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The Biotransformation Of Endobiotics By Sulfonation

磺化内生素的生物转化

基本信息

批准号：
7734675
负责人：
Charles A. Strott
金额：
$ 72.78万
依托单位：
EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7734675
关键词：
7-ketocholesterol Affinity Alkanesulfonates Amino Acid Sequence Amino Acids Arterial Fatty Streak Ascorbic Acid Attenuated Bile Acids Binding Biological Calcium Catecholamines Cations Cell Surface Receptors Cell Survival Cell surface Cells Cholesterol Cholesterol Homeostasis Class Classification Complementary DNA Condition Connective Tissue CpG dinucleotide Culture Media Cultured Cells Cytokeratin filaments Cytoplasmic Granules Cytosine DNA Sequence Rearrangement DNA analysis Development Disease Elasticity Employee Strikes Enzymes Epidermis Excision Exhibits Exons External Granular Layer Extracellular Matrix Proteins Family Fatty Acids Gene Expression Gene Expression Regulation Gene Order Genes Genomics Glycosaminoglycans Golgi Apparatus Growth Growth and Development function Histidine Hormones Human Hydration status Hydrogen Peroxide Hydroxycholesterols In Vitro Investigation Lead Lesion Ligase Localized Location MCF7 cell Macular degeneration Maintenance Membrane Messenger RNA Metabolic Metabolic Biotransformation Metabolic Pathway Methylation Modification Molecular Molecular Weight Mus Nuclear Orphan Receptor Nuclear Receptors Pathway interactions Pharmaceutical Preparations Physiological Play Post-Translational Protein Processing Pregnenolone Process Production Promoter Regions Protein Dephosphorylation Protein Isoforms Proteins Proteoglycan Proteolysis RNA Splicing Range Rattus Recovery Regulation Relative (related person)Reporter Resistance Retinal Retinoids Role SULT2B1 Signal Transduction Skin Small Interfering RNA Steroids Sterols System Time Tissues Toxic effect Transfection Vitamin D Xenobiotics cell injury cholesterol sulfotransferase cholesteryl sulfate cytotoxic cytotoxicity filaggrin in vivo interest keratinocyte knock-down mRNA Expression macromolecule methyl group monomer non-genomic promoter secretory protein selective expression stratum corneum basic protein precursor sulfotransferase

