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

描述(申请人提供)：烧伤每年夺走4000名美国平民的生命，另有500,000人求医。这相当于每年超过90万天的住院日，以及每年超过10亿美元的相关成本，包括生产力损失。因此，开发一种限制烧伤进展和促进创面愈合的疗法是一个未得到满足的需要，而FDA批准的任何疗法都没有解决这一问题。P12是一种小的纤维连接蛋白多肽，有望限制损伤的进展。该项目的总体目标是通过建立模拟烧伤周围组织受损条件的体外模型来研究P12促进细胞存活的机制。这是先前证明P12‘S与血小板衍生生长因子-BB协同作用以促进细胞存活的生化实验的合乎逻辑的延伸。我们的第一个假设是，P12通过增强PDGF-BB生存信号作为PDGF-BB辅助因子发挥作用。因此，AIM1旨在研究在营养缺乏条件下，P12对PDGF-BB刺激的AHDF信号转导通路，特别是PI3K/Akt通路的影响。为此，将使用营养剥夺系统来模拟烧伤周围组织的缺血状态。通过蛋白质印迹法检测PI3K-Akt通路上下游的PDGFR效应分子，以确定营养缺乏条件下P12‘S促生存活性的效应分子。的结构 P12与常用的蛋白转导结构域(PTD)非常相似，PTD促进结合的货物内化到哺乳动物细胞中。因此，P12可能将PDGF/PDGFR复合体带入细胞内。通过不同的内化途径进入细胞，PDGF可以产生不同的信号，导致迁移、增殖或生存。此外，当生长因子受体存在于内体和细胞膜之间时，它们会触发不同的信号复合体和信号通路。基于PDGF-BB与P12的结合，第二种假说认为P12既可促进PDGFR的内化，也可改变其转运，从而促进其促生存信号。因此，目标2是确定P12是否调节PDGF-BB/PDGFR-？内化/贩运。为此，将通过可逆生物素化来检测PDGF/P12诱导的PDGFR内化。然后，将使用不同内化途径的抑制剂来确定P12调节的途径。最后，将使用抑制肽来中断P12和PDGF-BB之间的结合，以确定它们的结合是否对P12促生存活性、下游信号事件和PDGFR内化重要。1

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