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

摘要 特定的人类第二类白细胞抗原(人类白细胞抗原)等位基因被发现是 许多皮肤和风湿性疾病，但潜在的联系机制尚不清楚。这个 40年前基于MHC限制范式的主流范式假设 自身或外来抗原的等位基因特异性表达是罪魁祸首；然而，这一假设仍然存在。 至今仍未得到证实。这里提出的非常规理论假设，除了提出 T细胞的多肽抗原，HLA-DR分子表达与表面相互作用的信号转导配体 受体并触发等位基因特有的信号事件。在一定的环境条件下或随机的 事件，这种相互作用会引发异常的细胞事件，可能会增加皮肤的风险和/或严重性 和风湿病。基于结构、功能和进化方面的考虑，我们在此建议 根据其晶体结构构象，人类白细胞抗原-DRβ链上的一个区域被称为‘尖点’-宿主 等位基因特异性信号转导配体。为了检验人类白细胞抗原尖点理论，我们建议执行一组 实验以验证这一新概念中的关键要素。在2年的发现期(R61阶段)中，我们将 对合成的多肽刺激的细胞进行RNA测序，这些多肽对应于由 具有代表性的HLA-DRB1等位基因。随后将进行生物信息学分析，以确定Cusp等位基因- 特定的签名转录本和基因本体论。此外，相同的等位基因特定的尖点多肽将 固定在固体基质上；细胞表面蛋白将被允许与配体和洗脱液相互作用 将通过质谱仪进行分析。感兴趣的蛋白质将被表达和纯化，他们的 与配体的相互作用将通过无细胞结合试验进行验证。发现候选路径 在为期两年的R61阶段，将在第三年(R33阶段)进行功能特征描述。给你，尖头- 激活的信号转导通路将被绘制出来，它们的生物学意义将在 翻译性实验设置。在这个为期3年的项目结束时，预计这部小说或之前 未知的、人类白细胞抗原-DRB1等位基因编码的尖端激活通路将被识别出来。由于 一方面，MHC参与了不同的生物过程，以及神秘的机制基础 一方面，它与许多健康特征和疾病的相关性，另一方面，对人类白细胞抗原Cusp的功能验证 理论可能会产生变革性的影响。

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.