CAREER: Mechanisms Regulating the Activity of the Pax-5 Transcription Factor During B-cell Development

职业：B 细胞发育过程中 Pax-5 转录因子活性的调节机制

• 批准号: 9874795
• 负责人: Patty Zwollo
• 金额: $ 40万
• 依托单位: College of William and Mary
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2004-04-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9874795&HistoricalAwards=false
• 关键词: CAREER; Mechanisms; Regulating; Activity; Pax

Zwollo, P The transcription factor Pax-5 is expressed during the early stages of B-cell development and influences the expression of several B cell-specific genes, most of which are involved in cell development and proliferation. Over the past grant period, the existence of alternatively spliced isoforms of the murine Pax-5 gene was investigated. In addition to the existing isoform (Pax-5a), three new isoforms, Pax-5b, 5d, and 5e, were isolated. In isoforms Pax-5b and Pax-5e, exon 2 has been spliced out, resulting in proteins with an incomplete DNA-binding domain. In Pax-5d and Pax-5e, the 3' region which encodes the transactivating domain, is deleted and replaced by a novel sequence with unknown function. Recent studies revealed that both Pax-5d and Pax-5a protein levels are high in normal B-cells, in contrast to cell lines of the B-cell lineage, where Pax-5d levels are very low. When normal B cells were activated by the B-cell mitogen lipopolysaccharide, the protein level of Pax-5d, but not 5a, decreased rapidly. The high protein levels of Pax-5d in resting B-cells, as well as its low levels in proliferating B-cells, would be in agreement with it functioning as a dominant negative regulator of proliferation or growth. Pax-5b protein, on the other hand, is only present during the later stages of B-cell development, including the mature B and plasma cell stages. The presence of Pax-5b so late in development was unexpected and is interesting in light of its possible regulatory roles during the terminal stages of B-cell development. In this project, the transcriptional activities of Pax-5 isoforms 5a, 5b, and 5d will be investigated. These studies are expected to lead to an increased understanding of specific mechanisms that regulate activity of the transcription factor Pax-5 during B-cell differentiation. Such mechanisms may include both the functional activities of its alternative isoforms as well as the differential regulation of their protein stability. The protein Pax-5a has been shown to play important roles during the development of antibody-producing cells. Pax-5 is a "regulator" protein that affects the amount of antibodies that are made in this cell type. Pax-5 also plays a role in cell proliferation, thus regulating the amount of "proliferation" determining proteins in the cells, but this process is not well understood. In this project, the main question will be: what is the role of Pax-5a, as well as two other Pax-5 like proteins (named Pax-5b and Pax-5d) in the development and activation of antibody producing cells. This research will provide insight into possible mechanisms that Pax-5a, 5b, and 5d use to make sure that cells produce antibodies at the right time and in the right place.

转录因子Pax-5在B细胞发育的早期阶段表达，影响几个B细胞特异性基因的表达，其中大部分参与细胞的发育和增殖。在过去的资助期间，研究了小鼠Pax-5基因的选择性剪接异构体的存在。除已有的Pax-5a亚型外，还分离到了Pax-5b、Pax-5d和Pax-5e三种新亚型。在异构体Pax-5b和Pax-5e中，外显子2被剪接，导致蛋白质的DNA结合域不完整。在Pax-5d和Pax-5e中，编码反式激活结构域的3‘区被缺失，并被一个新的功能未知的序列所取代。最近的研究表明，在正常的B细胞中，Pax-5d和Pax-5a蛋白水平都很高，而在B细胞系中，Pax-5d的水平非常低。当B细胞有丝分裂原脂多糖激活正常B细胞时，Pax-5d蛋白水平迅速下降，而5a蛋白水平下降不明显。静息B细胞中Pax-5d蛋白水平较高，而增殖中B细胞中Pax-5d蛋白水平较低，这与Pax-5d作为主要的增殖或生长负调控因子的作用相一致。另一方面，Pax-5b蛋白只存在于B细胞发育的后期，包括成熟的B细胞和浆细胞阶段。Pax-5b在发育后期的存在是意想不到的，考虑到它在B细胞发育的终末阶段可能起到的调节作用，这是很有趣的。在本项目中，将研究Pax-5亚型5a、5b和5d的转录活性。这些研究有望加深对B细胞分化过程中调节转录因子Pax-5活性的具体机制的理解。这种机制可能既包括其替代异构体的功能活性，也包括对其蛋白质稳定性的差异调节。Pax-5a蛋白已被证明在抗体产生细胞的发育过程中发挥重要作用。Pax-5是一种“调节性”蛋白质，可以影响这种细胞中产生的抗体数量。Pax-5还在细胞增殖中发挥作用，从而调节细胞中决定细胞增殖的蛋白质的数量，但这一过程尚不清楚。在这个项目中，主要的问题将是：Pax-5a以及另外两个类似Pax-5的蛋白(命名为Pax-5b和Pax-5d)在抗体产生细胞的发育和激活中扮演什么角色。这项研究将深入了解Pax-5a、5b和5d用来确保细胞在正确的时间和正确的地点产生抗体的可能机制。