Expression and Function of Human BRIGHT

人类免疫缺陷病X连锁α γ球蛋白血症（XLA）是由布鲁顿酪氨酸激酶（Btk）突变引起的，但尚不清楚该激酶的缺陷表达如何导致低血清免疫球蛋白水平和B淋巴细胞发育的阻断。具有Btk点突变的小鼠已经产生了用于研究X-连锁免疫缺陷的小鼠模型，但是小鼠中疾病的表型没有人中严重。Xid小鼠产生循环B淋巴细胞，尽管水平低于正常小鼠，并且B细胞发育的阻断是在不成熟的B细胞阶段而不是前B细胞到前B细胞阶段，小鼠和人中疾病表现差异的原因尚不清楚，但可能是由于B细胞发育的内在差异或Btk与其他蛋白质的差异相互作用。我们在小鼠中的研究表明，转录因子Bright（免疫球蛋白重链转录的B细胞调节因子）与Btk相互作用。Bright是一种70 kDa的蛋白质，与免疫球蛋白重链基因座的启动子和增强子序列结合，在那里它与转录的增加有关。尽管免疫球蛋白重链基因座的序列与转录增加有关。虽然xid细胞可以表达Bright蛋白，但它们缺乏Bright DNA结合活性。此外，虽然Bright和Btk从对照B淋巴细胞共沉淀，但它们在xid细胞中似乎不相互作用。 对人类Bright知之甚少，但我们的初步数据表明，它也与Btk相互作用，并且可以在正常外周血淋巴细胞中诱导。然而，我们已经观察到小鼠和人的类β-淀粉样蛋白之间的一些差异。为了更多地了解人类Bright及其在XLA中的潜在作用，有必要评估Bright表达和与Btk的相互作用。我们建议利用其他三个项目产生的信息，以评估光明的作用，在人类淋巴细胞生成。建议的具体目标是：（1）通过RT-PCR、Western印迹和分选亚群的迁移率变动测定来确定Bright在人淋巴细胞发育期间表达的位置，（2）使用共沉淀、等离子体共振、共转染和定向酵母双杂交测定来确定人Bright和Btk之间发生的分子相互作用，（3）使用相同的技术确定XLA突变如何影响这些相互作用，以及（4）使用经典方法和抗体促进的克隆鉴定Bright的人类基因靶标。这些研究将产生关于人类Bright及其与Btk相互作用的重要新信息，也可能导致对B细胞发育和XLA的深入了解。

The human immunodeficiency disease X-linked a gamma globulinemia (XLA) is caused by mutations in Bruton's tyrosine kinase (Btk) but it is not clear how defective expression of this kinase leads to low serum immunoglobulin levels and blocks in B lymphocyte development. Mice with a point mutation in Btk have produce a murine model for studying x- linked immunodeficiency, but the phenotype of the disease in mice is less severe than in man. Xid mice produce circulating B lymphocytes, albeit at lower levels than in normal mice, and the block in B cell development is at the immature B cell stage instead of the pro-B to pre-B cell stage, as occurs in man. The reasons for the differences in presentation of the disease in mouse and man are unknown, but could be due to intrinsic differences in B cell development or to differential interactions of Btk with additional proteins. Our studies in mice indicate that the transcription factor Bright (B cell regulator of immunoglobulin heavy chain transcription) interacts with Btk. Bright is a 70 kDa protein that binds to promoter and enhancer sequences of the immunoglobulin heavy chain locus where it has been associate with increases in transcription. Although sequences of the immunoglobulin heavy chain locus where it has been associated with increases in transcription. Although xid cells can express Bright protein, they are deficient in Bright DNA-binding activity. Furthermore, while Bright and Btk co-precipitate from control B lymphocytes, they do not appear to interact in xid cells. Little is known about human Bright, but our preliminary data suggest that it also interacts with Btk and that it can be induced in normal peripheral blood lymphocytes. However, we have observed a number of differences between mouse and human Bright-like proteins. To learn more about human Bright and its potential role in XLA, it will be necessary to evaluate Bright expression and interactions with Btk. We propose to utilize the information produced from the other three projects to assess Bright's role in human lymphopoiesis. The specific aims proposed are: (1) to define where Bright is expressed during human lymphocyte development by RT-PCR, Western blotting and mobility shift assay of sorted subpopulations, (2) to define the molecular interactions that occur between human Bright and Btk using co-precipitation, plasmon resonance, co-transfection and directed yeast two-hybrid assays, (3) to determine how XLA mutations affect these interactions using the same techniques, and (4) to identify human gene targets for Bright using classical methods and antibody facilitated cloning. These studies will yield important new information about human Bright and its interactions with Btk, and may also lead to insights into B cell development and XLA.