项目摘要

The gene for human SULT2B1, as a result of an alternative exon 1 and differential splicing, encodes for two mRNAs, i.e. SULT2B1a and SULT2B1. The use of exon 1A produces SULT2B1a, whereas to produce SULT2B1b exon 1B plus a portion of exon 1A is required. While SULT2B1a avidly sulfonates the steroid pregnenolone, SULT2B1b is the physiologic cholesterol sulfotransferase. SULT2B1b is selectively expressed in a tissue-specific manner e.g. skin, whereas SULT2B1a is essentially globally silenced. DNA analysis revealed that the proximal promoter regions of both SULT2B1 isoforms contain multiple CpG dinucleotides where cytosines are subject to methylation. SULT2B1a and SULT2B1b promoters in human cells that do not express these isoforms, are hypermethylated. In contrast, the proximal promoter of SULT2B1b in keratinocytes that do highly express this isoform, is completely unmethylated. Removal of the methyl groups leads to a striking induction of expression, whereas in vitro methylation of SULT2B1a and SULT2B1b promoter/reporter constructs markedly reduces promoter activity after transfection. Thus, expression of the SULT2B1 isoforms is regulated, at least in part, by methylation of CpG dinucleotides in their proximal promoter regions and suggests an explanation for both the global silencing of SULT2B1a as well as the tissue-specific expression of SULT2B1b. Similar to human and mouse genes the rat SULT2B1 gene consists of an alternative exon I; however, as a result of exonic rearrangement, the genic locations of exons IA and IB are reversed in the rat gene. Where exon IA is located downstream of exon IB in human and mouse SULT2B1 genes, in the rat SULT2B1 gene, exon IA is located upstream of exon IB. Furthermore, unlike the case with human and mouse SULT2B1 genes where differential splicing is necessitated since a portion of exon IA is fused with exon IB to complete the SULT2B1b mRNA, this step is not required with the rat gene. Especially interesting concerning the rearrangement of the rat SULT2B1 gene is that there is not just a relocation of exon IA to be upstream of exon IB, which is the reverse of the situation in human and mouse genes, but that only that portion of exon IA encoding for the unique amino terminus of the SULT2B1a isoform is relocated. This is opportune for otherwise the SULT2B1b protein would sustain a substantial amino acid deletion rendering it inactive. The part of exon IA encoding for common amino acid sequence of the two isoforms remains in the same relative gene position as it is in the human and mouse genes and becomes exon II in the rat gene. Cholesterol sulfate, which binds with high affinity to the retinoid-related orphan nuclear receptor alpha (RORalpha), induces expression for the barrier protein, filaggrin (a relatively small histidine-rich basic protein that is derived from profilaggrin, a large precursor containing 10-12 identical copies of the mature filaggrin protein arranged in tandem; profilaggrin is extensively phosphorylated and packaged into granules, which protects profilaggrin from proteolysis until filaggrin is needed for the aggregation of keratin filaments; dephosphorylation exposes profilaggrin to proteolysis resulting in the release of filaggrin monomers) when added to primary cultures of human keratinocytes (NHEK). Furthermore, RORalpha, SULT2B1b (cholesterol sulfotransferase) and filaggrin co-localize to the outer granular layer of the human epidermis suggesting a functional relationship. NHEK undergo terminal differentiation when subjected to an increased calcium concentration in the medium and under these conditions SULT2B1b, filaggrin and RORalpha are induced in a similar manner and time frame. Association of RORalpha with filaggrin production was suggested when expression of the gene for RORalpha by NHEK was inhibited by 95% using siRNA, which resulted in a parallel reduction in the expression of profilaggrin mRNA by 80%; furthermore, adding cholesterol sulfate to the medium failed to produce a recovery in the expression of profilaggrin mRNA. Additionally, knocking down the gene for SULT2B1b also led to a reduction in profilaggrin mRNA expression; however, in this case, profilaggrin expression could be successfully restored following addition of cholesterol sulfate to the medium. These studies strongly suggest that cholesterol sulfate produced by the SULT2B1b activates the gene for profilaggrin and does so via an interaction with RORalpha. This is the first demonstration of a molecular action for cholesterol sulfate that is reminiscent of a typical hormone. Oxysterols constitute a class of cholesterol derivatives that exhibit broad biological effects ranging from cytotoxicity to regulation of nuclear receptors such as LXR, which is involved in the regulation of genes engaged in fatty acid and cholesterol metabolism. The role of oxysterols such as 7-ketocholesterol (7-KC) in the development of retinal macular degeneration and atheromatous lesions is of particular interest but little is known of their metabolic fate. A major oxysterol found in atheromas as well as other tissues is 7-KC, which is known from cell culture studies to induce cell injury at concentrations present in vivo, and for this reason, there exists a particular focus on metabolic pathways that can lead to a reduction in its toxicity. We established that the steroid/sterol sulfotransferase, SULT2B1b, known to efficiently sulfonate cholesterol, also effectively sulfonates a variety of oxysterols including 7- KC. The cytotoxic effect of 7-KC on 293T cells was attenuated when these cells, which do not express SULT2B1b, were transfected with SULT2B1b cDNA. Importantly, protection from 7-KC-induced loss of cell viability with transfection correlated with synthesis of SULT2B1b protein and production of the 7-KC sulfoconjugate (7-KCS). Moreover, when 7-KCS was added to the culture medium of 293T cells in amounts equimolar to 7-KC no loss of cell viability occurred. Additionally, MCF-7 cells, which highly express SULT2B1b, were significantly more resistant to the cytotoxic effect of 7-KC. We extended the range of oxysterol substrates for SULT2B1b to include 7alpha/7beta-hydroxycholesterol and 5alpha,6alpha/5beta,6beta-epoxycholesterol as well as the 7alpha-hydroperoxide derivative of cholesterol. Thus, SULT2B1b by acting on a variety of oxysterols offers a potential pathway for modulating in vivo the injurious effects of these compounds. Importantly, we have also demonstrated by physiochemical means for the first time that the sulfoconjugate of 7-KC does, indeed, occur in vivo as demonstrated for human atheromatous tissue. It now appears that oxysterols have a broad range of biological effects and that SULT2B1b plays a significant role throughout the spectrum. SULT2B1b not only inactivates classes of oxysterols that are cytotoxic, it also inactivates classes of oxysterols involved in cell signaling.

