Systems analysis of protein modification and cellular responses induced by electrophile stress.
Systems analysis of protein modification and cellular responses induced by electrophile stress.
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DOI：
10.1021/ar900286y
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发表时间：
2010-05-18
影响因子：
18.3
通讯作者：
Marnett LJ
中科院分区：
文献类型：
--
作者：
Jacobs AT;Marnett LJ
生物亲电体由外源性化合物或内源性细胞成分的氧化代谢产生，并且它们有助于病理生理学，例如毒性和致癌性。亲电体的化学毒理学主要是通过与细胞内亲核体的共价加成作用。与DNA的反应导致加合物的产生，从而阻断复制或诱导突变。亲电试剂-DNA反应的化学和生物学已经被广泛研究，在许多情况下提供了对加合物结构和突变后果之间关系的详细理解。相比之下，蛋白质修饰和细胞反应之间的联系知之甚少。在本报告中，我们描述了我们使用脂质衍生的α，β-不饱和醛作为模型亲电体来定义蛋白质修饰的化学及其生物学后果的努力。在我们的全球方法中，分析了两个大的数据集：一个代表在广泛的亲电浓度范围内修饰的蛋白质的身份，第二个包括在类似条件下观察到的基因表达的变化。信息学工具显示理论上的联系，主要是基于转录因子假设共享的两个数据集，下游的加合蛋白质和上游的受影响的基因。这种方法突出了潜在的亲电敏感的信号通路和转录过程，以进一步评估。细胞磷脂的过氧化产生膜结合亲电体和扩散亲电体的复杂混合物。后者包括活性物质，如丙二醛、4-氧代壬烯醛和4-羟基壬烯醛（HNE）。富集HNE加合蛋白用于蛋白质组学分析是一项技术挑战，通过生成生物素标记的蛋白质加合物的点击化学解决了这一问题。为此，合成了带有末端叠氮化物或炔官能团的HNE类似物。首先将细胞裂解物暴露于单一类型的HNE类似物（叠氮基-或炔基-HNE），然后针对同源炔基-和叠氮基-生物素衍生物进行点击反应。捕获所得生物素标记的蛋白质并在链霉亲和素基质上富集，用于随后的质谱分析。因此，我们确定了大量的HNE目标。同时微阵列分析HNE引发的基因表达变化也产生了丰富的数据。这两个数据集的功能分析产生的假设，一个重要的细胞反应途径来自亲电修饰的蛋白伴侣，导致在释放的转录因子，是他们的客户。对蛋白质修饰和微阵列数据集的信息学分析确定了几种转录因子作为对HNE加合蛋白的细胞应答的潜在介质。其中，热休克因子1（HSF 1）被证实是一个敏感和强大的效应HNE诱导的基因表达的变化。HSF 1的活化部分地由Hsp 70和Hsp 90的亲电加合介导，其通常将HSF 1维持在无活性的胞质复合物中。HSF 1作为HSF 1下游生物效应的介体的鉴定为研究提供了新的机会，说明了我们基于系统的方法的潜力。因此，我们的特点是HSF 1介导的基因表达保护亲电诱导的毒性。在由HSF 1诱导的基因中，Bcl-2相关的产气基因3（BAG 3）因其通过稳定抗凋亡Bcl-2蛋白而促进细胞存活的作用而引人注目，似乎在介导细胞保护对抗亲电体诱导的死亡中具有关键作用。
Biological electrophiles result from oxidative metabolism of exogenous compounds or endogenous cellular constituents, and they contribute to pathophysiologies such as toxicity and carcinogenicity. The chemical toxicology of electrophiles is dominated by covalent addition to intracellular nucleophiles. Reaction with DNA leads to the production of adducts that block replication or induce mutations. The chemistry and biology of electrophile−DNA reactions have been extensively studied, providing in many cases a detailed understanding of the relation between adduct structure and mutational consequences. By contrast, the linkage between protein modification and cellular response is poorly understood. In this Account, we describe our efforts to define the chemistry of protein modification and its biological consequences using lipid-derived α,β-unsaturated aldehydes as model electrophiles. In our global approach, two large data sets are analyzed: one represents the identity of proteins modified over a wide range of electrophile concentrations, and the second comprises changes in gene expression observed under similar conditions. Informatics tools show theoretical connections based primarily on transcription factors hypothetically shared between the two data sets, downstream of adducted proteins and upstream of affected genes. This method highlights potential electrophile-sensitive signaling pathways and transcriptional processes for further evaluation. Peroxidation of cellular phospholipids generates a complex mixture of both membrane-bound and diffusible electrophiles. The latter include reactive species such as malondialdehyde, 4-oxononenal, and 4-hydroxynonenal (HNE). Enriching HNE-adducted proteins for proteomic analysis was a technical challenge, solved with click chemistry that generated biotin-tagged protein adducts. For this purpose, HNE analogues bearing terminal azide or alkyne functionalities were synthesized. Cellular lysates were first exposed to a single type of HNE analogue (azido- or alkynyl-HNE), and then click reactions were performed against the cognate alkynyl- and azido-biotin derivative. The resulting biotin-labeled proteins were captured and enriched over a streptavidin matrix for subsequent mass spectrometric analysis. We thereby identified a multitude of HNE targets. Simultaneous microarray analysis of changes in gene expression triggered by HNE also produced an abundance of data. Functional analysis of both data sets generated the hypothesis that an important pathway of cellular response derives from electrophile modification of protein chaperones, resulting in the release of transcription factors that are their clients. Informatic analysis of the protein modification and microarray data sets identified several transcription factors as potential mediators of the cellular response to HNE-adducted proteins. Among these, heat shock factor 1 (HSF1) was confirmed as a sensitive and robust effector of HNE-induced changes in gene expression. Activation of HSF1 appears, in part, to be mediated by the electrophilic adduction of Hsp70 and Hsp90, which normally maintain HSF1 in an inactive cytosolic complex. The identification of HSF1 as a mediator of biological effects downstream of HSF1 has provided new opportunities for research, illustrating the potential of our systems-based approach. Accordingly, we characterized HSF1-mediated gene expression in protecting against electrophile-induced toxicity. Among the genes induced by HSF1, Bcl-2- associated athanogene 3 (BAG3) is notable for its actions in promoting cell survival through stabilization of antiapoptotic Bcl-2 proteins, appearing to have a critical role in mediating cellular protection against electrophile-induced death.
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DOI：
10.1074/mcp.m800070-mcp200
发表时间：
2009-04
期刊：
Molecular & cellular proteomics : MCP
影响因子：
--
作者：
Codreanu SG;Zhang B;Sobecki SM;Billheimer DD;Liebler DC
通讯作者：
Liebler DC
影响因子：
7.4
作者：
Perluigi, M;Poon, HF;Butterfield, DA
通讯作者：
Butterfield, DA
影响因子：
64.5
作者：
Dai, Chengkai;Whitesell, Luke;Lindquist, Susan
通讯作者：
Lindquist, Susan
影响因子：
5.3
作者：
BALER, R;DAHL, G;VOELLMY, R
通讯作者：
VOELLMY, R
DOI：
10.1124/jpet.105.088088
发表时间：
2005-10-01
影响因子：
3.5
作者：
Carbone, DL;Doorn, JA;Petersen, DR
通讯作者：
Petersen, DR