POTENTIAL ROLE OF TFII-I IN IMMUNODEFICIENCY
POTENTIAL ROLE OF TFII-I IN IMMUNODEFICIENCY
批准号:
6044745
负责人:
Ananda L Roy
金额:
$28.72万
依托单位:
依托单位国家:
美国
项目类别:
财政年份:
2000
资助国家:
美国
项目状态:
已结题
起止时间:
2000-03-01 至 2005-02-28
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
中文摘要
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。
英文摘要
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.
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Role of TFII-I in B cell function
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批准号:8084528
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项目类别:
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资助金额:$39.63万
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财政年份:2010
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负责人:Ananda L Roy
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MOLECULAR ANALYSIS OF WILLIAMS SYNDROME
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Molecular Mechanisms of Acute Promyelocytic Leukemia
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资助金额:$7.93万
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Molecular Mechanisms of Acute Promyelocytic Leukemia
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资助金额:$7.93万
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MOLECULAR ANALYSIS OF WILLIAMS SYNDROME
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MOLECULAR ANALYSIS OF WILLIAMS SYNDROME
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Potential Role of TFII-I in Immunodeficiency
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资助金额:$30.0万
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依托单位:
POTENTIAL ROLE OF TFII-I IN IMMUNODEFICIENCY
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POTENTIAL ROLE OF TFII-I IN IMMUNODEFICIENCY
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批准号:6632087
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资助金额:$30.53万
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POTENTIAL ROLE OF TFII-I IN IMMUNODEFICIENCY
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批准号:6897411
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资助金额:$5.67万
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POTENTIAL ROLE OF TFII-I IN IMMUNODEFICIENCY
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批准号:6704707
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资助金额:$31.45万
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POTENTIAL ROLE OF TFII-I IN IMMUNODEFICIENCY
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MECHANISMS OF TF11/1 FUNCTION AND REGULATION
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依托单位:
海外基金