Purification and crystallization of human 12-LOX enzymes

人 12-LOX 酶的纯化和结晶

• 批准号: 6791645
• 负责人: Krishnarao Maddipati
• 金额: $ 10.07万
• 依托单位: BIOMIDE CORPORATION
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-01 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6791645
• 关键词: Escherichia coli; X ray crystallography; crystallization; enzyme activity; expression cloning; high performance liquid chromatography; lipoxygenase; microorganism culture; protein purification; protein quantitation /detection; spectrometry; technology /technique development

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.

描述（由申请人提供）：花生四烯酸通过12-脂氧合酶的代谢导致12（S）-羟基二十碳四烯酸的形成，其表现出深刻的生物活性，并在肿瘤细胞增殖、运动性、侵袭性、血管生成和细胞凋亡抑制中起重要作用，所有这些特性对于癌细胞的生长和转移都是必需的。花生四烯酸12-脂氧合酶以三种同种型存在，即，白细胞型、血小板型和表皮型。在这些亚型中，血小板型酶在许多类型的癌症中高度表达，并且在癌症病理生理学中起重要作用。在涉及122名患者的临床研究中，显示血小板型12-LOX mRNA表达的升高与人前列腺癌的晚期和低分化相关。鉴于血小板型12-脂氧合酶（因此12（S）-HETE）在癌症转移中的既定作用，从治疗观点来看，12-脂氧合酶的抑制是有吸引力的，并且同种型特异性12-脂氧合酶抑制剂将在化学预防治疗中提供重大突破。除了12-脂氧合酶外，还有两种其它主要的脂氧合酶，即，5-和15-脂氧合酶。所有的脂氧合酶都通过基本相同的机制催化多不饱和脂肪酸的氧化。基于植物脂氧合酶和人网织红细胞15-脂氧合酶的X射线晶体学研究的知识，很明显，这些酶的三维结构中的细微差异是负责氧化的区域特异性沿着脂肪酸链。虽然脂氧合酶的抑制剂已经存在了一段时间，但没有多少同工酶特异性抑制剂可用，并且开发此类抑制剂的努力受到了这些酶结构信息缺乏的严重阻碍。我们的长期目标是通过推导白细胞和血小板型12-脂氧合酶的三维结构来弥合这一信息差距，以开发血小板型12-脂氧合酶特异性抑制剂作为潜在的抗癌药物。在这个建议中，我们的目标是从细菌表达克隆中纯化多毫克量的两种酶，使其电泳均匀，并筛选几种方法以获得适合X射线晶体学研究的晶体。