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细胞表达对I类（CI）主要组织相容性复合物分子特异性的抑制性受体，其在健康细胞上普遍表达。由于通常在病毒感染的细胞或肿瘤细胞中发生的Cl表达的丧失，NK抑制性受体的参与的缺乏释放NK细胞的抑制，允许它们破坏这种改变的细胞。Ly 49是由小鼠和大鼠NK细胞表达的CI特异性受体的主要类别的大家族，但不是人类。CI与β-2微球蛋白（b2 m）结合，并结合和呈递来自细胞内蛋白水解事件的肽片段。我们和其他人已经发现，CI结合肽的身份可以决定Ly 49对CI的识别。目前尚不清楚的是健康细胞中调节NK细胞的内源性肽的性质，以及Ly 49对肽选择性的体内意义。我们的长期目标是了解Ly 49识别的肽选择性的分子基础。这项工作的重要性和独特性在于，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Ly 49的识别，可能会提供新的见解宿主抗感染，如汉坦病毒在小鼠中，并可能在马，一个物种，也有一个扩展的Ly 49基因家族的疾病。我们假设某些特定的内源性肽支持Ly 49对CI的识别，这将为Ly 49监测的目的提供一个窗口。我们还假设，肽的选择性决定Ly 49的亲和力阈值，并在体内的意义。为了检验这些假设，我们提出了以下目标： 1.评估控制Ly 49 C识别的内源性肽的多样性和同一性。质谱法的进展已经鉴定了与Cl结合的内源性肽。我们将研究与CI（Kb）结合的单个内源性肽，以支持Ly 49 C识别，包括它们的分子特性和起源蛋白（来自肽序列数据库匹配），有助于控制Ly 49 C识别。 2.定义Cl结合肽同一性在确定Ly 49亲和力中的影响。我们将通过表面等离子体共振测定Ly 49 C对不同Cl（Kb）-肽复合物的亲和力。这应该揭示是否存在由结合肽的身份控制的Ly 49 C相互作用的阈值亲和力。 3.确定Ly 49肽选择性对体内NK细胞活性的影响。通过在b2 m-/-背景上的转基因，我们将表达基因工程改造的单链肽-Cl复合物以确定不同的单个肽支持体内NK细胞的许可和功能的能力。

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.