Purification and crystallization of human 12-LOX enzymes

描述(申请人提供)：花生四烯酸在12-脂氧合酶作用下代谢生成12(S)-羟基二十碳四烯酸，它具有深刻的生物活性，在肿瘤细胞的增殖、移动、侵袭、血管生成和抑制凋亡方面发挥重要作用，所有这些特性对癌细胞的生长和转移都是必不可少的。花生四烯酸12-脂氧合酶有三种亚型，即白细胞型、血小板型和表皮型。在这些异构体中，血小板类型酶在许多类型的癌症中都高度表达，在癌症的病理生理学中发挥着重要作用。在一项涉及122名患者的临床研究中，研究表明，血小板型12-LOX mRNA表达升高与前列腺癌的晚期和低分化有关。鉴于血小板型12-脂氧合酶，即12(S)-HETE在肿瘤转移中的作用已被证实，从治疗的角度来看，抑制12-脂氧合酶是有吸引力的，而特异性的12-脂氧合酶抑制剂将在化学预防治疗方面提供重大突破。除了12-脂氧合酶外，哺乳动物还有另外两种主要的脂氧合酶，即5-脂氧合酶和15-脂氧合酶。所有脂肪氧合酶催化多不饱和脂肪酸氧化的机制基本相同。根据对植物脂肪氧合酶和人类网织红细胞15-脂氧合酶的X射线结晶学研究，很明显，这些酶的三维结构上的细微差异是导致脂肪酸链上氧化的区域特异性的原因。虽然脂氧合酶的抑制剂已经存在一段时间了，但同工酶特异性的抑制剂并不多，而且由于缺乏关于这些酶的结构信息，开发这种抑制剂的努力受到了严重的阻碍。我们的长期目标是通过推断白细胞和血小板型12-脂氧合酶的三维结构来弥合这一信息鸿沟，试图开发血小板型12-脂氧合酶特异性抑制剂作为潜在的抗癌药物。在这个方案中，我们的目标是从细菌表达克隆到电泳均一，纯化几毫克量的这两种酶，并筛选出几种方法来获得适合X射线结晶学研究的晶体。

DESCRIPTION (provided by applicant): Metabolism of arachidonic acid by 12-lipoxygenase results in the formation of 12(S)-hydroxy eicosatetraenoic acid, which exhibits profound biological activity and plays an important role in tumor cell proliferation, motility, invasiveness, angiogenesis, and inhibition of apoptosis, all properties essential for the growth and metastasis of cancer cells. Arachidonate 12-lipoxygenase exists in three isoforms, viz., leukocyte-type, platelet-type, and the epidermis-type. Of these isoforms the platelet-type enzyme is highly expressed in many types of cancers and plays an important role in cancer pathophysiology. In a clinical study involving 122 patients, it was shown that elevation of platelet-type 12-LOX mRNA expression correlates with advanced stage and poor differentiation of human prostate cancer. Given the established role of platelet-type 12-lipoxygenase, hence 12(S)-HETE, in cancer metastasis, inhibition of 12-lipoxygenase is attractive from a therapeutic standpoint and isoform-specific 12-lipoxygenase inhibitors would provide a major breakthrough in chemopreventive therapy. In addition to the 12-lipoxygenase, there are two other major lipoxygenases, viz., 5- and 15-lipoxygenases, of mammalian origin. All lipoxygenases catalyze the oxidation of polyunsaturated fatty acids by an essentially identical mechanism. Based on the knowledge from the X-ray crystallographic studies of plant lipoxygenases and human reticulocyte 15-lipoxygenase, it is evident that subtle differences in the three-dimensional structures of these enzymes are responsible for the regiospecificity of the oxidation along the fatty acid chain. While inhibitors for lipoxygenases have been available for some time, not many isozyme-specific inhibitors are available and efforts to develop such inhibitors have been severely hampered by the paucity of structural information on these enzymes. Our long-term objective is to bridge this information gap by deducing the three-dimensional structures of leukocyte and platelet-type 12-lipoxygenases in our attempts to develop platelet-type 12-lipoxygenase-specific inhibitors as potential anticancer agents. In this proposal, we aim to purify multi milligram quantities of both enzymes from bacterial expression clones to electrophoretic homogeneity and screen several methods to obtain crystals suitable for X-ray crystallographic studies.