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

基本信息

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
项目类别：
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

项目摘要

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、高尔基体蛋白聚糖生产和内质网局部修饰的限速前体我们的实验室表明，UGDH 不足会导致细胞内类固醇水平失控，并导致肿瘤细胞生长失调。目前尚不清楚胞质 UDP-葡萄糖醛酸如何在特定时间产物形成所需的高水平上分配其各自的命运，也不知道如何控制 UGDH 活性以限制与其他前列腺途径的竞争。 UGDH 的六聚体和二聚体单位感知细胞的代谢状态，并响应酶活性的增加或减少，部分通过二聚体-二聚体界面暴露时发生的差异蛋白质-蛋白质相互作用传递信息。我们将测试这个有两个目标：确定特定 UGDH 与雄激素消除途径成分相互作用的功能结果我们将直接测量 UGDH 与乙酰透明质酸合酶、高尔基体 UDP-木糖转运蛋白和 ER UDP- 的相互作用。葡萄糖醛酸转运蛋白，作为介导 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、类固醇-葡萄糖醛酸苷、 Notch 糖基化和透明质酸生成，分别报告 UGDH 的整体活性，以及雄激素消除的 ER、蛋白聚糖分泌的高尔基体或透明质酸合成的膜质的功能分布目标 2：使用无偏倚表征和验证 UGDH 相互作用组。我们将使用质谱法来鉴定通过尺寸排阻色谱法与我们充分表征的六聚体进行差异共分级的蛋白质。作为一种补充方法，我们将鉴定与六聚体与二聚体 UGDH 点突变体交联的蛋白质，这些蛋白质将光激活交联剂作为非天然氨基酸进行验证。可用于设计选择性分配 UDP-葡萄糖醛酸的策略，以有利于未来前列腺癌临床前研究中激素的消除。