Proteomic profiling of gamma-secretase substrates and mapping of substrate requirements.
Proteomic profiling of gamma-secretase substrates and mapping of substrate requirements.
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DOI：
10.1371/journal.pbio.0060257
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发表时间：
2008-10-21
期刊：
影响因子：
9.8
通讯作者：
Selkoe DJ
中科院分区：
文献类型：
--
作者：
Hemming ML;Elias JE;Gygi SP;Selkoe DJ
早老素/γ-分泌酶复合体是一种罕见的膜内天冬氨酸蛋白酶，在细胞信号转导和膜蛋白周转中发挥重要作用。它能够从细胞膜上释放大量的细胞内信号蛋白，并介导淀粉样蛋白-β蛋白(A-β)的分泌，使γ-分泌酶活性的调节成为癌症和阿尔茨海默病的治疗目标。尽管Notch和β-淀粉样前体蛋白(APP)的原型底物的蛋白质分解已经被深入研究，但底物的全谱和使跨膜蛋白成为底物的决定因素仍然不清楚。使用一种无偏见的底物鉴定方法，我们对人类细胞系蛋白质组进行了γ-分泌酶靶标的研究，发现了相对较少的新底物，它们都是I型跨膜蛋白，但具有不同的生物学作用。通过将这些底物与不受γ-分泌酶调控的I型蛋白进行比较，我们确定γ-分泌酶除了需要一个较短的胞外结构域外，还需要允许的跨膜和胞质结构域来结合和切割其底物。此外，我们提供了至少两种针对底物进行γ切割的机制的证据：一种是具有较短胞外结构域的底物被直接切割，而不依赖于脱落酶的结合；另一种是底物需要胞外结构域脱落来指导后续的γ分泌酶加工。这些发现扩大了我们对底物选择的机制以及γ分泌酶参与的不同细胞过程的理解。所有细胞都面临着从脂质双层中去除跨膜蛋白的挑战，以达到信号传递或降解的目的。解决这一问题的一个分子方案是多蛋白酶复合体γ-分泌酶，它能够在疏水的脂类环境中水解几种已知的跨膜蛋白。由于其在阿尔茨海默病发病机制中的核心作用，调节γ-分泌酶活性已成为治疗的目标。然而，可以被这种酶切割的蛋白质的数量和多样性仍然未知，并且针对这些蛋白质的γ-分泌酶的属性也不清楚。在这项研究中，我们使用一种无偏见的方法来鉴定底物并调查蛋白质组以寻找γ-分泌酶的靶标。在数以千计的可检测到的蛋白质中，只有相对较少的是γ-分泌酶的底物，所有这些蛋白质都是I型跨膜蛋白。除了验证其中几种新的底物外，我们还将它们与我们鉴定为非底物的其他蛋白质进行比较，并确定有特定的结构域可以激活或抑制γ分泌酶的处理。这些发现应该会促进我们对γ-分泌酶调控的许多细胞过程的理解，并可能为γ-分泌酶如何用于治疗目的提供见解。使用一种无偏的定量蛋白质组学方法，鉴定和分析了γ-分泌酶的新底物靶点，以确定哪些结构域能够切割它们。
The presenilin/γ-secretase complex, an unusual intramembrane aspartyl protease, plays an essential role in cellular signaling and membrane protein turnover. Its ability to liberate numerous intracellular signaling proteins from the membrane and also mediate the secretion of amyloid-β protein (Aβ) has made modulation of γ-secretase activity a therapeutic goal for cancer and Alzheimer disease. Although the proteolysis of the prototypical substrates Notch and β-amyloid precursor protein (APP) has been intensely studied, the full spectrum of substrates and the determinants that make a transmembrane protein a substrate remain unclear. Using an unbiased approach to substrate identification, we surveyed the proteome of a human cell line for targets of γ-secretase and found a relatively small population of new substrates, all of which are type I transmembrane proteins but have diverse biological roles. By comparing these substrates to type I proteins not regulated by γ-secretase, we determined that besides a short ectodomain, γ-secretase requires permissive transmembrane and cytoplasmic domains to bind and cleave its substrates. In addition, we provide evidence for at least two mechanisms that can target a substrate for γ cleavage: one in which a substrate with a short ectodomain is directly cleaved independent of sheddase association, and a second where a substrate requires ectodomain shedding to instruct subsequent γ-secretase processing. These findings expand our understanding of the mechanisms of substrate selection as well as the diverse cellular processes to which γ-secretase contributes. All cells face the challenge of removing transmembrane proteins from the lipid bilayer for the purpose of signaling or degradation. One molecular solution to this problem is the multiprotein enzyme complex γ-secretase, which is able to hydrolyze several known transmembrane proteins within the hydrophobic lipid environment. Due to its central role in the pathogenesis of Alzheimer disease, modulation of γ-secretase activity has become a therapeutic goal. However, the number and diversity of proteins that can be cleaved by this protease remain unknown, and the attributes that target these proteins to γ-secretase are unclear. In this study, we used an unbiased approach to substrate identification and surveyed the proteome for targets of γ-secretase. Of the thousands of proteins detectable, only a relative few were substrates of γ-secretase, all of which were type I transmembrane proteins. In addition to validating several of these novel substrates, we compared them to other proteins that we identified as nonsubstrates and determined that there are specific domains that can activate or inhibit γ-secretase processing. These findings should advance our understanding of the many cellular processes regulated by γ-secretase and may offer insights into how γ-secretase can be exploited for therapeutic purposes. Using an unbiased quantitative proteomics approach, novel substrate targets for the protease γ-secretase are identified and analyzed to determine which domains enable their cleavage.
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影响因子：
56.9
作者：
Cao, XW;Südhof, TC
通讯作者：
Südhof, TC
DOI：
10.1073/pnas.0404117101
发表时间：
2004-07-20
影响因子：
11.1
作者：
Ikeda, Y;Imai, Y;Kitamura, T
通讯作者：
Kitamura, T
影响因子：
7.7
作者：
Hebert, Sebastien S.;Serneels, Lutgarde;De Strooper, Bart
通讯作者：
De Strooper, Bart
影响因子：
4.4
作者：
Everley, PA;Bakalarski, CE;Gygi, SP
通讯作者：
Gygi, SP
影响因子：
4.8
作者：
Karlström, H;Bergman, A;Lundkvist, J
通讯作者：
Lundkvist, J