Identification and characterization of Brd4 interacting proteins relevant to oncogenic DNA viruses and human cancers

含有溴结构域的蛋白Brd4与多种人类癌症有关。一方面，Brd4是由致癌DNA肿瘤病毒编码的多种病毒蛋白的靶点，包括人乳头瘤病毒(HPV)、EB病毒(EBV)、人类疱疹病毒8(HHV8)以及默克尔细胞多瘤病毒(MCPV)。对于这些病毒，与Brd4的相互作用已被证明在维持病毒基因组以及病毒和细胞基因的转录调控中发挥作用。另一方面，基因组易位(T15；19)导致Brd4与NUT(睾丸核蛋白)基因融合，导致一种侵袭性的鳞状细胞癌，称为NUT-中线癌(NMC)。在三分之二的NMC病例中，NUT基因与Brd4融合，导致Brd4-NUT融合蛋白的表达。虽然可以假设在病毒感染或与NUT融合后，Brd4的一些功能可能被保留，但由于细胞生理条件的改变，其他功能可能会改变、丧失甚至产生。我推测，在这些病理条件下发现的Brd4功能的变化也将反映在与Brd4结合的细胞蛋白质的变化上。因此，我使用蛋白质组学方法，通过质谱学和随后的CompPASS软件分析，在存在和不存在不同病毒蛋白的情况下识别细胞Brd4相互作用伙伴。我的初步数据确定了几个新的Brd4交互合作伙伴。有趣的是，这些蛋白质包括仅在特定病毒蛋白存在时才与Brd4结合的蛋白质，以及在病毒蛋白存在时失去与Brd4结合能力的蛋白质。这些蛋白是值得进一步分析的候选蛋白，因为病毒蛋白表达对Brd4相互作用的解除调控可能有助于致病。因此，我将验证这些新发现的相互作用，并研究它们在特定病毒感染期间的生理相关性。此外，我将扩展我的基于质谱学的相互作用研究，并使用类似的方法来识别在NMC中Brd4与NUT融合后显示与Brd4结合行为发生变化的蛋白质。这两项研究将为癌症和感染特定致癌病毒后的特定Brd4功能和功能障碍提供新的见解。

The Bromodomain-containing protein Brd4 has been implicated in a variety of human cancers. On the one hand, Brd4 is targeted by a number of viral proteins encoded by oncogenic DNA tumor viruses including human papillomaviruses (HPV), Epstein-Barr-virus (EBV), human herpesvirus 8 (HHV8) as well as Merkel cell polyomavirus (MCPV). For these viruses the interaction with Brd4 has been demonstrated to have roles in the maintenance of viral genomes as well as transcriptional regulation of both viral and cellular genes. On the other hand, a genomic translocation (t15;19), that results in a gene fusion of Brd4 with the NUT (nuclear protein in testis) gene leads to an aggressive form of squamous cell carcinoma called NUT-midline carcinoma (NMC). In two thirds of NMC cases the NUT gene is fused with Brd4, resulting in the expression of a Brd4-NUT fusion protein. While it can be assumed that some Brd4 functions may be retained after viral infections or fusion with NUT, it is likely that other functions might be altered, lost or even created due to the modified cellular physiological conditions. I hypothesize that the alterations in Brd4 functions found under these pathological conditions will also be reflected by a change in cellular proteins that bind to Brd4. I thus performed a proteomic approach to identify cellular Brd4 interaction partners in the presence and absence of different viral proteins by mass-spectrometry and subsequent CompPASS software analysis. My preliminary data identified several new Brd4 interaction partners. Interestingly, these proteins include proteins that only bind to Brd4 in the presence of a specific viral protein as well as proteins that lost their binding ability towards Brd4 in the presence of a viral protein. These proteins are interesting candidates for further analysis as deregulation of Brd4 interactions by viral protein expression potentially contributes to pathogenesis. I will therefore validate these newly identified interactions and study their physiological relevance during specific viral infections. Additionally, I will expand my mass-spectrometry based interaction studies and use a similar approach to identify proteins that show altered binding behavior towards Brd4 upon the fusion of Brd4 to NUT in NMC. Both studies will provide new insights into specific Brd4 functions and dysfunctions in cancer and after infection by specific oncogenic viruses.