POTENTIAL ROLE OF TFII-I IN IMMUNODEFICIENCY

TFII-I 在免疫缺陷中的潜在作用

• 批准号: 6632087
• 负责人: Ananda L Roy
• 金额: $ 30.53万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-03-01 至 2005-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6632087
• 关键词: B cell receptor; B lymphocyte; Williams syndrome; biological signal transduction; gene deletion mutation; hypogammaglobulinemia; laboratory mouse; mass spectrometry; molecular pathology; phosphorylation; point mutation; protein localization; protein sequence; protein tyrosine kinase; site directed mutagenesis; transcription factor; transfection

TFII-II is an important multi-functional transcription factor that links events to transcription in several genes. TFII-I is constitutively associated with Bruton's tyrosine kinase (Btk), a non-receptor tyrosine kinase that is essential for normal B cell function, as its mutation causes X-linked agammaglobulinemia (XLA) in humans and X-linked immune deficiency (xid) in mice. We propose that TFII-I is an important and novel component in linking Btk-mediated signaling to transcription in B cells. Furthermore, the TFII-I gene gets deleted in William's syndrome (WS) which is a neuro-developmental disorder with multi-system manifestations, including supravalvar aortic stenosis, hypercalcemia in infancy, mental retardation and cognitive defects. Thus, TFII-I appears to be involved in two genetic disorders: William's Syndrome and X-linked agammaglobulinemia (XLA). Knowledge gained from these studies may help us better understand a critical Btk dependent pathway that links B cell receptor mediated signal transduction to B cell specific transcription. These studies may also ultimately help identify potential target gene(s) that are affected by mutations in Btk. Importantly, these studies may establish possible connections between the neuro-developmental disorders (as in WS) and immuno-developmental disorders (as in XLA). Toward a better understanding of TFII-I function in Btk mediated immune response, we will first map the region(s) in TFII-I important for its physical and functional interactions with BTK. We will determine by deletion and point mutation the region(s) in TFII-I that is important for its interaction with Btk, followed by mapping the sites in TFII-I that are tyrosine phosphorylated by Btk in vitro and in vivo by a combination of site directed mutagenesis, phosphopeptide, finger printing, and mass spectrometric analysis. We will also analyze these mutants in functional transient transfection assays. To determine the functions of TFII-I and its biochemical interactions with Btk in B cells, we will employ in vivo transcriptional analysis. To determine the functions of TFII-I and its biochemical interactions with Btk in B cells, we will employ in vivo transcriptional analysis followed by the interaction studies by co- immunoprecipitation and ectopic expression of mutant forms of TFII-I in B cells. We will also stably express wild type and mutant forms of TFII-I, and Btk in B cell lines, and genetically delete TFII-I from chicken B cells. Finally, to ascertain the localization of TFII-I in the absence and in the presence of non-activated versus activated Btk, first, we will co-express various mutants of TFII-I with Btk in COS cells. Subsequently, we will employ freshly isolated primary splenic B cells derived from wild type, xid and Btk-/- mice and study the localization and tyrosine phosphorylation of TFII-I in the absence and in presence of B cell receptor signaling.

TFII-II是一种重要的多功能转录因子，它将事件与几种基因的转录联系起来。TFII-I - i与布鲁顿酪氨酸激酶（Btk）构成相关，布鲁顿酪氨酸激酶是一种非受体酪氨酸激酶，对正常B细胞功能至关重要，因为它的突变导致人类x -连锁无球蛋白血症（XLA）和小鼠x -连锁免疫缺陷（xid）。我们认为TFII-I是连接btk介导的信号传导与B细胞转录的重要新成分。此外，TFII-I基因在William's综合征（WS）中缺失，WS是一种多系统表现的神经发育障碍，包括瓣上主动脉瓣狭窄、婴儿期高钙血症、智力低下和认知缺陷。因此，TFII-I似乎与两种遗传疾病有关：威廉氏综合征和x连锁无球蛋白血症（XLA）。从这些研究中获得的知识可以帮助我们更好地理解连接B细胞受体介导的信号转导到B细胞特异性转录的关键Btk依赖途径。这些研究也可能最终有助于确定受Btk突变影响的潜在靶基因。重要的是，这些研究可能在神经发育障碍（如WS）和免疫发育障碍（如XLA）之间建立可能的联系。为了更好地了解TFII-I在Btk介导的免疫应答中的功能，我们将首先绘制TFII-I中与Btk物理和功能相互作用重要的区域。我们将通过缺失和点突变来确定TFII-I中与Btk相互作用重要的区域，然后通过位点定向突变、磷酸化肽、指纹和质谱分析相结合，绘制TFII-I中被Btk体外和体内酪氨酸磷酸化的位点。我们还将在功能瞬时转染试验中分析这些突变体。为了确定TFII-I在B细胞中的功能及其与Btk的生化相互作用，我们将采用体内转录分析。为了确定TFII-I在B细胞中的功能及其与Btk的生化相互作用，我们将采用体内转录分析，然后通过共免疫沉淀和突变形式的TFII-I - i在B细胞中的异位表达进行相互作用研究。我们还将在B细胞系中稳定表达野生型和突变型TFII-I和Btk，并从鸡B细胞中基因删除TFII-I。最后，为了确定TFII-I在Btk未激活和激活情况下的定位，首先，我们将TFII-I与Btk在COS细胞中共表达各种突变体。随后，我们将利用野生型、xid和Btk-/-小鼠新鲜分离的原代脾B细胞，研究在没有和存在B细胞受体信号的情况下TFII-I的定位和酪氨酸磷酸化。