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

描述（由申请人提供）：烧伤每年夺去4000名美国平民的生命，另有50万人寻求医疗照顾。这造成每年超过90万个住院日和每年超过10亿美元的相关费用，包括生产力损失。因此，开发一种限制烧伤进展并促进稳健伤口愈合的疗法是一种未满足的需求，任何FDA批准的疗法都没有解决这一问题。P12是一种小的纤连蛋白肽，具有限制损伤进展的前景。本项目的总体目标是研究P12促进细胞存活的机制，通过建立体外模型来模拟烧伤周围组织中的受损条件。这是先前生物化学实验的逻辑延伸，证明了P12与血小板衍生生长因子-BB（PDGF-BB）的协同作用以促进细胞存活。我们的第一个假设是P12通过作为PDGF-BB辅因子增强PDGF-BB存活信号而起作用。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来模拟烧伤周围组织中的缺血状况。将通过蛋白质印迹法探测PDGFR效应子上游和下游PI3K-Akt通路以确定在营养剥夺下P12的促存活活性的效应子。的结构 P12非常类似于常用的蛋白转导结构域（PTD），其促进结合的货物内化到哺乳动物细胞中。因此，P12有可能将PDGF/PDGFR复合物带入细胞内。通过不同的内化途径进入细胞，PDGF可以产生不同的信号传导，导致迁移，增殖或存活。此外，当生长因子受体存在于内体和细胞膜区室中时，它们触发不同的信号传导复合物和信号传导途径。基于PDGF-BB和P12之间的结合，第二种假设是P12增强PDGFR的内化或改变其运输，从而其促存活信号传导。因此，目的2是确定P12是否调节PDGF-BB/PDGFR-β内化/运输。为此目的，将通过可逆生物素化来测量PDGF/P12诱导的PDGFR内化。然后，将采用不同内化途径的抑制剂来确定P12调节的途径。最后，将使用抑制性肽来中断P12和PDGF-BB之间的结合，以确定它们的结合对于P12促存活活性、下游信号传导事件和PDGFR内化是否重要。1 公共卫生相关性：在美国，烧伤每年夺去4000人的生命，另有50万人需要就医。烧伤每年还造成90多万住院日，每年的相关费用超过10亿美元，包括生产力损失。这项补助金的目的是调查机制， 一种新的生物活性肽，作为限制烧伤进展和促进稳健伤口愈合的疗法，这是任何FDA批准的疗法都没有解决的。

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 PUBLIC HEALTH RELEVANCE: Burn injuries claim the lives of 4000 people in the United States each year and send another 500,000 to seek medical attention. Burn injuries also account for over 900,000 hospital days per year and more than $1 billion per year in associated costs, including loss of productivity. The purpose of this grant is to investigate the mechanism of a novel bioactive peptide as a therapy to limit burn injury progression and promote robust wound healing which is not addressed by any FDA approved therapy.