates for Y649C compared to WT h12-LOX at physiological or lower temperatures. Both proteins exhibited similar melting temperatures, metal content, and oligomerization state. Liposome binding for both proteins was also dependent upon the presence of calcium, temperature, and liposome composition; however, the Y649C variant was found to have lowered binding capacity to liposomes compared to WT at physiological temperatures. Further, hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments revealed a regional defined enhancement in the peptide mobility caused by the mutation. This increased instability for the mutation stemmed from a change in an interaction with an arched helix that lines the substrate binding site, located ≥15 Å from the mutation site. Finally, differential scanning calorimetry demonstrated a reduced protein (un)folding enthalpy, consistent with the HDX results. Taken together, these results demonstrate remarkable similarity between the mutant and WT h12-LOX, and yet, subtle changes in activity, membrane affinity and protein stability may be responsible for the significant physiological changes that the Y649C SNP manifests in platelet biology.
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影响因子：
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作者：
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DOI：
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影响因子：
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