由于替代外显子 1 和差异剪接，人类 SULT2B1 基因编码两种 mRNA，即 SULT2B1a 和 SULT2B1。使用外显子 1A 产生 SULT2B1a，而要产生 SULT2B1b，需要外显子 1B 加上外显子 1A 的一部分。 SULT2B1a 强烈磺化类固醇孕烯醇酮，而 SULT2B1b 是生理性胆固醇磺基转移酶。 SULT2B1b 以组织特异性方式选择性表达，例如皮肤，而 SULT2B1a 基本上是全局沉默的。 DNA 分析显示，两种 SULT2B1 同工型的近端启动子区域均含有多个 CpG 二核苷酸，其中胞嘧啶会发生甲基化。不表达这些同种型的人类细胞中的 SULT2B1a 和 SULT2B1b 启动子是高度甲基化的。相比之下，高度表达该亚型的角质形成细胞中 SULT2B1b 的近端启动子完全未甲基化。甲基的去除会导致表达的显着诱导，而 SULT2B1a 和 SULT2B1b 启动子/报告基因构建体的体外甲基化会显着降低转染后的启动子活性。因此，SULT2B1同工型的表达至少部分地受到其近端启动子区域CpG二核苷酸甲基化的调节，并为SULT2B1a的整体沉默以及SULT2B1b的组织特异性表达提供了解释。与人类和小鼠基因类似，大鼠 SULT2B1 基因由一个替代外显子 I 组成；然而，由于外显子重排，外显子 IA 和 IB 的基因位置在大鼠基因中发生了逆转。在人和小鼠SULT2B1基因中，外显子IA位于外显子IB的下游，在大鼠SULT2B1基因中，外显子IA位于外显子IB的上游。此外，与人类和小鼠 SULT2B1 基因的情况不同，因为外显子 IA 的一部分与外显子 IB 融合以完成 SULT2B1b mRNA，因此需要差异剪接，而大鼠基因不需要此步骤。关于大鼠 SULT2B1 基因的重排特别有趣的是，不仅外显子 IA 重新定位到外显子 IB 的上游（这与人类和小鼠基因中的情况相反），而且只有编码 SULT2B1a 同工型独特氨基末端的外显子 IA 部分被重新定位。这是有利的，否则 SULT2B1b 蛋白将持续大量氨基酸缺失，从而使其失活。编码两种亚型的共同氨基酸序列的外显子IA部分保留在与人类和小鼠基因中相同的相对基因位置，并成为大鼠基因中的外显子II。硫酸胆固醇以高亲和力与类维生素A相关的孤儿核受体α（RORα）结合，诱导屏障蛋白丝聚合蛋白（一种相对较小的富含组氨酸的碱性蛋白，源自丝聚合蛋白原，丝聚合蛋白原是一种大型前体，含有串联排列的成熟丝聚合蛋白的10-12个相同拷贝；丝聚合蛋白原是广泛磷酸化并包装成颗粒，保护丝聚合蛋白原免受蛋白水解，直到需要丝聚合蛋白来聚集角蛋白丝；当添加到人角质形成细胞 (NHEK) 的原代培养物中时，去磷酸化会使丝聚合蛋白原暴露于蛋白水解，从而导致丝聚合蛋白单体的释放。此外，RORα、SULT2B1b（胆固醇磺基转移酶）和聚丝蛋白共定位于人表皮的外颗粒层，表明存在功能关系。当培养基中钙浓度增加时，NHEK 会经历终末分化，并且在这些条件下，SULT2B1b、聚丝蛋白和 RORalpha 以类似的方式和时间范围被诱导。当使用 siRNA 将 NHEK 的 RORα 基因表达抑制 95% 时，表明 RORα 与丝聚合蛋白产生相关，从而导致丝聚合蛋白原 mRNA 的表达同时减少 80%；此外，向培养基中添加硫酸胆固醇未能使聚丝蛋白原mRNA的表达恢复。此外，敲低 SULT2B1b 基因也会导致聚丝蛋白原 mRNA 表达减少。然而，在这种情况下，向培养基中添加硫酸胆固醇后，聚丝蛋白原的表达可以成功恢复。这些研究强烈表明，SULT2B1b 产生的硫酸胆固醇可激活聚丝蛋白原基因，并通过与 RORalpha 相互作用来实现这一目的。这是硫酸胆固醇分子作用的首次演示，它让人想起典型的激素。氧甾醇是一类胆固醇衍生物，具有广泛的生物效应，从细胞毒性到对核受体（如 LXR）的调节，LXR 参与脂肪酸和胆固醇代谢基因的调节。 7-酮胆固醇（7-KC）等氧甾醇在视网膜黄斑变性和动脉粥样硬化病变发展中的作用特别令人感兴趣，但对其代谢命运知之甚少。在动脉粥样硬化和其他组织中发现的一种主要氧甾醇是 7-KC，从细胞培养研究中得知，7-KC 在体内存在的浓度下会诱导细胞损伤，因此，人们特别关注可以降低其毒性的代谢途径。我们确定，已知能有效磺化胆固醇的类固醇/甾醇磺基转移酶 SULT2B1b 也能有效磺化包括 7-KC 在内的多种氧甾醇。当这些不表达SULT2B1b的细胞被SULT2B1b cDNA转染时，7-KC对293T细胞的细胞毒性作用减弱。重要的是，通过转染防止 7-KC 诱导的细胞活力丧失与 SULT2B1b 蛋白的合成和 7-KC 磺基缀合物 (7-KCS) 的产生相关。此外，当将7-KCS以与7-KC等摩尔的量添加到293T细胞的培养基中时，没有发生细胞活力的损失。此外，高度表达 SULT2B1b 的 MCF-7 细胞对 7-KC 的细胞毒性作用具有明显更强的抵抗力。我们扩展了 SULT2B1b 的氧化甾醇底物范围，包括 7α/7β-羟基胆固醇和 5α,6α/5β,6β-环氧胆固醇以及胆固醇的 7α-氢过氧化物衍生物。因此，SULT2B1b 通过作用于多种氧甾醇，提供了在体内调节这些化合物的有害作用的潜在途径。重要的是，我们还首次通过物理化学手段证明，7-KC 的磺基结合物确实存在于体内，正如人类动脉粥样硬化组织所证明的那样。现在看来，氧甾醇具有广泛的生物效应，而 SULT2B1b 在整个范围内发挥着重要作用。 SULT2B1b 不仅使具有细胞毒性的氧甾醇类失活，还使参与细胞信号传导的氧甾醇类失活。