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

纤连蛋白肽 P12 的机制研究：PDGF-BB 的辅助因子

• 批准号: 8497633
• 负责人: RICHARD August CLARK
• 金额: $ 16.63万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-07-01 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8497633
• 关键词: Accounting; Address; Adult; Affinity; Amino Acids; Area; Binding; Biochemical; Biotinylation; Blood Vessels; Burn injury; Cell Death; Cell Survival; Cell membrane; Cells; Cicatrix; Comb animal structure; Complex; Connective Tissue Cells; Data; Dermal; Development; Dose; Event; FDA approved; Family suidae; Fibroblasts; Fibronectins; Goals; Grant; Growth Factor; Growth Factor Receptors; Hypoxia; Injury; Length of Stay; Mammalian Cell; Measures; Modeling; Molecular; Nutrient; PDGFRB gene; PI3K/AKT; Pathway interactions; Peptides; Pharmaceutical Preparations; Platelet-Derived Growth Factor; Plug-in; Proteins; Rattus; Reporting; Role; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Site; Stress; Structure; System; TNF gene; Temperature; Tissues; United States; Western Blotting; Work; Wound Healing; base; cost; deprivation; in vitro Model; inhibitor/antagonist; injured; macromolecule; medical attention; migration; novel; platelet-derived growth factor BB; productivity loss; research study; synergism; trafficking; uptake

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

描述（由申请人提供）：烧伤每年夺走 4000 名美国平民的生命，并导致另外 500,000 人寻求医疗救助。这导致每年超过 900,000 个住院天数以及每年超过 10 亿美元的相关成本，包括生产力损失。因此，开发一种治疗方法来限制烧伤进展并促进伤口愈合是一个未满足的需求，而 FDA 批准的任何治疗方法都没有解决这一问题。 P12 是一种小纤连蛋白肽，有望限制损伤进展。该项目的总体目标是通过模拟烧伤周围组织受损状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) 具有协同作用，可促进细胞存活。我们的第一个假设是 P12 作为 PDGF-BB 辅助因子通过增强 PDGF-BB 存活信号发挥作用。因此，目的是确定营养剥夺下 AHDF 中 P12 对 PDGF-BB 刺激的信号转导途径（特别是 PI3K/Akt 途径）的影响。为此，将采用营养剥夺系统来模拟烧伤周围组织的缺血状况。通过蛋白质印迹法探测 PI3K-Akt 通路上游和下游的 PDGFR 效应子，以确定营养剥夺下 P12 促生存活性的效应子。的结构 P12 与常用的蛋白质转导结构域 (PTD) 非常相似，可促进结合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，从而导致迁移、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，它们会触发不同的信号复合物和信号传导途径。基于 PDGF-BB 和 P12 之间的结合，第二个假设是 P12 增强内化或改变 PDGFR 的运输，从而发挥其促生存信号传导。因此，目标 2 是确定 P12 是否调节 PDGF-BB/PDGFR-¿ 内化/运输。为此，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，抑制肽将用于中断 P12 和 PDGF-BB 之间的结合，以确定它们的结合对于 P12 促生存活性、下游信号传导事件和 PDGFR 内化是否重要。 1