The Biotransformation Of Endobiotics By Sulfonation

人类SULT2B1基因，由于另一个外显子1和差异剪接，编码两个mrna，即SULT2B1a和SULT2B1。使用外显子1A产生SULT2B1a，而产生SULT2B1b则需要外显子1B加上部分外显子1A。虽然SULT2B1a能有效地将类固醇孕烯醇酮磺化，但SULT2B1b是生理性胆固醇磺化转移酶。SULT2B1b以组织特异性的方式选择性表达，例如皮肤，而SULT2B1a基本上是全局沉默的。DNA分析显示，两个SULT2B1亚型的近端启动子区域含有多个CpG二核苷酸，其中胞嘧啶受到甲基化。人类细胞中不表达这些亚型的SULT2B1a和SULT2B1b启动子被超甲基化。相反，在高度表达该亚型的角化细胞中，SULT2B1b的近端启动子完全未甲基化。去除甲基会显著诱导表达，而转染后，SULT2B1a和SULT2B1b启动子/报告基因构建的体外甲基化会显著降低启动子活性。因此，SULT2B1亚型的表达至少在一定程度上受到其近端启动子区域CpG二核苷酸甲基化的调节，这可能解释了SULT2B1a的全局沉默以及SULT2B1b的组织特异性表达。与人类和小鼠基因相似，大鼠SULT2B1基因由另一个外显子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被逆转。在人和小鼠SULT2B1基因中，IA外显子位于IB外显子的下游，而在大鼠SULT2B1基因中，IA外显子位于IB外显子的上游。此外，与人和小鼠SULT2B1基因不同，由于IA外显子的一部分与IB外显子融合以完成SULT2B1b mRNA，因此需要进行差异剪接，而大鼠基因则不需要进行这一步骤。关于大鼠SULT2B1基因的重排，特别有趣的是，不仅外显子IA被重新定位到外显子IB的上游，这与人类和小鼠基因的情况相反，而且只有编码SULT2B1a亚型独特氨基末端的外显子IA部分被重新定位。这是合适的，否则SULT2B1b蛋白将维持大量氨基酸缺失，使其失去活性。编码两种同工异构体共同氨基酸序列的IA外显子部分与人和小鼠基因保持相同的相对基因位置，在大鼠基因中成为II外显子。硫酸胆固醇与类视黄酮相关的孤儿核受体（rorα）高亲和力结合，诱导了屏障蛋白聚丝蛋白（一种相对较小的富含组氨酸的碱性蛋白，来源于侧聚蛋白，一个大的前体，含有10-12个串联排列的成熟聚丝蛋白的相同拷贝）的表达；聚丝蛋白被广泛磷酸化并包装成颗粒，保护聚丝蛋白免受蛋白质水解，直到角蛋白丝聚集需要聚丝蛋白；当添加到人角质形成细胞（NHEK）的原代培养物中时，去磷酸化使聚丝蛋白暴露于蛋白水解，导致聚丝蛋白单体的释放。此外，rorα、SULT2B1b（胆固醇硫转移酶）和聚丝蛋白共同定位于人类表皮的外颗粒层，表明它们之间存在功能关系。当培养基中钙浓度增加时，NHEK会发生终末分化，在这些条件下，SULT2B1b、聚丝蛋白和rorα以类似的方式和时间框架被诱导。当使用siRNA抑制NHEK表达rorα基因95%时，表明rorα与聚丝蛋白的产生有关，这导致侧聚蛋白mRNA的表达平行减少80%；此外，在培养基中加入硫酸胆固醇也不能恢复profilaggrin mRNA的表达。此外，敲除SULT2B1b基因也会导致侧聚蛋白mRNA表达减少；然而，在这种情况下，在培养基中加入硫酸胆固醇后，可以成功地恢复profilaggrin的表达。这些研究强烈表明，SULT2B1b产生的硫酸胆固醇激活了侧聚蛋白基因，并通过与rorα的相互作用来激活。这是第一次证明了硫酸胆固醇的分子作用，使人想起一种典型的激素。氧化甾醇是一类胆固醇衍生物，具有广泛的生物学作用，从细胞毒性到核受体（如LXR）的调节，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是人们特别感兴趣的，但对它们的代谢命运知之甚少。在动脉粥样硬化和其他组织中发现的一种主要的氧固醇是7-KC，从细胞培养研究中得知，在体内存在浓度的情况下，它会诱导细胞损伤，因此，人们特别关注可导致其毒性降低的代谢途径。我们发现，类固醇/甾醇磺酸转移酶SULT2B1b能够有效地将胆固醇磺化，同时也能有效地将包括7-KC在内的多种氧甾醇磺化。当不表达SULT2B1b的293T细胞转染SULT2B1b cDNA后，7-KC对293T细胞的细胞毒作用减弱。重要的是，通过转染保护7-KC诱导的细胞活力丧失与SULT2B1b蛋白的合成和7-KC硫偶联物（7-KCS）的产生相关。另外，在293T细胞的培养基中加入7-KCS，其量等于7-KC，细胞活力未发生损失。此外，高表达SULT2B1b的MCF-7细胞对7-KC的细胞毒性作用具有更强的抵抗力。我们扩大了SULT2B1b的氧甾醇底物范围，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一种潜在的途径来调节这些化合物在体内的有害作用。重要的是，我们也首次通过物理化学手段证明了7-KC的硫缀合物确实在体内发生，正如在人类动脉粥样硬化组织中所证明的那样。现在看来，氧化甾醇具有广泛的生物效应，而SULT2B1b在整个光谱中起着重要作用。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。

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.