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）：12-脂氧合酶对蛛网膜酸的代谢导致形成12（S） - 羟基二羟基二十二烯酸酸，它表现出深刻的生物学活性，并且在肿瘤细胞的增殖，运动性，无敌性，象征性，血管生成，血管生成和癌细胞的癌症中都具有重要的癌症和抑制作用。三种同工型，即白细胞型，血小板类型和表皮型中存在三种同工型中。在这些同工型中，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。在一项涉及122例患者的临床研究中，结果表明，血小板型12-LOX mRNA表达的升高与人类前列腺癌的晚期阶段和不良分化相关。鉴于血小板型12-脂氧合酶的确定作用，因此在癌症转移中12（s） - hete，从治疗的角度来看，抑制12-脂氧合酶在化学治疗中可以为化学治疗带来重大突破。除12-脂氧酶外，还有另外两个主要的脂氧酶，即哺乳动物起源的5-和15-脂氧酶。所有脂氧酶都通过基本相同的机制催化多不饱和脂肪酸的氧化。根据植物脂氧酶和人网状细胞15-二氧合酶的X射线晶体学研究的知识，很明显，这些酶的三维结构的细微差异是沿脂肪酸链氧化的氧化造成的。虽然已经有一段时间的脂氧酶抑制剂可用，但没有太多的同工酶特异性抑制剂可用，开发此类抑制剂的努力受到这些酶的结构信息的匮乏，从而严重阻碍了这些抑制剂。我们的长期目标是通过推断白细胞和血小板型12-脂氧酶的三维结构来弥合这一信息差距，以在我们尝试开发血小板型12-脂氧合酶特异性抑制剂作为潜在抗癌药物的尝试中。在此提案中，我们旨在将两种酶从细菌表达克隆到电泳同质性纯化，并筛选几种方法以获取适合X射线晶体学研究的晶体。