Recognition of Fibrinogen by Leukocyte Integrins

项目摘要/摘要 白细胞整合素aMb2(CD11b/CD18，Mac-1)在正常保护性炎症中起关键作用 反应和病理性炎症。这种受体具有惊人的粘附性和信号传递能力。 这使得它成为东道主防守中的头号主力。它也是一种潜在的治疗靶点 许多疾病中，炎症起着重要作用，包括心血管疾病。这个 归因于aMb2的不同功能和活动源于它在结构上结合多种 不同的配体。然而，使aMb2表现出广泛的配体识别的机制仍然是 人们对此知之甚少。我们之前对aMb2配体纤维蛋白原原型的研究提供了初步的见解 受体的Ami结构域识别其配体的机制。在过去的资助期 我们已经解决了一致的AMI领域识别基序，我们称之为IRM。IRM的一个关键功能是 由碱性和疏水氨基酸残基的特定组合组成的小核心，普遍存在于 许多aMb2配体。IRM的特征与aMb2识别广谱的能力是一致的 不相关序列的多样性，从而形成Mb2配体结合混杂的分子基础。 特异的Aim1是为了进一步表征aMb2广泛识别特异性的机制。 将使用组合多肽文库和突变分析来阐明其结构特征 IRM。质谱仪将被用来确定炎症相关蛋白的作用 对IRM功能的修改。我们的初步研究显示，中性粒细胞分泌产物 都富含IRMS，这使得他们的预测成为一类新的aMb2配体。我们已经找到了那个 其中，人中性粒细胞天冬氨酸多肽LL-37能有效结合aMb2，并产生一种有效的aMb2- 依赖的迁徙反应。基于这一发现，我们提出了LL-37和其他中性粒细胞来源的 蛋白质/多肽通过与单核/巨噬细胞结合aMb2发挥其强大的免疫调节作用。 具体目标2是通过表征依赖于Mb2的单核细胞反应来检验这一假设 LL-37。IL-37对aMb2信号和迁移功能的影响将通过aMb2- 在体内动物模型中表达和aMb2缺陷的细胞。过去资助期的研究 整合素aDb2被鉴定为一种多配体受体，其特异性与aMb2相似，并揭示其 炎症巨噬细胞的上调抑制了它们的迁移。具体目标3是描述 巨噬细胞上含量最丰富、粘附性最强的两种整合素aMb2和aDb2在巨噬细胞迁移中的作用 炎症消退过程中炎症部位的细胞。巨噬细胞的外流通过 将在野生型和整合素缺陷小鼠身上研究淋巴管引流。总体而言，这些研究将 增加对aMb2配基识别原理的理解，将给出新的 对aMb2和aDb2生物学的洞察可能有助于设计新的治疗策略。

PROJECT SUMMARY/ABSTRACT Leukocyte integrin aMb2 (CD11b/CD18, Mac-1) plays a pivotal role in normal protective inflammatory response and pathological inflammation. This receptor has prodigious adhesive and signaling capabilities which allowed it to become the premier workhorse in host defense. It is also a potential therapeutic target in many diseases in which inflammation plays an essential role, including cardiovascular diseases. The diverse functions and activities ascribed to aMb2 arise from its ability to bind a multitude of structurally diverse ligands. However, the mechanisms which allow aMb2 to exhibit broad ligand recognition are still poorly understood. Our previous studies with a prototype aMb2 ligand fibrinogen provided initial insight into the mechanism by which the aMI-domain of the receptor recognizes its ligands. In the past funding period we have solved the consensus aMI-domain recognition motif, we termed IRM. A key feature of IRM is a small core consisting of specific combinations of basic and hydrophobic amino acid residues ubiquitous in many aMb2 ligands. The characteristics of IRM are consistent with the capacity of aMb2 to recognize a wide variety of unrelated sequences and, thus, form a molecular basis for aMb2 ligand binding promiscuity. Specific Aim1 is to further characterize the mechanism underlying broad recognition specificity of aMb2. Combinatorial peptide libraries and mutational analyses will be used to clarify the structural features of IRM. Mass spectrometry will be used to determine the effect of inflammation-associated protein modifications on the function of IRM. Our preliminary studies revealed that neutrophil secretion products are enriched in IRMs which allowed their prediction as a new class of aMb2 ligands. We have found that one of them, human neutrophil cathelicidin peptide LL-37, effectively binds aMb2 and unduces a potent aMb2- dependent migratory response. Based on this finding we propose that LL-37 and other neutrophil-derived proteins/peptides exert their potent immunomodulatory effects by binding aMb2 on monocyte/macrophages. Specific Aim 2 is to test this hypothesis by characterizing aMb2-dependent monocyte responses elicited by LL-37. The effect of LL-37 on signaling and migratory functions of aMb2 will be determined using aMb2- expressing and aMb2-deficient cells and in the in vivo animal model. Studies over the past funding period identified integrin aDb2 as a multiligand receptor with specificity similar to that of aMb2 and revealed that its upregulation on inflammatory macrophages inhibits their migration. Specific Aim 3 is to characterize the role of aMb2 and aDb2, two most abundant and adhesive integrins on macrophages, in emigration of these cells from the inflammatory site during the resolution of inflammation. The efflux of macrophages by draining lymphatics will be investigated in wild-type and integrin-deficient mice. Overall, these studies will lead to an increased understanding of the principles which govern ligand recognition by aMb2, will give new insights into the biology of aMb2 and aDb2 and may be useful in the design of novel therapeutic strategies.