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(包括人乳头瘤病毒（HPV）、爱泼斯坦-巴尔病毒（EBV）、人疱疹病毒8 （HHV8）和默克尔细胞多瘤病毒（MCPV）编码的许多病毒蛋白的靶标。对于这些病毒，与Brd4的相互作用已被证明在病毒基因组的维持以及病毒和细胞基因的转录调节中起作用。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，导致一种称为NUT-中线癌（NMC）的侵袭性鳞状细胞癌。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，其他功能可能被改变、丢失甚至产生。我推测，在这些病理条件下发现的Brd4功能的改变也将反映在与Brd4结合的细胞蛋白的变化上。因此，我采用了蛋白质组学方法，通过质谱分析和随后的CompPASS软件分析，在存在和不存在不同病毒蛋白的情况下，鉴定细胞Brd4相互作用伙伴。我的初步数据确定了几个新的Brd4互动伙伴。有趣的是，这些蛋白质包括仅在特定病毒蛋白存在时与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有趣候选蛋白，因为通过病毒蛋白表达解除Brd4相互作用可能有助于发病机制。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，我将扩展我基于质谱的相互作用研究，并使用类似的方法来鉴定在NMC中Brd4与NUT融合后对Brd4的结合行为发生改变的蛋白质。这两项研究都将为癌症和特定致癌病毒感染后的特定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.