Biochemistry of ETS Factors Involved in Prostate Cancer

前列腺癌相关 ETS 因子的生物化学

• 批准号: 8000610
• 负责人: Krista D Meyer
• 金额: $ 2.77万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-30 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8000610
• 关键词: Affect; Affinity; Biochemical; Biochemistry; Biological Assay; Cells; Chromosomal Rearrangement; Chromosomal translocation; DNA Binding; DNA Binding Domain; Drug Design; ETS Domain; ETS Family Protein; ETV1 gene; ETV4 gene; Ewings sarcoma; Family; Gene Expression; Genes; Genetic Transcription; Goals; Housekeeping; Human; In Vitro; Malignant Neoplasms; Malignant neoplasm of prostate; Mass Spectrum Analysis; Measures; Mediating; Nature; Nuclear; Nuclear Extract; Oncogenic; Property; Proteins; Proteolysis; Reporter; Structure; Techniques; Technology; Therapeutic; Tissues; Work; design; mutant; public health relevance; research study; transcription factor; tumor progression

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.

描述（由申请人提供）：转录因子ETS家族调节数千个基因的表达，包括管家基因和组织特异性基因。ETS DNA结合结构域，或简称ETS结构域，在整个家族中是保守的;然而，在ETS结构域之外，该家族是多样的和特化的，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结合亲和力的自抑制机制以及与控制基因表达的辅因子的相互作用。最近在前列腺癌中的发现揭示了ETS蛋白的子集（ERG、ETV 1、ETV 4或ETV 5）在50%的这些癌症中经历染色体易位，并且这些ETS因子的失调对于转化和癌症进展是重要的。本提案的目标是揭示前列腺癌中涉及的ETS因子的独特特征，不仅进一步加深我们对ETS家族的理解，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对前列腺癌中ETS因子的变构调节机制的透彻理解，以及对核辅因子与这些致癌蛋白相互作用的发现和表征。尚不清楚自身抑制是否控制ERG、ETV 1、ETV 4或ETV 5，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。这可以通过使用EMSA评估DNA结合亲和力来轻松测量。将产生截短突变体，以确定参与抑制的区域，并以生物化学方式评估自抑制的性质。最后，将使用完善的NMR技术在结构上表征抑制结构域。结合，将使用无偏的方法从细胞核提取物中捕获细胞内和体外的相互作用伴侣。质谱技术将允许ETS相互作用伙伴的阳性鉴定，这将进一步使用生物化学进行验证。有限的蛋白水解将确定特定的结构域，介导的相互作用和简单的报告分析将用于探测细胞内的相互作用的功能。为了实现理解新辅因子的全部潜力，这些界面的结构将通过NMR技术来确定。 公共卫生相关性：影响ETS因子的人类染色体重排与尤文氏肉瘤和前列腺癌有关。完成拟议的实验将提供控制前列腺癌，ERG，ETV 1，ETV 4和ETV 5中涉及的ETS因子的调控机制的详细图片，包括抑制特性和辅因子相互作用。从ETS家族的角度来看，这两个界面都很重要，但也可以用于合理的药物设计，治疗前列腺癌。