Mechanistic studies of fibronectin peptide P12: a co-factor of PDGF-BB

描述（由申请人提供）：每年有4000名美国平民因烧伤而丧生，另有50万人需要就医。这相当于每年90多万个住院日，包括生产力损失在内的相关费用每年超过10亿美元。因此，开发一种限制烧伤进展和促进伤口愈合的治疗方法是一个未满足的需求，这是任何FDA批准的治疗方法都没有解决的问题。P12是一种小的纤维连接蛋白肽，具有限制损伤进展的前景。该项目的总体目标是通过模拟烧伤周围组织受损情况的体外模型来研究P12促进细胞存活的机制。这是先前生化实验的逻辑延伸，证明P12与血小板衍生生长因子- bb （PDGF-BB）协同作用可促进细胞存活。我们的第一个假设是P12通过增加PDGF-BB生存信号作为PDGF-BB辅助因子发挥作用。因此，目的1是确定P12对营养剥夺下AHDF中pdgf - bb刺激的信号转导通路，特别是PI3K/Akt通路的影响。为此，将采用营养剥夺系统模拟烧伤周围组织的缺血情况。通过western blot检测PI3K-Akt通路上下游的PDGFR效应因子，以确定营养剥夺条件下P12促生存活性的影响因子。的结构

DESCRIPTION (provided by applicant): Burn injuries claim the lives of 4000 United States civilians each year and send another 500,000 to seek medical attention. This accounts for over 900,000 hospital days per year and more than $1 billion per year in associated costs, including loss of productivity. Thus it is an unmet need to develop a therapy to limit burn injury progression and promote robust wound healing which is not addressed by any FDA approved therapy. P12 is a small fibronectin peptide with shown promise limiting injury progression. The overall goal of this project is to investigate the mechanism of P12 promoting cell survival by in vitro models created to mimic the compromised condition in peri-burn tissue. This is a logical extension of previous biochemical experiments demonstrating P12's synergistic effects with platelet derived growth factor-BB (PDGF-BB) to promote cell survival. Our first hypothesis is that P12 functions through augmenting PDGF-BB survival signals as a PDGF- BB co-factor. Thus Aim1 is to determine the effect of P12 on PDGF-BB-stimulated signal transduction pathways, particularly the PI3K/Akt pathway, in AHDF under nutrient deprivation. For this aim, nutrient deprivation system will be employed to mimic the ischemic condition in the peri-burn tissue. PDGFR effectors upstream and downstream PI3K-Akt pathway will be probed by western blot to determine the effectors of P12's pro-survival activity under nutrient deprivation. The structure of P12 is very similar to commonly used protein transduction domains (PTDs) that promote internalization of bound cargoes into mammalian cells. So it is possible that P12 can bring PDGF /PDGFR complex into the cell. By entering the cell through different internalization pathways, PDGF can produce different signaling leading to migration, proliferation or survival. Also, growth factor receptors trigger different signaling complex and signaling pathways when they are present in endosomal and cell membrane compartments. Based on the binding between PDGF-BB and P12, the second hypothesis is that P12 either enhances internalization or changes trafficking of PDGFR, thereby its pro-survival signaling. Thus Aim 2 is to determine whether P12 modulates PDGF-BB/PDGFR-¿ Internalization/ trafficking. For this aim, PDGF/P12 induced PDGFR internalization will be measured by reversible biotinylation. Then, Inhibitors for different internalization pathways will be employed to determine the pathway the P12 modulates. Finally, a inhibitory peptide will be used to interrupt the binding between P12 and PDGF-BB to determine whether their binding is important for P12 pro-survival activity, downstream signaling events, and PDGFR internalization. 1