Empirical validation of a novel HLA-disease association theory in skin and rheumatic diseases

皮肤和风湿病中新型 HLA 疾病关联理论的实证验证

• 批准号: 9464174
• 负责人: Joseph Holoshitz
• 金额: $ 38.79万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9464174
• 关键词: Adolescent; Affinity; Affinity Chromatography; Algorithms; Alleles; Antigens; Binding; Bioinformatics; Biological Assay; Cell Differentiation process; Cell Surface Proteins; Cell Surface Receptors; Cells; Code; Complement; Complementarity Determining Regions; Crystallization; Disease; Elements; Event; Gene Expression Profile; Genes; Goals; HLA Antigens; Health; Human; Immobilization; Inflammatory; Inflammatory Arthritis; Intervention; Knowledge; Ligands; Maps; Mass Spectrum Analysis; Molecular Conformation; Mus; Myositis; Ontology; Pathway interactions; Peptides; Peripheral Blood Mononuclear Cell; Phase; Process; Proteins; Quantitative Reverse Transcriptase PCR; Rheumatism; Risk; Role; Severities; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Sjogren' s Syndrome; Solid; Structure; Surface; Systemic Lupus Erythematosus; T-Lymphocyte; Time; Transgenic Mice; Validation; base; crosslink; experimental study; genetic risk factor; interest; macrophage; magnetic beads; novel; novel therapeutic intervention; receptor; skin disorder; synthetic peptide; tandem mass spectrometry; theories; trait; transcriptome; transcriptome sequencing; translational study

ABSTRACT Particular class II human leukocyte antigen (HLA) alleles have been found to be major genetic risk factors in many skin and rheumatic diseases, but the mechanisms underlying the associations are unknown. The prevailing paradigm, which had been based on the MHC restriction paradigm four decades ago, postulates that allele-specific presentation of self or foreign antigens is the culprit; however, this hypothesis remains unverified to this day. The unconventional theory presented here postulates that in addition to presenting peptidic antigens to T cells, HLA-DR molecules express signal transduction ligands that interact with surface receptors and trigger allele-specific signaling events. Under certain environmental conditions or stochastic events, such interactions provoke aberrant cellular events that may increase the risk and/or severity of skin and rheumatic diseases. Based on structural, functional and evolutionary considerations, we propose here that a region on the HLA-DR beta chain, called `cusp' - based on its crystal structure conformation - hosts allele-specific signal transduction ligands. To examine the HLA Cusp theory, we propose to perform a set of experiments to validate key elements in this novel concept. In the 2-year discovery period (R61 phase) we will perform RNA sequencing of cells stimulated by synthetic peptides corresponding to cusp regions coded by representative HLA-DRB1alleles. This will be followed by bioinformatics analyses to determine cusp allele- specific signature transcriptomes and gene ontologies. Additionally, the same allele-specific cusp peptides will be immobilized on solid matrices; cell surface proteins will be allowed to interact with the ligands and eluates will be analyzed by mass spectrometry. Proteins of interest will be expressed and purified, and their interaction with the ligands will be validated by cell-free binding assays. Candidate pathways discovered during the two-year R61 phase will be functionally characterized during year-3 (R33 phase). Here, cusp- activated signal transduction pathways will be mapped, and their biologic significance will be determined in translational experimental settings. At the end of this 3-year project, it is expected that novel, or previously unrecognized, HLA-DRB1allele-coded cusp-activated pathways will have been identified. Due to the involvement of the MHC in diverse biologic processes on the one hand, and the enigmatic mechanistic basis of its association with many health traits and diseases on the other, functional validation of the HLA Cusp theory could have a transformative effect.

摘要 已发现特定的II类人类白细胞抗原（HLA）等位基因是遗传性白细胞增多症的主要遗传风险因素。 许多皮肤和风湿性疾病，但相关机制尚不清楚。的 40年前基于MHC限制性范式的流行范式假设， 自身或外源抗原的等位基因特异性呈递是罪魁祸首;然而，这一假设仍然存在。 至今未被证实。这里提出的非传统理论假设，除了提出 HLA-DR分子表达与T细胞表面抗原相互作用的信号转导配体， 受体并触发等位基因特异性信号传导事件。在一定的环境条件下或随机 在一些情况下，这种相互作用引起异常细胞事件，其可增加皮肤损害的风险和/或严重性。 和风湿性疾病。基于结构，功能和进化的考虑，我们建议在这里， HLA-DR β链上的一个区域，根据其晶体结构构象被称为“尖端”， 等位基因特异性信号转导配体。为了检验HLA Cusp理论，我们建议执行一组 实验来验证这一新概念的关键要素。在两年的探索期（R61阶段），我们将 对由合成肽刺激的细胞进行RNA测序，所述合成肽对应于由 代表性HLA-DRB 1等位基因。随后将进行生物信息学分析，以确定尖点等位基因- 特定的签名转录组和基因本体。此外，相同的等位基因特异性尖肽将 固定在固体基质上;细胞表面蛋白质将与配体和洗脱液相互作用 将通过质谱分析。将表达和纯化感兴趣的蛋白质，并将它们的 与配体的相互作用将通过无细胞结合试验来验证。发现的候选途径 在两年的R61阶段，将在第3年（R33阶段）进行功能鉴定。在这里，尖- 将绘制激活的信号转导通路，并确定其生物学意义， 翻译实验设置。在这个为期3年的项目结束时，预计小说，或以前 未被识别的HLA-DRB 1等位基因编码的尖端激活通路将被识别。由于 一方面，MHC参与各种生物过程，另一方面， 另一方面，HLA Cusp的功能验证， 理论可能会产生变革性的影响。