Novel temperature-responsive fusions of clathrin and caveolin to explore TGF-beta mediated fibrosis

网格蛋白和小窝蛋白的新型温度响应性融合物探索 TGF-β 介导的纤维化

• 批准号: 9788043
• 负责人: David Ryan Tyrpak
• 金额: $ 3.86万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2020-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9788043
• 关键词: Affect; Angiotensin II Receptor; Behavior; Biological Assay; Biotin; Caveolae; Caveolins; Cell Culture Techniques; Cell Line; Cell Surface Receptors; Centrifugation; Chemicals; Chimeric Proteins; Cholera Toxin; Clathrin; Clathrin Light Chains; Communities; Development; Disease; Dominant-Negative Mutation; Dynamin; Elastin; Endocytosis; Fibrosis; Genetic; Health; Heating; Hepatic Stellate Cell; Knowledge; Label; Liver; Liver Fibrosis; Liver diseases; MADH2 gene; Mediating; Mediator of activation protein; Membrane; Membrane Microdomains; Mentors; Mus; Nystatin; Organelles; Pathway interactions; Pharmaceutical Preparations; Pharmacology; Phase; Phenotype; Phosphorylation; Physiological; Physiology; Plasminogen Activator Inhibitor 1; Play; Primary carcinoma of the liver cells; Proteins; Pulmonary Fibrosis; Receptor Signaling; Recombinant Proteins; Research; Role; Route; Scientist; Signal Transduction; Sucrose; Surface; TGFB1 gene; TGFBR1 gene; TGFBR2 gene; Temperature; Therapeutic; Time; Transformed Cell Line; Transforming Growth Factor beta; Transforming Growth Factor beta Receptors; United States National Institutes of Health; Western Blotting; Work; biocompatible polymer; caveolin 1; cellular imaging; chronic liver disease; confocal imaging; density; inhibitor/antagonist; innovation; kidney fibrosis; knock-down; nanomedicine; new technology; novel; novel strategies; pathogen; polypeptide; prevent; receptor; receptor internalization; receptor-mediated signaling; response; self assembly; tool; trafficking

PROJECT SUMMARY This proposal studies Transforming Growth Factor Beta Receptors (TGFBRs) using novel fusion proteins for rapid, reversible, and precise inhibition of Clathrin mediated endocytosis (CME) and activation of lipid raft/Caveolin mediated endocytosis (CAVME). CME and CAVME are the predominant mechanisms for receptor internalization, and their deregulation is implicated in myriad diseases, including fibrosis of the kidney and liver. Transforming Growth Factor Beta 1 (TGF-B1) is a master regulator of liver fibrosis; therefore, signaling will be evaluated using primary mouse hepatic stellate cells, along with transformed cell lines. TGF- B1 fibrotic signaling is mediated through its two cell surface receptors, TGFBR1 and TGFBR2. Endocytosis plays a major regulatory role in TGFBR mediated signaling, as TGF-B1/TGFBR internalization via CME leads to pro-fibrotic SMAD signaling while internalization via CAVME downregulates this signaling. Despite the importance of endocytosis in fibrosis and normal physiology, distinguishing if cargo (e.g. a receptor, a drug, a pathogen) is selectively internalized via CME or CAVME remains challenging, as scientists cannot exclusively manipulate one pathway at a time. This proposal aims to overcome the limitations of currently employed endocytic inhibitors/activators by using temperature sensitive recombinant proteins composed of Clathrin light chain (CLC) or Caveolin 1 (CAV1) and a thermally responsive Elastin-like polypeptide (ELP). I hypothesize that ELP-mediated assembly of CLC will inhibit CME and down regulate TGF-B1 mediated fibrotic signaling, while CAV1-ELP assembly will activate CAVME but also down regulate this signaling. In summary, this proposal employs the TGF-B1/TGFBR pathway and primary hepatic stellate cells to further characterize CAV1-ELP and CLC-ELP fusion proteins. Completion of this project will provide the scientific community with: 1) new tools to rapidly and reversibly manipulate endocytosis; 2) a better understanding of how TGFBR trafficking contributes to a fibrotic cellular phenotype; 3) higher-fidelity knowledge about how endocytosis regulates HSC activation; and 4) new experimental assays that may identify targets for anti-fibrotic therapeutics.

项目摘要 该提案使用新型融合蛋白研究转化生长因子β受体（TGFBR）， 快速、可逆和精确地抑制网格蛋白介导的内吞作用（CME）和脂质活化 筏/小窝蛋白介导的内吞作用（CAVME）。CME和CAVME是主要的机制， 受体内化，以及它们的失调与包括肾纤维化在内的无数疾病有关 和肝脏。转化生长因子β 1（TGF-β 1）是肝纤维化的主要调节因子;因此， 使用原代小鼠肝星状细胞以及转化的细胞系沿着评价信号传导。转化生长因子 B1纤维化信号通过其两种细胞表面受体TGFBR 1和TGFBR 2介导。内吞 在TGFBR介导的信号传导中起主要调节作用，因为TGF-B1/TGFBR通过CME内化导致 促纤维化SMAD信号传导，而通过CAVME的内化下调该信号传导。尽管 内吞作用在纤维化和正常生理学中的重要性，如果货物（例如受体、药物、 病原体）通过CME或CAVME选择性内化仍然具有挑战性，因为科学家不能完全 一次只操纵一条通道该提案旨在克服目前就业的局限性， 通过使用由网格蛋白轻链组成的温度敏感性重组蛋白的内吞抑制剂/激活剂 链（CLC）或小窝蛋白1（CAV 1）和热响应弹性蛋白样多肽（ELP）。我假设 ELP介导的CLC组装将抑制CME并下调TGF-β 1介导的纤维化信号传导， CAV 1-ELP组装将激活CAVME，但也下调该信号传导。总而言之，这一提议 采用TGF-B1/TGFBR途径和原代肝星状细胞进一步表征CAV 1-ELP， CLC-ELP融合蛋白。该项目的完成将为科学界提供：1）新的 快速和可逆地操纵内吞作用的工具; 2）更好地理解TGF-β R如何 贩运有助于纤维化细胞表型; 3）关于如何更高保真的知识 内吞作用调节HSC活化;和4）新的实验测定，可以确定的目标， 抗纤维化治疗。