POTENTIAL ROLE OF TFII-I IN IMMUNODEFICIENCY

TFII-I 在免疫缺陷中的潜在作用

• 批准号: 6044745
• 负责人: Ananda L Roy
• 金额: $ 28.72万
• 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/6044745
• 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，它将事件与几个基因的转录联系起来。TFII-I与布鲁顿酪氨酸激酶（Btk）组成性相关，Btk是一种对正常B细胞功能至关重要的非受体酪氨酸激酶，因为其突变导致人类X连锁无丙种球蛋白血症（XLA）和小鼠X连锁免疫缺陷（xid）。我们认为TFII-I是连接Btk介导的信号转导与B细胞转录的重要和新的组分。此外，TFII-I基因在威廉综合征（WS）中缺失，这是一种具有多系统表现的神经发育障碍，包括瓣膜上主动脉瓣狭窄、婴儿期高钙血症、智力迟钝和认知缺陷。因此，TFII-I似乎涉及两种遗传疾病：威廉氏综合征和X连锁无丙种球蛋白血症（XLA）。从这些研究中获得的知识可以帮助我们更好地理解一个关键的Btk依赖性途径，该途径将B细胞受体介导的信号转导与B细胞特异性转录联系起来。这些研究也可能最终帮助确定受Btk突变影响的潜在靶基因。重要的是，这些研究可能建立神经发育障碍（如WS）和免疫发育障碍（如XLA）之间的可能联系。为了更好地理解TFII-I在Btk介导的免疫应答中的功能，我们将首先绘制TFII-I中对其与BTK的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中对其与Btk相互作用重要的区域，然后通过定点突变、磷酸肽、指纹图谱和质谱分析的组合，在体外和体内绘制TFII-I中被Btk酪氨酸磷酸化的位点。我们还将在功能性瞬时转染试验中分析这些突变体。为了确定TFII-I的功能及其在B细胞中与Btk的生物化学相互作用，我们将采用体内转录分析。为了确定TFII-I的功能及其与B细胞中Btk的生物化学相互作用，我们将采用体内转录分析，然后通过免疫共沉淀和突变形式的TFII-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形式的TFII-I和Btk，并从鸡B细胞中遗传删除TFII-I。最后，为了确定TFII-I在不存在和存在非活化Btk与活化Btk的情况下的定位，首先，我们将在COS细胞中共表达TFII-I的各种突变体与Btk。随后，我们将使用来自野生型、xid和Btk-/-小鼠的新鲜分离的原代脾B细胞，并研究在存在和不存在B细胞受体信号传导的情况下TFII-I的定位和酪氨酸磷酸化。