Modulation of B cell tolerance checkpoints by distinct Ras/Erk Pathways

通过不同的 Ras/Erk 通路调节 B 细胞耐受检查点

• 批准号: 8446910
• 负责人: Andre Limnander
• 金额: $ 7.84万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-01-10 至 2015-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8446910
• 关键词: 1,2-diacylglycerol; Address; Age; Alleles; Antibody-Producing Cells; Antigens; Apoptosis; Apoptotic; Autoantibodies; Autoantigens; Autoimmune Diseases; Autoimmune Process; Autoimmunity; B cell repertoire; B-Cell Development; B-Lymphocytes; Biochemical; Biochemistry; Biological Assay; Bone Marrow; Cell Line; Cells; Cessation of life; Defect; Development; Diglycerides; Disease; EF Hand Motifs; Ensure; Etiology; Event; Failure; Goals; Immature Bone; Immune; Immunocompetent; Immunoglobulin D; Immunoglobulin M; In Vitro; Lead; Lymphocyte; Lymphopenia; Mature B-Lymphocyte; Mediating; Minor; Molecular; Mus; Mutation; Output; Pathway interactions; Phenotype; Phosphorylation; Phosphorylation Site; Physiological; Point Mutation; Production; Property; Protein Biochemistry; Protein Isoforms; Proteins; Relative (related person); Retroviridae; Role; Serine; Signal Pathway; Signal Transduction; Site; Son of Sevenless Proteins; Specificity; Staging; Structure; System; Systemic Lupus Erythematosus; T-Cell Development; T-Lymphocyte; Testing; Tissues; cell type; in vivo; insight; lupus-like; mouse model; mutant; novel; overexpression; public health relevance; receptor; research study; response; sensor; therapeutic target

DESCRIPTION (provided by applicant): Establishment of a proper B cell repertoire that is immunocompetent but not autoimmune depends on critical developmental checkpoints that edit, silence or delete autoreactive cells. The properties of B cells change as they progress through distinct stages in development, but the signaling mechanisms by which antigen drives the different selection checkpoints are incompletely defined. We have recently described a novel Ca2+- dependent Erk signaling pathway in developing B cells that is pro-apoptotic and mediates B cell negative selection. This pathway requires PKC¿ and RasGRP proteins and loss of this pathway in PKCg-deficient mice results in increased survival of B cells during negative selection and subsequent development of an SLE-like disease with lymphoproliferation and autoantibody production. RasGRP1-deficient mice have a substantial developmental block in T cell development that results in T cell lymphopenia, but as they age they also develop an SLE-like disease with B cell lymphoproliferation and autoantibody production, the etiology of which is not well understood. In addition, a recently identified RasGRP1Anaef allele, which carries a point mutation in the second EF-hand of RasGRP1, also causes an SLE-like disease with distinct effects on T cell development from those observed in the RasGRP1-deficient mice. The first goal of this proposal is to determine whether the SLE-like phenotype in these RasGRP1 mouse models is B cell intrinsic, and if it is due to loss of pro-apoptotic Erk signaling during B cell development. Secondly, because we have identified Serine 332 on RasGRP1 as a putative PKCg target phosphosite that is required for the activation of this novel Ca2+-Erk pathway, I will use in vitro biochemistry experiments to determine the effect of this phosphorylation on the function and specificity of RasGRP1. Finally, I will develop retroviruses encoding mutant S332 RasGRP1 to determine the relevance of this phospho-site in B cell development in vivo. Successful completion of these studies will greatly advance our understanding of Ras/Erk signaling in B cell development in normal and pathological settings. Such insight is essential to define the molecular mechanisms that confer functional specificity to different Ras/Erk pathways, which in turn may pinpoint events that can serve as therapeutic targets while having minor or no consequences on closely related but functionally distinct pathways.

描述（由申请方提供）：具有免疫活性但非自身免疫性的适当B细胞库的建立取决于编辑、沉默或删除自身反应性细胞的关键发育检查点。B细胞的特性随着它们在发育中的不同阶段的进展而改变，但是抗原驱动不同选择检查点的信号传导机制还不完全确定。我们最近描述了一种新的钙依赖性ERK信号通路在发展中的B细胞，是促凋亡和介导B细胞负选择。这一途径需要PKC？和RasGRP蛋白质，并且在PKC g缺陷型小鼠中该途径的缺失导致阴性选择期间B细胞存活增加，并且随后发展为具有淋巴细胞增殖和自身抗体产生的SLE样疾病。RasGRP 1缺陷型小鼠在T细胞发育中具有实质性的发育阻滞，这导致T细胞淋巴细胞减少症，但是随着它们年龄的增长，它们也发展出具有B细胞淋巴细胞增殖和自身抗体产生的SLE样疾病，其病因尚不清楚。此外，最近鉴定的RasGRP 1Anaef等位基因，其在RasGRP 1的第二EF-手中携带点突变，也导致SLE样疾病，其对T细胞发育的影响与RasGRP 1缺陷小鼠中观察到的不同。该建议的第一个目标是确定这些RasGRP 1小鼠模型中的SLE样表型是否是B细胞固有的，以及是否是由于B细胞发育期间促凋亡Erk信号传导的丧失。其次，因为我们已经确定了RasGRP 1上的丝氨酸332作为一个假定的PKC β靶磷酸化位点，这是激活这种新的Ca 2 +-ERK途径所必需的，我将使用体外生物化学实验来确定这种磷酸化对RasGRP 1的功能和特异性的影响。最后，我将开发编码突变S332 RasGRP 1的逆转录病毒，以确定该磷酸化位点在体内B细胞发育中的相关性。这些研究的成功完成将极大地推进我们对正常和病理条件下B细胞发育中Ras/Erk信号传导的理解。这样的洞察力是必不可少的定义的分子机制，赋予功能特异性不同的Ras/Erk途径，这反过来可能会查明事件，可以作为治疗靶点，而有轻微的或没有后果密切相关，但功能不同的途径。