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（由适用提供）：UDP-葡萄糖脱氢酶（UGDH）是一种独特的，必不可少的酶，其核心作用是提供UDP-葡萄糖醛酸，这是质膜膜的质量限制前体，用于质膜膜的HydraRoNan综合，Golgi Proteoglycan生产，以及磨性的Hormized Hormentized Hormentized Hormentific for Hormentife hormitification for Hormitification for hermitification for hormitification。我们的实验室表明，UGDH的不足导致了对细胞内立体水平的控制丧失，并且前列腺癌的肿瘤细胞生长速率失调。尚不清楚胞质UDP-葡萄糖醛酸是如何在特定定时产物形成所需的高水平上分配给其相对命运的，或者如何控制UGDH活性以限制与其他途径的竞争。我们对该提案的假设是，UGDH感官代谢状态的六聚体和二聚体单位，并随着酶活性的增加或减少做出反应。分子传感的信息通过在暴露于二聚体二聚体界面时发生的差异蛋白 - 蛋白质相互作用来传达。我们将以两个目标进行测试。目标1：确定特定UGDH相互作用与雄激素演化途径的组件的功能结果。我们将直接测量UGDH与Hydouronan合酶，Golgi UDP- Xylose Transporter和ER UDP-葡萄糖醛酸转运蛋白的相互作用，作为介导UDP-葡萄糖醛酸碳氧酸盐需求的三种蛋白质。我们将量化UDP-葡萄糖醛酸酯，类固醇 - 葡萄糖醛酸，Notch糖基化和透明质酸的产生，它们将分别报告整体UGDH活性，以及对ER的功能分布，用于ER的雄激素消除，GOLGI，蛋白质CaS proteoglyca分泌的GOLGI，或用于Yyaluronuronan hyaluronaran consathessessessess的plasmamemmmmmmmmmmembrane。 AIM 2：使用公正的方法来表征和验证UGDH Interactome。我们将使用质谱法来鉴定通过大小排除色谱法与我们特征良好的六聚体和二聚体点突变体的蛋白质不同。作为一种完整的方法，我们将鉴定与六聚体与二聚体UGDH点突变体交联的蛋白质，该突变体将可相动的交联链链接作为非天然氨基酸。经过验证的相互作用和/或翻译后修饰将用于设计UDP-葡萄醛酸酯选择性分区的策略，以在前列腺癌的未来临床前研究中偏爱霍斯奈人的排放。