Miles Rodney R, Crockett David K, Lim Megan S, Elenitoba-Johnson Kojo S J
Department of Pathology, University of Utah School of Medicine, Salt Lake City, Utah 84132, USA.
Mol Cell Proteomics. 2005 Dec;4(12):1898-909. doi: 10.1074/mcp.M500112-MCP200. Epub 2005 Sep 6.
B-cell lymphoma 6 (BCL6) is a 95-kDa nuclear phosphoprotein and member of the Pox virus zinc finger/bric-a-brac, tramtrack, broad complex (POZ/BTB) family of transcription factors. BCL6 is a transcriptional repressor required for germinal center formation, and the gene encoding it is frequently altered in diffuse large B-cell and follicular lymphomas. The dysregulation of BCL6 has therefore been implicated in lymphomagenesis. A limited number of proteins is known to interact with BCL6 and modulate its activity or participate in its role in transcriptional regulation. Identification of additional BCL6-binding proteins could reveal potential signaling targets and previously undescribed functional roles for BCL6. We used a functional proteomic approach to determine the identity of proteins that interact with BCL6. Proteins were isolated by co-immunoprecipitation with an anti-BCL6 antibody and identified using MS/MS. We identified 61 proteins in the BCL6 immunocomplex from the following Gene Ontology categories: transcription regulator activity (n = 18), binding activity (n = 11), signal transducer activity (n = 10), catalytic activity (n = 8), structural molecule activity (n = 3), enzyme regulator activity (n = 3), transporter activity (n = 2), motor activity (n = 2), chaperone activity (n = 1), and unknown function (n = 3). Importantly we identified BCL6 and several previously reported BCL6-interacting proteins in the BCL6 immunocomplex. The remaining proteins have not been shown previously to be associated with BCL6. MS/MS results were validated on four proteins using immunoprecipitation and Western blotting. Two of these protein interactions were further confirmed by reciprocal immunoprecipitation. This study demonstrates the utility of antibody immunoprecipitation and subsequent peptide identification by MS/MS for the elucidation of BCL6-binding proteins. Many of the novel proteins identified in this study suggest additional functional roles for BCL6 beyond transcriptional repression.
B细胞淋巴瘤6（BCL6）是一种95千道尔顿的核磷蛋白，属于痘病毒锌指/布里克-布拉克、轨蛋白、宽复合体（POZ/BTB）转录因子家族成员。BCL6是生发中心形成所必需的转录抑制因子，编码它的基因在弥漫性大B细胞淋巴瘤和滤泡性淋巴瘤中经常发生改变。因此，BCL6的失调与淋巴瘤的发生有关。已知有少数蛋白质与BCL6相互作用并调节其活性或参与其在转录调控中的作用。鉴定其他BCL6结合蛋白可能揭示潜在的信号靶点以及BCL6以前未被描述的功能作用。我们采用功能蛋白质组学方法来确定与BCL6相互作用的蛋白质的身份。通过用抗BCL6抗体进行共免疫沉淀分离蛋白质，并使用串联质谱（MS/MS）进行鉴定。我们从以下基因本体论类别中鉴定出BCL6免疫复合物中的61种蛋白质：转录调节活性（n = 18）、结合活性（n = 11）、信号转导活性（n = 10）、催化活性（n = 8）、结构分子活性（n = 3）、酶调节活性（n = 3）、转运活性（n = 2）、运动活性（n = 2）、伴侣活性（n = 1）和未知功能（n = 3）。重要的是，我们在BCL6免疫复合物中鉴定出了BCL6和几种先前报道的与BCL6相互作用的蛋白质。其余蛋白质以前尚未显示与BCL6相关。使用免疫沉淀和蛋白质印迹法对四种蛋白质的串联质谱结果进行了验证。其中两种蛋白质相互作用通过相互免疫沉淀进一步得到证实。这项研究证明了抗体免疫沉淀和随后通过串联质谱鉴定肽段用于阐明BCL6结合蛋白的实用性。本研究中鉴定出的许多新蛋白质表明BCL6除转录抑制作用外还有其他功能作用。
