Defining aberrant steroid elimination in castration resistant prostate cancer

 描述（由申请方提供）：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，UDP-葡萄糖醛酸是质膜透明质酸合成、高尔基体蛋白聚糖产生和激素ER定位修饰的限速前体，用于消除。我们的实验室已经表明，UGDH不足导致细胞内类固醇水平失控，以及前列腺癌中肿瘤细胞生长速率失调。目前尚不清楚胞质UDP-葡糖醛酸盐如何以特定时间产物形成所需的高水平分配至其各自的命运，也不知道UGDH活性如何控制以限制与其他途径的竞争。我们对此建议的假设是，UGDH的六聚体和二聚体单位感测细胞的代谢状态，并以增加或减少的酶活性作出反应。分子传感的信息部分地通过暴露于二聚体-二聚体界面时发生的差异蛋白质-蛋白质相互作用来传达。我们将以两个目标来检验这一点。目的1：确定特异性UGDH与雄激素消除途径组分相互作用的功能结果。我们将直接测量UGDH与透明质酸合酶、高尔基UDP-木糖转运蛋白和ER UDP-葡萄糖醛酸转运蛋白的相互作用，这三种蛋白质介导UDP-葡萄糖醛酸通量的需求。我们将定量UDP-葡萄糖醛酸、类固醇-葡萄糖醛酸苷、notch糖基化和透明质酸的产生，分别报告总体UGDH活性，以及雄激素消除的ER、蛋白多糖分泌的高尔基体或透明质酸合成的质膜的功能分布。目的2：使用无偏方法表征和验证UGDH相互作用组。我们将使用质谱鉴定通过尺寸排阻色谱与我们充分表征的六聚体和二聚体点突变体差异共断裂的蛋白质。作为一种补充方法，我们将鉴定与六聚体相对于二聚体UGDH点突变体交联的蛋白质，所述点突变体掺入可光活化的交联剂作为非天然氨基酸。经验证的相互作用和/或翻译后修饰将用于设计UDP-葡萄糖醛酸选择性分配的策略，以在未来的前列腺癌临床前研究中促进激素消除。

 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.