Molecular and functional investigation of the role of HLA-F in immune regulation

项目摘要/摘要 HL A-F是一种非经典的I类MHC(Ib)分子，已发现在多种癌症中表达， 被证明在HIV和腺病毒感染，神经系统自身免疫性疾病ALS中起作用，并表达 在整个怀孕期间。尽管这种蛋白质在这些情况下具有潜在的重要性，但人们对此知之甚少。 这种分子在其功能方面，甚至在何种构象状态下表达。我们最近做了 研究表明，除了表达为重链状态或开放构象(HL A-FOC)外，HL A-F 也可以表达为一个与β2M亚基(PhLA-F)相关的成键多肽递呈分子。 多肽以一种非常规的方式呈现，N-末端不固定在凹槽内， 以多肽锚定为特征的翻译后修饰的可能性。尽管取得了这些进展，但 这些构象状态是如何调节的，它是如何与各种受体结合的，目前还不清楚 在这些构象状态中的每一个，以及人类白细胞抗原-F在其不同的肿瘤监测环境中的作用， 自身免疫和生殖。因此，这项提案的目标侧重于解决这些问题，包括： 目的1：从结构和功能两个方面研究人类白细胞抗原-F分子在体内的各种构象状态。 人类的健康和疾病。我们将继续进行人类白细胞抗原F亚型的结构研究，以了解这些亚型是如何 两种状态彼此不同。使用构象特异性抗体，我们将确定哪些细胞类型表达 哪一种(或两者)形成，以及健康细胞和疾病细胞之间的差异。我们还将进行多肽洗脱 来自一系列人类来源的研究，以确定多肽谱系是否根据细胞来源而变化 或疾病。目的2：鉴定和分析调节人类白细胞抗原-1产生或交换的因素。 细胞内的F构象和剪接形式。我们将探索在转换过程中可能起作用的细胞因素 人类白细胞抗原F位于多肽和人类白细胞抗原FOC之间，以及一种有趣的未知功能的人类白细胞抗原F的剪接变异体。 最后，在目标3中，我们试图建立使人类白细胞抗原-F参与其各种构象的受体谱系 状态，确定其缔合的分子基础，并研究其功能后果 它们的约束力。我们将利用亚当斯实验室的结构、生物物理和功能专业知识来确定 结合这些构象状态的HLA-F的受体谱系，并在功能和 分子水平。

Project Summary/Abstract HLA-F is a nonclassical class I MHC (Ib) molecule that has been found expressed on a variety of cancers, shown to play a role in HIV and adenoviral infection, the neurological autoimmune disease ALS and is expressed throughout pregnancy. Despite the potential importance of this protein in these conditions, little is known about this molecule in terms of its function or even in which conformational state it is expressed. We have recently shown that, in addition to being expressed as a heavy chain only state, or open conformer (HLA-FOC), HLA-F can also be expressed as a bon fide peptide presenting molecule, associated with the β2m subunit (pHLA-F). Peptides are presented in an unconventional way, with the N-terminus not anchored within the groove and the potential for post-translational modifications featuring in peptide anchoring. Despite these advances, there remains much unknown about how these conformer states are regulated, how it engages its various receptors in each of these conformer states, and the role of HLA-F in its various environments of tumor surveillance, autoimmunity and reproduction. Thus, the aims of this proposal focus on addressing these questions and are: Aim 1: To investigate, structurally and functionally, the various conformer states that HLA-F adopts in human health and disease. We will pursue structural studies of the HLA-F isoforms to understand how these two states differ from each other. Using conformer-specific antibodies, we will determine what cell types express which (or both) forms and how this differs between healthy and disease cells. We will also pursue peptide elution studies from a range of human sources to determine if the peptide repertoire shifts depending on cellular origin or disease. Aim 2: To identify and analyze the factors that regulate the production or interchange of HLA- F conformers and splice forms in a cell. We will explore the cellular factors that may play a role in switching HLA-F between peptide-loaded and HLA-FOC as well as an intriguing splice variant of HLA-F of unknown function. Finally, in Aim 3 we seek to establish the receptor repertoire that engage HLA-F in its various conformer states, determine the molecular basis for their association and study the functional consequences of their binding. We will employ the structural, biophysical and functional expertise of the Adams lab to determine the receptor repertoire that engage these conformer states of HLA-F and study them at the functional and molecular level.