Defining aberrant steroid elimination in castration resistant prostate cancer

去势抵抗性前列腺癌中异常类固醇消除的定义

• 批准号: 8881763
• 负责人: JOSEPH J BARYCKI
• 金额: $ 18.24万
• 依托单位: UNIVERSITY OF NEBRASKA LINCOLN
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-05-19 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8881763
• 关键词: Address; Amino Acid Substitution; Amino Acids; Androgen Receptor; Androgens; Biological Assay; Biopsy; Cancer Patient; Cell membrane; Cell physiology; Cells; Chondroitin Sulfate Proteoglycan; Chondroitin Sulfates; Competitive Binding; Critical Pathways; Crosslinker; Data; Drug Metabolic Detoxication; Enzymes; Equilibrium; Excision; Excretory function; Fractionation; Future; Glucuronates; Glucuronides; Glucuronosyltransferase; Glycosaminoglycans; Goals; Golgi Apparatus; Hepatotoxicity; High Pressure Liquid Chromatography; Hormones; Human; Hyaluronan; Immunoblotting; Immunoprecipitation; In Vitro; LNCaP; Laboratories; Ligands; Malignant Neoplasms; Malignant neoplasm of prostate; Mass Spectrum Analysis; Measures; Mediating; Membrane; Metabolic; Modification; Molecular; Molecular Sieve Chromatography; Organelles; PC3 cell line; Pathway interactions; Phenotype; Post-Translational Protein Processing; Production; Prostate; Proteins; Proteoglycan; Regulation; Reporting; Role; Signal Pathway; Solutions; Steroids; Structure; Testing; Time; Translating; Tumor Cell Line; Uridine Diphosphate Glucose Dehydrogenase; Uridine Diphosphate Xylose; Variant; Xenobiotics; Xenograft procedure; cancer risk; cancer therapy; castration resistant prostate cancer; cell growth; crosslink; deprivation; design; dimer; enzyme activity; functional outcomes; glycosylation; hyaluronan synthase 1; improved; inhibitor/antagonist; lentivirally transduced; link protein; liquid chromatography mass spectrometry; meetings; mortality; mutant; neoplastic cell; notch protein; novel; preclinical study; prevent; prostate cancer cell; protein protein interaction; public health relevance; response; tumor progression

 DESCRIPTION (provided by applicant): UDP-glucose dehydrogenase (UGDH) is a unique, essential enzyme with the central role of providing UDP- glucuronate, a rate-limiting precursor for plasma membrane hyaluronan synthesis, Golgi proteoglycan production, and ER-localized modification of hormones for elimination. Our laboratory has shown that insufficiency of UGDH contributes to loss of control of intracellular steroid levels, and dysregulated tumor cell growth rate in prostate cancer. It is not known how the cytosolic UDP-glucuronate is partitioned to its respective fates in the high levels needed for specifically timed product formation, nor how UGDH activity is controlled to limit competition with other pathways. Our hypothesis for this proposal is that hexameric and dimeric units of UGDH sense metabolic status of the cell and respond with increased or decreased enzymatic activity. Information for molecular sensing is conveyed partly through differential protein-protein interactions that occur upon exposure of the dimer-dimer interface. We will test this with two aims. Aim 1: Determine the functional outcome of specific UGDH interactions with components of the androgen elimination pathway. We will directly measure interactions of UGDH with hyaluronan synthase, the Golgi UDP-xylose transporter, and the ER UDP-glucuronate transporter, as the three proteins that mediate the demands for UDP-glucuronate flux. We will quantify UDP-glucuronate, steroid-glucuronide, notch glycosylation and hyaluronan production, which will respectively report overall UGDH activity, and functional distribution to the ER for androgen elimination, the Golgi for proteoglyca secretion, or the plasma membrane for hyaluronan synthesis. Aim 2: Characterize and validate the UGDH interactome using an unbiased approach. We will use mass spectrometry to identify proteins that differentially co-fractionate by size exclusion chromatography with our well- characterized hexameric and dimeric point mutants. As a complementary approach, we will identify proteins that cross-link with hexameric versus dimeric UGDH point mutants that incorporate a photo-activatable crosslinker as a non-natural amino acid. Validated interactions and/or post-translational modifications will be used to design strategies for selective partitionin of UDP-glucuronate to favor hormone elimination in future preclinical studies of prostate cancer.

 描述(由申请人提供)：UDP-葡萄糖脱氢酶(UGDH)是一种独特的必需酶，其核心作用是提供UDP-葡萄糖醛酸，这是质膜透明质酸合成、高尔基蛋白多糖合成和激素内质网局部修饰的限速前体。我们的实验室已经证明，UGDH的不足导致前列腺癌细胞内类固醇水平的失控，以及肿瘤细胞生长速度的失调。目前尚不清楚胞质UDP-葡萄糖醛酸在特定时间产物形成所需的高水平下如何分配给各自的命运，也不知道UGDH活性如何受到控制以限制与其他途径的竞争。我们对这一提议的假设是，UGDH的六聚体和二聚体单位感知细胞的新陈代谢状态，并以酶活性的增加或减少做出反应。分子传感的信息部分是通过在二聚体-二聚体界面暴露时发生的不同蛋白质-蛋白质相互作用来传递的。我们将通过两个目标来测试这一点。目的1：确定UGDH与雄激素消除途径组分的特异性相互作用的功能结果。我们将直接测定UGDH与透明质酸合成酶、高尔基UDP-木糖转运体和ER UDP-葡萄糖醛酸转运体的相互作用，作为调节UDP-葡萄糖醛酸通量需求的三种蛋白质。我们将量化UDP-葡萄糖醛酸酸、类固醇-葡萄糖醛酸苷、缺口糖基化和透明质酸产量，它们将分别向ER报告总体UGDH活性和功能分布，以消除雄激素，高尔基体用于蛋白多糖分泌，或质膜用于透明质酸合成。目的2：用一种公正的方法描述和验证UGDH互动组。我们将使用质谱仪，通过尺寸排除层析与我们特征良好的六聚体和二聚体突变体鉴定差异共分的蛋白质。作为一种补充方法，我们将识别与六聚体UGDH点突变体和二聚体UGDH点突变体交联的蛋白质，这些突变体将光激活交联剂作为非天然氨基酸。经过验证的相互作用和/或翻译后修饰将被用来设计策略，在未来的前列腺癌临床前研究中选择性地分割UDP-葡萄糖醛酸，以有利于激素消除。