Determinants of Natural Killer Cell Ly49 Receptor Specificity

自然杀伤(NK)细胞是淋巴细胞，作为抵御病毒和肿瘤的第一道防线，摧毁病理改变的细胞。NK细胞表达针对健康细胞上普遍表达的I类(CI)主要组织相容性复合体分子的抑制性受体。由于感染病毒的细胞或肿瘤细胞通常发生的CI表达的丧失，缺乏NK抑制受体的参与，释放了对NK细胞的抑制，使它们能够破坏这种改变的细胞。Ly49是由小鼠和大鼠NK细胞表达的CI特异性受体大家族，但不表达人类。CI与β-2微球蛋白(B2M)结合，结合并呈递来自细胞内蛋白降解事件的多肽片段。我们和其他人发现，CI结合肽的特性可以决定Ly49对CI的识别。目前尚不清楚的是，健康细胞中调节NK细胞的内源性多肽的性质，以及Ly49对多肽的选择性在体内的意义。我们的长期目标是了解Ly49识别多肽选择性的分子基础。这项工作的意义和独特之处在于，它直接解决了NK细胞识别健康细胞的分子基础这一非常基本的生物学问题。此外，了解Ly49的识别，可能为宿主对感染的抵抗力提供新的见解，例如小鼠中的Hanta病毒，以及可能在马中的疾病，一个也具有扩大的Ly49基因家族的物种。我们假设某些特定的内源性多肽支持Ly49识别CI，对Ly49的识别将为Ly49监测的目的提供一个窗口。我们还假设，多肽的选择性决定了Ly49的亲和力阈值，并具有体内意义。为了验证这些假设，我们提出了以下目标：*1.评估控制Ly49C识别的内源性多肽的多样性和同一性。质谱学的进展已经确定了与CI结合的内源性多肽。我们将研究与CI(KB)结合的单个内源性多肽对Ly49C识别的支持，包括它们的分子性质和起源蛋白(来自肽序列数据库匹配)，以帮助控制Ly49C识别。*2.确定CI结合多肽同一性在确定Ly49亲和力中的影响。我们将用表面等离子体共振法测定Ly49C与不同CI(KB)-多肽复合物的亲和力。这将揭示Ly49C相互作用是否存在受结合肽特性控制的阈值亲和力。*3.确定Ly49肽选择性对体内NK细胞活性的影响。通过在B2M-/-背景上转基因，我们将表达基因工程单链多肽-CI复合体，以确定不同的单肽在体内支持NK细胞许可和功能的能力。

Natural killer (NK) cells are lymphocytes that act as a first line of defense against viruses and tumours, destroying pathologically altered cells. NK cells express inhibitory receptors specific for class I (CI) major histocompatiblity complex molecules ubiquitously expressed on healthy cells. Lack of engagement of NK inhibitory receptors, due to loss of CI expression that typically occurs with virus-infected cells or tumour cells, releases inhibition of NK cells, allowing them to destroy such altered cells. Ly49 are large families of major class of CI-specific receptors expressed by mouse and rat NK cells, but not humans. CI associate with beta-2 microglobulin (b2m) and bind and present peptide fragments from intracellular proteolytic events. We, and others, have found that the identity of the CI-bound peptide can determine Ly49 recognition of CI. What is unclear, is the nature of endogenous peptides in healthy cells that regulate NK cells, and the in vivo significance of peptide selectivity by Ly49. Our long-term goal is to understand the molecular basis of peptide selectivity of Ly49 recognition. The significance and uniqueness of this undertaking is that it directly addresses the very fundamental biological question of the molecular basis of healthy cell recognition by NK cells. Furthermore, understanding Ly49 recognition, may provide novel insights into host resistance to infections such as hanta viruses in mice, and possibly diseases in horses, a species which also has an expanded Ly49 gene family. We hypothesize that certain and specific endogenous peptides support CI recognition by Ly49, identification of which will provide a window into the purpose of Ly49 surveillance. We also hypothesize that peptide selectivity determines Ly49 affinity thresholds, and has in vivo significance. To test these hypotheses, we propose the following objectives:***1. Assess diversity and identity of endogenous peptides controlling Ly49C recognition. Advances in mass spectrometry have identified endogenous peptides bound to CI. We will examine individual endogenous peptides bound to CI (Kb) in supporting Ly49C recognition, including their molecular properties and proteins of origin (from peptide sequence database matching), in contributing to the control of Ly49C recognition.****2. Define the influence of CI bound peptide identity in determining Ly49 affinity. We will determine the affinities of Ly49C for different CI (Kb)-peptide complexes by surface plasmon resonance. This should reveal whether there are threshold affinities for Ly49C interaction controlled by identities of bound peptides.***3. Determine the influence of Ly49 peptide selectivity on NK cell activity in vivo. By transgenesis on a b2m-/- background we will express genetically engineered single chain peptide-CI complexes to determine the ability of distinct individual peptides to support licensing and function of NK cells in vivo.**