Role and regulation of the splicing factor ASF/SF2 in human T lymphocyte physiolo

描述(由申请者提供)：这份K01申请书旨在帮助申请者具备建立系统性红斑狼疮(SLE)研究的独立研究计划所需的技能。系统性红斑狼疮是一种原因不明的自身免疫性疾病，主要影响育龄妇女，影响身体的多个器官系统。细胞内信号事件的异常改变导致的T细胞功能障碍被认为是该病发病机制的核心。一种称为CD3 Zeta链的关键信号分子在SLE患者的T细胞中异常低表达，这一缺陷导致SLE T细胞的功能低下。半衰期较差的CD3 Zeta m RNA的异常选择性剪接(AS)形式的表达是SLE T细胞Zeta链表达减少的原因之一。异常剪接是系统性红斑狼疮的常见特征，涉及T细胞信号转导、迁移和细胞因子产生的其他关键基因，这些基因的调控尚不清楚。为了了解CD3 Zeta选择性剪接的调节，申请人使用了一种发现方法，并鉴定了几种与CD3 Zeta mRNA结合的蛋白质。在这些蛋白质中，一个很有希望的候选蛋白是丝氨酸精氨酸(SR)家族成员选择性剪接因子/剪接因子2(ASF/SF2)。申请人发表，在来自健康个体的T细胞中，ASF/SF2通过抑制AS亚型的产生来调节CD3 Zeta链的表达。对SLE患者T细胞的检测显示，患者ASF/SF2的表达降低，提示ASF/SF2的异常表达可能与其T细胞功能缺陷有关。此外，ASF/SF2的表达水平似乎与SLE患者的疾病活动性呈负相关。申请人的初步研究表明，剪接调节因子ASF/SF2在T细胞生理学中具有潜在的作用 SLE病理生理学。根据初步证据，本项目的假设是ASF/SF2调节人类T细胞功能，可能是SLE T细胞缺陷的贡献者。对于这一假说，本项目旨在了解ASF/SF2在人类T淋巴细胞中的作用，并确定ASF/SF2是否可能代表SLE的疾病标志物。方法将包括对人类T细胞进行生化分析，并开发一种细胞系，以实现ASF/SF2的可控表达。该项目的具体目标是1)确定TCR刺激是否调节人类T淋巴细胞中ASF/SF2的表达和活性；2)确定ASF/SF2如何调节T细胞功能；3)确定SLE T细胞中ASF/SF2的表达水平是否代表疾病标志物。为了实现这些目标，申请者需要扩展她在细胞和分子免疫学方面的技术和智力技能，并精通SLE患者研究的设计、解释和分析。贝丝以色列女执事医疗中心和哈佛医学院系统的其他附属机构的研究环境将为申请人提供资源，以在5年内实现她的目标。

DESCRIPTION (provided by applicant): This K01 application is designed to prepare the applicant with the skills necessary to establish an independent research program on the study of systemic lupus erythematosus (SLE). SLE is an autoimmune disease of unknown cause which mainly afflicts women in their child bearing years and affects multiple organ systems of the body. T cell dysfunction due to abnormally altered intracellular signaling events is thought to be central in the pathogenesis of this disease. A critical signaling molecule known as the CD3 zeta chain is expressed in abnormally low amounts in the T cells of SLE patients, and this defect contributes to the poor functional capacity of the SLE T cells. Expression of an abnormal alternatively spliced (AS) form of the CD3 zeta mRNA with poor half life is a contributor to the reduced expression of zeta chain in SLE T cells. Aberrant splicing is a common feature observed in SLE and involves other critical genes involved in T cell signaling, migration, and cytokine production and the regulation of these genes has not been characterized. To understand the regulation of the CD3 zeta alternative splicing, the applicant used a discovery approach and identified several proteins binding to the CD3 zeta mRNA. Among these proteins, a promising candidate is the serine arginine (SR) family member Alternative Splicing Factor/Splicing Factor 2 (ASF/SF2). The applicant published that in T cells from healthy individuals, ASF/SF2 regulates CD3 zeta chain expression by suppressing production of the AS isoform. Examination of T cells from SLE patients revealed that patients had reduced expression of ASF/SF2 suggesting that aberrant ASF/SF2 expression may contribute to their defective T cell function. Additionally, the ASF/SF2 expression levels appeared to inversely correlate with the disease activity of SLE patients. The applicant's preliminary studies have shown a novel role for the splicing regulator ASF/SF2 in T cell physiology with a potential role in SLE pathophysiology. Based on the preliminary evidence, the hypothesis of this project is that ASF/SF2 regulates human T cell function and may represent a contributor to the SLE T cell defect. Toward the hypothesis, this project is aimed to understand the role of ASF/SF2 in human T lymphocytes and to determine whether ASF/SF2 may represent a disease marker in SLE. Methods will include biochemical analyses of human T cells and the development of a cell line to enable controlled expression of ASF/SF2. The specific aims of the project are 1) To determine whether TCR stimulation regulates ASF/SF2 expression and activity in human T lymphocytes 2) To determine how ASF/SF2 regulates T cell function and 3) To determine whether ASF/SF2 expression levels in SLE T cells may represent a disease marker. To accomplish these aims, the applicant will need to expand her technical and intellectual skills in cellular and molecular immunology and become proficient in the design, interpretation and analysis of studies in SLE patients. The research environment at Beth Israel Deaconess Medical Center and the other affiliates of the Harvard Medical School system will provide the applicant with the resources to reach her goals within 5 years.