FUNCTIONAL ANALYSIS OF A PDGF-INDUCIBLE CYTOKINE

单核细胞趋化蛋白-1（MCP-1）是近年来发现的一种新的趋化因子， 定义的趋化因子家族称为“趋化因子”， 以其吸引特定白细胞亚群的能力而闻名。 MCP-1， 最初克隆为PDGF诱导型JE基因，吸引单核细胞， 记忆性T淋巴细胞排斥其他白细胞，如 中性粒细胞 由于它的引诱特性和它的模式， MCP-1的表达，MCP-1已被假设在一个发病机制中发挥作用。 具有单核细胞炎性成分的多种疾病，例如 动脉粥样硬化 然而，到目前为止， MCP-1参与疾病的因果关系。 在这项赠款的赞助下， MCP-1的化学引诱活性所需的区域，证明 MCP-1作为二聚体，并产生显性抑制突变体。 此外，过表达MCP-1的转基因小鼠提供了证据， MCP-1参与抵抗胞内病原体如L. 单核细胞增多症和M.结核 本补助金续期旨在：（1） 将结构/活性分析扩展到MCP-1及其 受体的相互作用位点;和（2）创建MCP-1缺陷小鼠 来验证MCP-1与疾病有关的假设。 以下 为实现这些目标制定了具体目标： 特定目标1：先前定义为对以下关键的MCP-1区域： 将通过扫描诱变分析活性，以鉴定特异性 参与受体相互作用的氨基酸。 以前未检查的区域 MCP-1也将被分析。 MCP-1受体B的区域也将是 通过取代IL-8受体1的胞外结构域和通过 定点诱变。 基于这些发现的肽将是 测试它们的抑制或激活特性。 具体目标2：我的实验室最近开发了MCP-1缺陷小鼠 通过靶向基因破坏。 将对这些小鼠的 基线表型及其对免疫学和感染性 挑战 MCP-1在动脉粥样硬化中的作用将通过 使这些小鼠接受致动脉粥样硬化饮食， 等位基因转化为动脉粥样硬化倾向的遗传背景。 特定目的3：许多趋化因子具有重叠的特性，这表明 这种冗余可能会阻止一种异常表型的出现， MCP-1缺陷小鼠。 由于趋化因子基因簇， 同时“敲除”在技术上难以通过 育种 相反，表达显性抑制基因的转基因小鼠 将构建趋化因子变体。 它们的交配会产生老鼠 多种趋化因子的多重同时破坏。

Monocyte chemoattractant protein-1 (MCP-1) is a member of a recently defined family of chemotactic factors called "chemokines," which are notable for their ability to attract specific leukocyte subsets. MCP-1, originally cloned as the PDGF-inducible JE gene, attracts monocytes and memory T lymphocytes to the exclusion of other leukocytes, such as neutrophils. Because of its attractant properties and its patterns of expression, MCP-1 has been hypothesized to play a pathogenetic role in a variety of diseases having a monocyte inflammatory component, such as atherosclerosis. So far, however, there have been no direct demonstrations of MCP-1's causal participation in disease. Under the auspices of this grant, structure/activity analyses defined regions of MCP-1 required for its chemoattractant activity, demonstrated that MCP-1 acts as a dimer, and generated a dominant suppressor mutant. In addition, transgenic mice overexpressing MCP-1 provided evidence that MCP-1 is involved in resistance to intracellular pathogens such as L. monocytogenes and M. tuberculosis. This grant renewal seeks: (1) to extend the structure/activity analyses to test regions of MCP-1 and its receptor for sites of interaction; and (2) to create MCP-1-deficient mice to test hypotheses about MCP-1's involvement in disease. The following specific aims are designed to accomplish these goals: SPECIFIC AIM 1: Regions of MCP-1 previously defined as critical for activity will be analyzed by scanning mutagenesis to identify specific amino acids involved in receptor interaction. Previously unexamined areas of MCP-1 will also be analyzed. Regions of MCP-1 receptor B will also be tested by substituting extracellular domains of IL-8 receptor 1 and by site-directed mutagenesis. Peptides based on these findings will be tested for their inhibitory or activating properties. SPECIFIC AIM 2: My laboratory has recently developed MCP-1-deficient mice by targeted gene disruption. These mice will be characterized for their baseline phenotype and their responses to immunologic and infectious challenges. MCP-1's role in atherosclerosis will be investigated by subjecting these mice to atherogenic diets and by breeding the disrupted alleles into an atherosclerosis-prone genetic background. SPECIFIC AIM 3: Many chemokines have overlapping properties, suggesting that redundancy might prevent the appearance of an abnormal phenotype in MCP-1-deficient mice. Since chemokine genes cluster, multiple simultaneous "knockouts" would be technically difficult to construct by breeding. Instead, transgenic mice expressing dominant suppressor chemokine variants will be constructed. Their mating will produce mice multiple simultaneous disruption of several chemokines.