Biochemistry of ETS Factors Involved in Prostate Cancer

描述（由申请人提供）：ETS转录因子家族调节数千种基因的表达，包括内务管理和组织特异性。ETS DNA结合域，或简称ETS结构域，在整个家族中是保守的；然而，在ETS领域之外，这个家族是多样化和专业化的，允许ETS因子以独特的方式靶向和控制转录。这些包括控制DNA结合亲和力的自身抑制机制和与控制基因表达的辅助因子的相互作用。最近在前列腺癌中的一项发现表明，ETS蛋白亚群（ERG、ETV1、ETV4或ETV5）在50%的这些癌症中发生染色体易位，这些ETS因子的失调对转化和癌症进展很重要。本研究的目的是揭示与前列腺癌相关的ETS因子的独特特征，不仅进一步了解ETS家族，而且为设计前列腺癌治疗方法提供重要的生化细节。这项工作的成功完成将包括全面了解前列腺癌中ETS因子的变构调节机制，以及发现和表征核辅助因子与这些致癌蛋白的相互作用。目前尚不清楚自体抑制是否控制ERG、ETV1、ETV4或ETV5，然而，整个ETS家族的证据表明，通过抑制结构域调节DNA结合是调节ETS DNA结合的可行机制。这可以很容易地通过使用emsa评估DNA结合亲和力来测量。将产生截断突变体，以便查明参与抑制的区域，并从生化角度评估自抑制的性质。最后，抑制结构域将使用完善的核磁共振技术进行结构表征。同时，一种无偏倚的方法将用于捕获细胞内和体外细胞核提取物的相互作用伙伴。质谱技术将允许对ETS相互作用的伙伴进行正面鉴定，这将进一步通过生物化学进行验证。有限的蛋白水解将识别介导相互作用的特定区域，简单的报告基因检测将用于探测细胞内相互作用的功能。为了充分发挥理解新辅因子的潜力，这些界面的结构将通过核磁共振技术确定。

DESCRIPTION (provided by applicant): The ETS family of transcription factors regulates the expression of thousands of genes, both housekeeping and tissue-specific. The ETS DNA binding domain, or simply, the ETS domain, is conserved throughout the family; however, outside of the ETS domain the family is diverse and specialized allowing the ETS factors to target and control transcription in unique ways. These include autoinhibitory mechanisms that control DNA binding affinity and interactions with co-factors that control gene expression. A recent discovery in prostate cancer revealed that a sub-set of ETS proteins (ERG, ETV1, ETV4, or ETV5) undergo chromosomal translocations in 50% of these cancers and the misregulation of these ETS factors is important for transformation and cancer progression. The goal of this proposal is to reveal the unique features of the ETS factors involved in prostate cancer to not only further our understanding of the ETS family, but to provide biochemical details important for designing prostate cancer therapeutics. Successful completion of the work will include a thorough understanding of allosteric regulatory mechanisms of ETS factors involved in prostate cancer and discovery and characterization of nuclear co-factor interactions with these oncogenic proteins. It is unknown if autoinhibition controls ERG, ETV1, ETV4 or ETV5, however, evidence throughout the ETS family suggests modulating DNA binding through inhibitory domains is a feasible mechanism for regulating ETS DNA binding. This can easily be measured by assessing DNA binding affinities using EMSAs. Truncation mutants will be generated in order to pinpoint regions involved in the inhibition and to biochemically assess the nature of the autoinhibition. Finally, inhibitory domains will be characterized structurally using well established NMR techniques. In conjunction, an unbiased approach will be used for capturing interacting partners both within cells and in vitro from cellular nuclear extracts. Mass spectrometry technologies will allow positive identifications of ETS interacting partners, which will be further, validated using biochemistry. Limited proteolysis will identify specific domains that mediate the interaction and simple reporter assays will be used to probe for the function of the interaction within cells. To realize the full potential of understanding new co-factors, the structures of these interfaces will be determined by NMR techniques. PUBLIC HEALTH RELEVANCE: Human chromosomal rearrangements that affect ETS factors are involved in both Ewing's sarcoma and prostate cancer. The completion of the proposed experiments will provide a detailed picture of the regulatory mechanisms controlling the ETS factors involved in prostate cancer, ERG, ETV1, ETV4 and ETV5, including both inhibitory properties and co-factor interactions. Both of these interfaces are important to understand from an ETS family perspective, but can also be exploited for rational drug design in the treatment of prostate cancer.