The Biotransformation Of Endobiotics By Sulfonation

磺化内生素的生物转化

• 批准号: 7594118
• 负责人: Charles A. Strott
• 金额: $ 67.21万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 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/7594118
• 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

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.

人类Sult2B1的基因是替代外显子1和差异剪接的结果，编码了两个mRNA，即SULT2B1A和SULT2B1。外显子1a的使用产生sult2b1a，而生产Sult2B1B外显子1B则需要外显子1a的一部分。尽管Sult2B1A狂热地将类固醇孕酮磺化，而Sult2B1b是生理胆固醇磺胺硫代转移酶。 Sult2b1b以组织特异性的方式选择性表达，例如皮肤，而Sult2B1A本质上是全球沉默的。 DNA分析表明，两个SULT2B1同工型的近端启动子区域均包含多种CpG二核苷酸，其中细胞固醇受到甲基化的影响。人类细胞中未表达这些同工型的Sult2B1A和Sult2B1b启动子是高甲基化的。相反，在高度表达这种同工型的角质形成细胞中Sult2b1b的近端启动子是完全未甲基化的。甲基的去除导致表达的引人注目，而在体外甲基化SULT2B1A和SULT2B1B启动子/报告基因构建体构造显着降低了转染后启动子活性。因此，至少部分通过CpG二核苷酸在其近端启动子区域的甲基化来调节SULT2B1同工型的表达，并提出了SULT2B1A的全局沉默以及SULT2B1B的组织特异性表达的全局沉默。与人类和小鼠基因相似，大鼠sult2b1基因由替代外显子I组成。但是，由于外显子重排的结果，外显子Ia和Ib的遗传位置在大鼠基因中逆转。外显子Ia位于人和小鼠Sult2B1基因的外显子IB的下游，而大鼠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，需要进行差剪接，因为一部分外显子IA与外显子IB融合以完成SULT2B1B mRNA，因此不需要大鼠基因的步骤。对于大鼠Sult2B1基因的重排特别有趣的是，不仅可以重新定位外显子IB上游，这是人类和小鼠基因中情况的逆转，而且只有外显子IA编码的部分编码用于Sult2b1a Isoform的独特氨基末端。否则，Sult2B1b蛋白将维持大量的氨基酸缺失，使其不活跃。编码两种同工型的共同氨基酸序列的外显子IA的一部分保持与人和小鼠基因相同的相对基因位置，并在大鼠基因中变成外显子II。硫酸胆固醇与高亲和力与类维生素类似相关的孤儿核受体α（Roralpha）结合，诱导屏障蛋白Filaggrin（一种相对较小的组氨酸基本蛋白）的表达，该蛋白源自profilaggrin，含有大型前体，含有10-12个相同的copie in Isefirag in fipirag filaggrig filaggrin fillaggrin fillaggrin filgrin fillig fillig fillig Grand Grand Grand Grand Grand Grand Grand rand part rand part lactrin;广泛的磷酸化并包装到颗粒中，该颗粒免受蛋白水解的影响，直到需要丝状蛋白丝的聚集需要丝状蛋白，从而将profilaggrin暴露于蛋白水解中，导致蛋白水解导致filaggrin Monomers的释放）。此外，Roralpha，Sult2b1b（胆固醇亚硫代转移酶）和Filaggrin共定位于人表皮的外颗粒层提示功能关系。在培养基中钙浓度升高时，NHEK会经历末端分化，在这些条件下，Sult2B1b，Filaggrin和Roralpha以类似的方式和时间范围诱导。当使用siRNA抑制95％的roralpha基因对rolalpha的基因的表达，这导致profilaggrin mRNA的表达平行降低了80％时，提出了罗拉帕与菲拉格林产生的关联。此外，将硫酸胆固醇添加到培养基中，无法在profilaggrin mRNA的表达中恢复。另外，击倒Sult2b1b的基因也导致profilaggrin mRNA表达降低。但是，在这种情况下，在将硫酸胆固醇添加到培养基中后，可以成功恢复叶片蛋白表达。这些研究强烈表明，由Sult2B1b产生的硫酸胆固醇激活了叶子蛋白的基因，并通过与Roralpha的相互作用进行。这是硫酸胆固醇分子作用的首次演示，硫酸胆固醇使人联想到典型激素。氧化酚构成一类胆固醇衍生物，其表现出广泛的生物学作用，从细胞毒性到调节核受体（例如LXR），这些作用参与了从事脂肪酸和胆固醇代谢的基因的调节。氧化酚（例如7-酮胆脂酯（7-kC））在视网膜黄斑变性和动脉瘤性病变中的作用尤其令人感兴趣，但对它们的代谢命运知之甚少。在动脉瘤和其他组织中发现的主要氧化酚是7-kc，从细胞培养研究中知道，在体内诱导细胞损伤中诱导细胞损伤，因此，存在特定的重点对代谢途径的关注，这可能导致其毒性降低。我们确定，已知可以有效地磺酸胆固醇的类固醇/固醇磺胺转移酶Sult2b1b也有效地磺化了多种氧化甲醇，包括7- KC。当这些未表达SULT2B1B的细胞被SULT2B1B cDNA转染时，7-KC对293T细胞的细胞毒性作用被衰减。重要的是，通过转染的7-KC诱导的细胞活力丧失的保护与SULT2B1B蛋白的合成以及7-KC硫磺酸盐（7-KCS）的生产有关。此外，当将7-KCS添加到293T细胞的培养基中，含量等摩尔到7-KC，不会发生任何细胞活力的损失。另外，高度表达SULT2B1B的MCF-7细胞对7-KC的细胞毒性作用具有更大的抵抗力。我们扩展了Sult2B1B的氧蛋白酶底物的范围，包括7Alpha/7beta-羟基胆固醇和5alpha，6alpha/5beta，6Beta-EpoxyCholesterol以及7Alpha-Hydroperoxide of Cholesteroloxide。因此，SULT2B1B通过作用于多种氧甲醇提供了一种潜在的途径，可以在体内调节这些化合物的损害作用。重要的是，我们还首次通过生理化学手段证明了7-KC的硫二轭物确实发生在体内，如人类动脉瘤组织所证明的那样。现在看来，氧甲醇具有广泛的生物学作用，而Sult2B1b在整个光谱中起着重要作用。 Sult2B1b不仅使细胞毒性的氧蛋白酶类别灭活，而且还使参与细胞信号传导的氧甲甲醇类别都失活。