REDOX CONTROL OF HELIX LOOP HELIX PROTEIN ACTIVITY

描述：本提案的主要长期目标是了解 控制螺旋-环-螺旋（bHLH）蛋白的氧化还原调节机制 在哺乳动物细胞中的活性。 事实表明，成员的活动 bHLH蛋白的E蛋白家族的蛋白质的形成是由一个 分子间二硫键，这是稳定 同源二聚体形成、DNA结合和转录激活。 同源二聚体在淋巴样细胞中稳定，但在非淋巴样细胞中不稳定。 由于存在可还原该二硫化物的活性， 邦德 因此，同源二聚体的形成可能有助于改变 B细胞分化过程中的基因表达。 具体目标是 建议：1）进一步探索E蛋白的二硫键模式 通过确定E蛋白单体之间的肽连接是否可以 绕过对分子间二硫键的要求;以确定 如果E蛋白家族（E2-2）的成员缺乏保守的 二硫化物形成所需的半胱氨酸可以作为同型二聚体， 以形成交联。 2)为了纯化和克隆与以下相关的蛋白质： 还原分子间二硫化物所需的活性 邦德 蛋白质纯化将通过标准的生物化学方法完成。 是指使用凝胶迁移率变动测定来跟踪活性， 洁净. 这些目标有助于我们进一步了解 转录控制机制在这种情况下淋巴细胞 通过HLH蛋白的E蛋白家族进行分化。

DESCRIPTION: The major long term goal of this proposal is to understand the redox regulatory mechanisms which control helix-loop-helix (bHLH) protein activity in mammalian cells. It has been shown that the activity of members of the E protein family of bHLH proteins is regulated by the formation of an intermolecular disulfide bond which is absolutely required for stable homodimer formation, DNA binding, and transcriptional activation. Homodimers are stable in lymphoid cells but are unstable in non-lymphoid cells due to the presence of an activity which can reduce this disulfide bond. Therefore, homodimer formation probably contributes to changes in gene expression during B cell differentiation. The specific aims of this proposal are: 1) to further explore the disulfide model of E protein regulation by determining if a peptide link between E protein monomers can bypass the requirement for the intermolecular disulfide bond; to determine if a member of the E protein family (E2-2) which lacks the conserved cysteine required for disulfide formation can act as a homodimer if forced to form a crosslink. 2) to purify and clone the protein(s) associated with the activity required for the reduction of the intermolecular disulfide bond. Protein purification will be accomplished by standard biochemical means using a gel mobility shift assay to follow activity through purification. These aims serve to further our understanding of transcriptional control mechanisms in this case of lymphoid cell differentiation by the E protein family of HLH proteins.