Subproteomic analysis of metal-interacting proteins in human B cells
Subproteomic analysis of metal-interacting proteins in human B cells
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DOI：
10.1002/pmic.200401215
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发表时间：
2005-09-01
期刊：
影响因子：
3.4
通讯作者：
Thierse, HJ
中科院分区：
文献类型：
--
作者：
Heiss, K;Junkes, C;Thierse, HJ
金属-蛋白质相互作用在所有生物体中至关重要。金属蛋白质，包括结构蛋白质和代谢酶，参与能量转移和氧化还原反应或在金属运输中充当金属伴侣。在金属相关疾病中，T细胞介导的对镍（Ni）的过敏是人类接触性超敏反应的最常见形式。为了阐明疾病的潜在机制，如镍特异性T细胞活化，我们启动了一个蛋白质组学的方法来确定镍相互作用的蛋白质在人类B细胞。作为抗原呈递细胞，B细胞能够将MHC相关的Ni表位呈递给T细胞，这是半抗原特异性T细胞活化的先决条件。通过金属亲和富集、双向电泳和质谱分析，共鉴定出22个镍离子相互作用蛋白。除了已知的镍结合分子，如微管蛋白，肌动蛋白或cullin-2，我们意外地发现，这22个蛋白质中至少有9个属于胁迫诱导的热休克蛋白或伴侣蛋白。富集对于异源寡聚TRiC/CCT复合物特别有效，其参与MHC I类加工。蓝色天然/SDS电泳分析显示，Ni-NTA-珠特异性地保留了完整的蛋白质机制，包括相关的伴侣蛋白底物微管蛋白。热休克蛋白的镍亲和力表明，这些分子在人类镍过敏的一个新的功能，通过连接先天性和适应性免疫反应。
Metal-protein interactions are vitally important in all living organisms. Metalloproteins, including structural proteins and metabolic enzymes, participate in energy transfer and redox reactions or act as metallochaperones in metal trafficking. Among metal-associated diseases, Tcell mediated allergy to nickel (Ni) represents the most common form of human contact hypersensitivity. With the aim to elucidate disease-underlying mechanisms such as Ni-specific T cell activation, we initiated a proteomic approach to identify Ni-interacting proteins in human B cells. As antigen presenting cells, B cells are capable of presenting MHC-associated Ni-epitopes to T cells, a prerequisite for hapten-specific Tcell activation. Using metal-affinity enrichment, 2-DE and MS, 22 Ni-interacting proteins were identified. In addition to known Ni-binding molecules such as tubulin, actin or cullin-2, we unexpectedly discovered that at least nine of these 22 proteins belong to stress-inducible heat shock proteins or chaperonins. Enrichment was particularly effective for the hetero-oligomeric TRiC/CCT complex, which is involved in MHC class I processing. Blue Native/SDS electrophoresis analysis revealed that Ni-NTA-beads specifically retained the complete protein machinery, including the associated chaperonin substrate tubulin. The apparent Ni-affinity of heat shock proteins suggests a new function of these molecules in human Ni allergy, by linking innate and adaptive immune responses.