Expression and Function of Human BRIGHT

人类免疫缺陷疾病X-linked a - 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血清免疫球蛋白水平降低并阻碍B淋巴细胞发育。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，但是该疾病在小鼠中的表型没有在人类中严重。Xid小鼠产生循环B淋巴细胞，尽管其水平低于正常小鼠，并且B细胞发育的阻滞发生在未成熟B细胞阶段，而不是像人类那样发生在前B细胞到前B细胞阶段。小鼠和人的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与其他蛋白质的不同相互作用。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，与免疫球蛋白重链位点的启动子和增强子序列结合，在那里它与转录增加有关。尽管免疫球蛋白重链位点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淋巴细胞中共同沉淀，但它们似乎不会在氧化细胞中相互作用。我们对人类Bright知之甚少，但我们的初步数据表明，它也能与Btk相互作用，并能在正常外周血淋巴细胞中被诱导。然而，我们已经观察到小鼠和人类Bright-like蛋白之间的许多差异。为了进一步了解人类Bright及其在XLA中的潜在作用，有必要对Bright的表达及其与Btk的相互作用进行研究。我们建议利用其他三个项目产生的信息来评估Bright在人类淋巴系统中的作用。建议的具体目标是：(1)通过RT-PCR、Western blotting和分类亚群的迁移转移试验确定Bright在人类淋巴细胞发育过程中的表达位置；(2)通过共沉淀、等离子体共振、共转染和定向酵母双杂交试验确定人类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。(4)利用经典方法和抗体克隆技术鉴定Bright的人类基因靶点。这些研究将提供关于人类Bright及其与Btk相互作用的重要新信息，也可能有助于对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.