Elucidation of a Eukaryotic Chemorepulsion Mechanism

真核细胞的定向运动远离化学排斥物的来源似乎起到了促进作用。 在发育和炎症的解决中起主要作用，但对真核生物知之甚少。 以及它们如何引导细胞运动。从分泌型细胞中化学排斥网骨藻细胞 称为AprA的蛋白质是一种模型，其中人们可以将生物化学和遗传学联合收割机结合起来以阐明化学排斥。 使用可用的突变体和初步的遗传筛选，我们鉴定了AprA受体， AprA信号转导通路的组成部分，并发现AprA化学排斥涉及一个 与化学吸引力有着根本不同的机制。 预测AprA与人分泌蛋白二肽基肽酶IV（DPPIV）结构相似， 并与DPPIV具有相同的功能特性。我们发现DPPIV对人和小鼠具有化学排斥作用， 中性粒细胞，发现G蛋白偶联受体PAR2介导DPPIV化学排斥，并发现， 小分子PAR 2激动剂作为中性粒细胞化学排斥剂。初步研究表明， AprA和PAR 2激动剂化学排斥机制之间的强烈相似性。 急性呼吸窘迫综合征（ARDS）是一种无法治愈的疾病，涉及损害肺部 导致中性粒细胞进入肺部中性粒细胞进一步损害肺部，导致更多的中性粒细胞 进入正反馈循环，这导致每年约20万例ARDS患者中约40%的死亡 在美国.对可能的治疗方法的一个令人兴奋的见解是，当DPPIV或PAR 2激动剂被用于治疗时， 在两种ARDS小鼠模型中，吸入肺中的它们诱导中性粒细胞离开肺。关键 将其应用于临床的障碍是，我们需要知道PAR2激动剂的化学排斥作用是什么， 机制是预测潜在的药物相互作用和副作用。 为了深入了解诱导中性粒细胞离开组织的基本机制和方法，我们 建议利用的力量阐明AprA和PAR2激动剂的化学排斥机制 Dictyosteoblasts来领导这项工作。我们将严格测试新发现的Dictyosteoblastoma化学排斥途径 组件，并确定它们在通路中的功能。我们将完成网骨藻的遗传 筛选，以进一步了解化学排斥，然后使用这些信息来指导检查 可能与中性粒细胞的机制相似。在研究人类中性粒细胞化学排斥时，我们观察到， 男性和女性中性粒细胞之间的显着差异，我们将描绘的程度和分子 这种差异背后的机制。总之，拟议的工作将阐明一个鲜为人知的 基本机制，阐明先天免疫系统中意想不到的性别差异，并帮助 阐明PAR2激动剂如何在临床上用于将过量的中性粒细胞从组织中排出， 可能对男性和女性有不同的治疗。

The directed movement of eukaryotic cells away from a source of a chemorepellent appears to play a major role in development and the resolution of inflammation, but very little is known about eukaryotic chemorepellents and how they direct cell motility. Chemorepulsion of Dictyostelium cells from a secreted protein called AprA is a model where one can combine biochemistry and genetics to elucidate chemorepulsion. Using available mutants and a preliminary genetic screen, we identified the AprA receptor, identified several components of the AprA signal transduction pathway, and found that AprA chemorepulsion involves a fundamentally different mechanism from chemoattraction. AprA has predicted structural similarity to the human secreted protein dipeptidyl peptidase IV (DPPIV), and shares functional properties with DPPIV. We found that DPPIV is a chemorepellent for human and mouse neutrophils, found that the G protein-coupled receptor PAR2 mediates DPPIV chemorepulsion, and found that small molecule PAR2 agonists act as neutrophil chemorepellents. Preliminary studies indicate that there are strong similarities between the AprA and PAR2 agonist chemorepulsion mechanisms. Acute respiratory distress syndrome (ARDS) is an untreatable disease involving damage to the lungs causing neutrophils to enter the lungs. The neutrophils further damage the lungs, causing more neutrophil entry in a positive feedback loop, and this results in the death of ~40% of the ~200,000 ARDS patients per year in the US. An exciting insight into a possible therapeutic approach is that when DPPIV or PAR2 agonists are aspirated into the lungs, they induce neutrophils to leave the lungs in two mouse models of ARDS. The key roadblock to moving this into the clinic is that we need to know what the PAR2 agonist chemorepulsion mechanism is to anticipate potential drug interactions and side effects. To gain insights into a fundamental mechanism, and ways to induce neutrophils to leave a tissue, we propose to elucidate the AprA and PAR2 agonist chemorepulsion mechanisms using the power of Dictyostelium to lead the work. We will rigorously test newly identified Dictyostelium chemorepulsion pathway components, and determine where they function in the pathway. We will complete the Dictyostelium genetic screens to gain further insight into chemorepulsion, and then use this information to guide an examination of possibly similar mechanisms in neutrophils. While examining human neutrophil chemorepulsion, we observed a significant difference between male and female neutrophils, and we will delineate the extent and molecular mechanism underlying this difference. Together, the proposed work will elucidate a poorly understood fundamental mechanism, elucidate an unexpected sex difference in the innate immune system, and help elucidate how PAR2 agonists could be used clinically to drive excessive neutrophils out of a tissue, with possibly different treatments for men and women.