Quantitative membrane proteomics reveals new cellular targets of viral immune modulators.
Quantitative membrane proteomics reveals new cellular targets of viral immune modulators.
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DOI：
10.1371/journal.ppat.0020107
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发表时间：
2006-10
期刊：
影响因子：
6.7
通讯作者：
Früh K
中科院分区：
文献类型：
--
作者：
Bartee E;McCormack A;Früh K
病原体的免疫调节剂经常影响多个细胞靶标，从而阻止不同免疫细胞的识别。例如，来自卡波西肉瘤相关疱疹病毒的K5免疫识别调节剂（MIR 2）通过下调主要组织相容性复合体（MHC）I类分子、MHC样分子CD 1、细胞粘附分子ICAM-1和PECAM以及共刺激分子B7.2来防止细胞毒性T细胞、自然杀伤细胞和自然杀伤T细胞的活化。K5属于病毒和细胞跨膜RING泛素连接酶家族。虽然有限数量的跨膜蛋白已被证明是该家族降解的靶点，但尚不清楚是否存在其他靶点。我们现在描述了一种定量蛋白质组学方法来确定这个蛋白质家族的新靶点。使用稳定的同位素标记的氨基酸，我们比较了血浆，高尔基体和内质网膜的蛋白质组中存在和不存在的K5。质谱蛋白质鉴定揭示了在K5表达细胞的质膜中始终代表不足的四种蛋白质：MHC I（如预期的）、骨髓基质抗原2（BST-2，CD 316）、活化的白细胞粘附分子（ALCAM，CD 166）和突触融合蛋白-4。这些蛋白质中的每一种的下调通过用特异性抗体的免疫印迹独立地证实。我们进一步证明ALCAM是K5和粘液瘤病毒同源物M153 R的真正靶点。在离开内质网后，ALCAM在野生型K5存在下被泛素化，但不存在RING缺陷或酸性基序缺陷K5，并通过多泡体途径靶向溶酶体降解。由于ALCAM是CD 6的配体，CD 6是T细胞免疫突触的成员，因此病毒免疫调节剂对其的去除意味着CD 6在T细胞识别病原体中的作用。因此，无偏的全球蛋白质组分析揭示了病原体蛋白质的新的免疫调节功能。病毒免疫调节剂通常靶向多种细胞蛋白进行破坏。据推测，这种策略使病毒病原体能够优化多重免疫应答的逃避。为了以无偏的方式系统地鉴定这些宿主细胞靶标，Bartee等人应用最近开发的定量蛋白质组学方法来鉴定K5的新靶标。K5属于在γ-疱疹病毒和痘病毒中发现的病毒泛素连接酶家族，其靶向多个细胞跨膜蛋白进行破坏。使用稳定同位素标记结合串联质谱法，作者比较了表达K5的细胞和不表达K5的细胞的膜制备物中蛋白质的丰度。在他们的实验中，三种新的膜蛋白（BST-2，Syntaxin-4和ALCAM）在表达K5的细胞中的丰度一直较低。重要的是，作者能够通过独立的实验和独立的方法证实所有这些蛋白质的K5依赖性下调。选择ALCAM进行更深入的分析，以坚定地证明该蛋白质以与其他已知靶标相似的方式被K5下调。这项原理验证研究表明，可以用定量蛋白质组学鉴定病毒免疫调节剂的新靶点。
Immunomodulators of pathogens frequently affect multiple cellular targets, thus preventing recognition by different immune cells. For instance, the K5 modulator of immune recognition (MIR2) from Kaposi sarcoma–associated herpesvirus prevents activation of cytotoxic T cells, natural killer cells, and natural killer T cells by downregulating major histocompatibility complex (MHC) class I molecules, the MHC-like molecule CD1, the cell adhesion molecules ICAM-1 and PECAM, and the co-stimulatory molecule B7.2. K5 belongs to a family of viral- and cellular-membrane-spanning RING ubiquitin ligases. While a limited number of transmembrane proteins have been shown to be targeted for degradation by this family, it is unknown whether additional targets exist. We now describe a quantitative proteomics approach to identify novel targets of this protein family. Using stable isotope labeling by amino acids, we compared the proteome of plasma, Golgi, and endoplasmic reticulum membranes in the presence and absence of K5. Mass spectrometric protein identification revealed four proteins that were consistently underrepresented in the plasma membrane of K5 expression cells: MHC I (as expected), bone marrow stromal antigen 2 (BST-2, CD316), activated leukocyte cell adhesion molecule (ALCAM, CD166) and Syntaxin-4. Downregulation of each of these proteins was independently confirmed by immunoblotting with specific antibodies. We further demonstrate that ALCAM is a bona fide target of both K5 and the myxomavirus homolog M153R. Upon exiting the endoplasmic reticulum, ALCAM is ubiquitinated in the presence of wild-type, but not RING-deficient or acidic motif–deficient, K5, and is targeted for lysosomal degradation via the multivesicular body pathway. Since ALCAM is the ligand for CD6, a member of the immunological synapse of T cells, its removal by viral immune modulators implies a role for CD6 in the recognition of pathogens by T cells. The unbiased global proteome analysis therefore revealed novel immunomodulatory functions of pathogen proteins. Viral immune modulators often target multiple cellular proteins for destruction. Presumably, this strategy enables viral pathogens to optimize evasion of multiple immune responses. To systematically identify such host cell targets in an unbiased fashion, Bartee et al. applied recently developed quantitative proteomics methods to identify novel targets for K5. K5 belongs to a family of viral ubiquitin ligases found in gamma-herpesviruses and poxviruses that target multiple cellular transmembrane proteins for destruction. Using stable isotope labeling combined with tandem mass spectrometry, the authors compared the abundance of proteins in membrane preparations from cells that expressed K5 to that in cells without K5. In their experiments, three novel membrane proteins (BST-2, Syntaxin-4, and ALCAM) were consistently found in lower abundance in K5-expressing cells. Importantly, the authors were able to confirm the K5-dependent downregulation of all of these proteins in independent experiments and by independent methods. ALCAM was chosen for a more in-depth analysis to firmly demonstrate that this protein is downregulated by K5 in a manner similar to other known targets. This proof-of-principle study demonstrates that novel targets of viral immune modulators can be identified with quantitative proteomics.
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